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  • Small molecule inhibition of dipeptidyl peptidase-4 enhances bone marrow progenitor cell function and angiogenesis in diabetic wounds. Translational research : the journal of laboratory and clinical medicine Whittam, A. J., Maan, Z. N., Duscher, D., Barrera, J. A., Hu, M. S., Fischer, L. H., Khong, S., Kwon, S. H., Wong, V. W., Walmsley, G. G., Giacco, F., Januszyk, M., Brownlee, M., Longaker, M. T., Gurtner, G. C. 2018

    Abstract

    In diabetes, stromal cell-derived factor-1 (SDF-1) expression and progenitor cell recruitment are reduced. Dipeptidyl peptidase-4 (DPP-4) inhibits SDF-1 expression and progenitor cell recruitment. Here we examined the impact of the DPP-4 inhibitor, MK0626, on progenitor cell kinetics in the context of wound healing. Wildtype (WT) murine fibroblasts cultured under high-glucose to reproduce a diabetic microenvironment were exposed to MK0626, glipizide, or no treatment, and SDF-1 expression was measured with ELISA. Diabetic mice received MK0626, glipizide, or no treatment for 6 weeks and then were wounded. Immunohistochemistry was used to quantify neovascularization and SDF-1 expression. Gene expression was measured at the RNA and protein level using quantitative polymerase chain reaction and ELISA, respectively. Flow cytometry was used to characterize bone marrow-derived mesenchymal progenitor cell (BM-MPC) population recruitment to wounds. BM-MPC gene expression was assayed using microfluidic single cell analysis. WT murine fibroblasts exposed to MK0626 demonstrated increased SDF-1 expression. MK0626 treatment significantly accelerated wound healing and increased wound vascularity, SDF-1 expression, and dermal thickness in diabetic wounds. MK0626 treatment increased the number of BM-MPCs present in bone marrow and in diabetic wounds. MK0626 had no effect on BM-MPC population dynamics. BM-MPCs harvested from MK0626-treated mice exhibited increased chemotaxis in response to SDF-1 when compared to diabetic controls. Treatment with a DPP-4 inhibitor significantly improved wound healing, angiogenesis, and endogenous progenitor cell recruitment in the setting of diabetes.

    View details for PubMedID 30452888

  • Pathway Analysis of Gene Expression in Murine Fetal and Adult Wounds ADVANCES IN WOUND CARE Hu, M. S., Hong, W., Januszyk, M., Walmsley, G. G., Luan, A., Maan, Z. N., Moshrefi, S., Tevlin, R., Wan, D. C., Gurtner, G. C., Longaker, M. T., Lorenz, H. 2018
  • Pathway Analysis of Gene Expression of E14 Versus E18 Fetal Fibroblasts ADVANCES IN WOUND CARE Hu, M. S., Borrelli, M. R., Januszyk, M., Luan, A., Malhotra, S., Walmsley, G. G., Hong, W., Tevlin, R., Gurtner, G. C., Longaker, M. T., Lorenz, H. P. 2018; 7 (1): 1–10

    Abstract

    Objective: Fetuses early in gestation heal skin wounds without forming scars. The biological mechanisms behind this process are largely unknown. Fibroblasts, however, are cells known to be intimately involved in wound healing and scar formation. We examined fibroblasts in different stages of development to characterize differences in gene expression that may result in the switch from regenerative wound repair to repair with scarring. Approach: Fibroblasts were isolated and cultured from the back skin of BALB/c wild-type mouse fetuses at embryonic day (E)14 and E18 (n = 10). The fibroblast total RNA was extracted, and microarray analysis was conducted using chips containing 42,000 genes. Significance analysis of microarrays was performed to identify genes with greater than twofold expression difference and a false discovery rate of less than two. Identified genes subsequently underwent enrichment analysis to detect differentially expressed pathways. Results: Two hundred seventy-five genes were differentially expressed between E14 and E18 in fetal fibroblasts. Thirty genes were significantly downregulated and 245 genes were significantly upregulated at E18 compared with E14. Ingenuity pathway analysis identified the top 20 signaling pathways differentially activated in fetal fibroblasts between the E18 and E14 time points. Innovation: To our knowledge, this work represents the first instance where differentially expressed genes and signaling pathways between fetal fibroblasts at E14 and E18 have been studied. Conclusion: The genes and pathways identified here potentially underlie the mechanism behind the transition from fetal wound healing via regeneration to wound healing by repair, and may prove to be key targets for future therapeutics.

    View details for PubMedID 29344429

  • Pathway Analysis of Gene Expression in Murine Fetal and Adult Wounds. Advances in wound care Hu, M. S., Hong, W. X., Januszyk, M., Walmsley, G. G., Luan, A., Maan, Z. N., Moshrefi, S., Tevlin, R., Wan, D. C., Gurtner, G. C., Longaker, M. T., Lorenz, H. P. 2018; 7 (8): 262–75

    Abstract

    Objective: In early gestation, fetal wounds heal without fibrosis in a process resembling regeneration. Elucidating this remarkable mechanism can result in tremendous benefits to prevent scarring. Fetal mouse cutaneous wounds before embryonic day (E)18 heal without scar. Herein, we analyze expression profiles of fetal and postnatal wounds utilizing updated gene annotations and pathway analysis to further delineate between repair and regeneration. Approach: Dorsal wounds from time-dated pregnant BALB/c mouse fetuses and adult mice at various time points were collected. Total RNA was isolated and microarray analysis was performed using chips with 42,000 genes. Significance analysis of microarrays was utilized to select genes with >2-fold expression differences with a false discovery rate of <2. Enrichment analysis was performed on significant genes to identify differentially expressed pathways. Results: Our analysis identified 471 differentially expressed genes in fetal versus adult wounds following injury. Utilizing enrichment analysis of significant genes, we identified the top 20 signaling pathways that were upregulated and downregulated at 1 and 12 h after injury. At 24 h after injury, we discovered 18 signaling pathways upregulated in adult wounds and 11 pathways upregulated in fetal wounds. Innovation: These novel target genes and pathways may reveal repair mechanisms of the early fetus that promote regeneration over fibrosis. Conclusion: Our microarray analysis recognizes hundreds of possible genes as candidates for regulators of scarless versus scarring wound repair. Enrichment analysis reveals 109 signaling pathways related to fetal scarless wound healing.

    View details for PubMedID 30087802

  • Is Distraction Osteogenesis of the Irradiated Craniofacial Skeleton Contraindicated? JOURNAL OF CRANIOFACIAL SURGERY Momeni, A., Januszyk, M., Wan, D. C. 2017; 28 (5): 1236–41

    Abstract

    Craniofacial distraction osteogenesis (DO) is a common treatment modality today. Despite its numerous advantages, however, concerns have been expressed regarding the use of DO in the irradiated setting.A systematic review was performed to identify all published reports of patients who underwent DO of the irradiated craniofacial skeleton. The following parameters were of particular interest: postoperative complications, specifically, insufficient bone formation, fracture, and hardware exposure (intraoral and cutaneous), as well as the need for additional bone grafting.The initial search retrieved a total of 183 articles of which 20 articles (38 patients) met predetermined inclusion criteria. The most common site of distraction was the mandible (76.3%). The median radiation dose was 50.7 Gy (range, 30-70 Gy). Bone defects ranged from 30 to 80 mm (median, 42.5 mm). Complications were encountered in 19 patients (50%), with insufficient bone formation being most common (9 patients; 23%). The overall incidence of complications was not significantly associated with radiation dosage (P = 0.79). The remaining procedural and demographic variables also failed to meet statistical significance when compared against the overall complication rate (P = 0.27-0.97).The complication rate associated with craniofacial DO of the irradiated skeleton does not appear to be substantially higher than what is reported for DO in the nonirradiated setting. As such, patients should be offered this treatment modality, particularly in light of the fact, that it offers the option to decrease patient morbidity as well as treatment complexity.

    View details for PubMedID 28665865

  • Isolation of CD248-expressing stromal vascular fraction for targeted improvement of wound healing. Wound repair and regeneration Brett, E., Zielins, E. R., Chin, M., Januszyk, M., Blackshear, C. P., Findlay, M., Momeni, A., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2017

    Abstract

    Wound healing remains a global issue of disability, cost, and health. Addition of cells from the stromal vascular fraction (SVF) of adipose tissue has been shown to increase the rate of full thickness wound closure. This study aimed to investigate the angiogenic mechanisms of CD248+ SVF cells in the context of full thickness excisional wounds. Single cell transcriptional analysis was used to identify and cluster angiogenic gene-expressing cells, which was then correlated with surface marker expression. SVF cells isolated from human lipoaspirate were FACS sorted based on the presence of CD248. Cells were analyzed for angiogenic gene expression and ability to promote microvascular tubule formation in vitro. Following this, 6mm full thickness dermal wounds were created on the dorsa of immunocompromised mice and then treated with CD248+, CD248-, or unsorted SVF cells delivered in a pullalan-collagen hydrogel or the hydrogel alone. Wounds were measured every other day photometrically until closure. Wounds were also evaluated histologically at 7 and 14 days post-wounding and when fully healed to assess for reepithelialization and development of neovasculature. Wounds treated with CD248+ cells healed significantly faster than other treatment groups, and at 7 days, had quantitatively more reepithelialization. Concurrently, immunohistochemistry of CD31 revealed a much higher presence of vascularity in the CD248+ SVF cells treated group at the time of healing and at 14 days post-op, consistent with a pro-angiogenic effect of CD248+ cells in vivo. Therefore, using CD248+ pro-angiogenic cells obtained from SVF presents a viable strategy in wound healing by promoting increased vessel growth in the wound.

    View details for DOI 10.1111/wrr.12542

    View details for PubMedID 28464475

  • Comparison of the Hydroxylase Inhibitor Dimethyloxalylglycine and the Iron Chelator Deferoxamine in Diabetic and Aged Wound Healing. Plastic and reconstructive surgery Duscher, D., Januszyk, M., Maan, Z. N., Whittam, A. J., Hu, M. S., Walmsley, G. G., Dong, Y., Khong, S. M., Longaker, M. T., Gurtner, G. C. 2017; 139 (3): 695e-706e

    Abstract

    A hallmark of diabetes mellitus is the breakdown of almost every reparative process in the human body, leading to critical impairments of wound healing. Stabilization and activity of the transcription factor hypoxia-inducible factor (HIF)-1α is impaired in diabetes, leading to deficits in new blood vessel formation in response to injury. In this article, the authors compare the effectiveness of two promising small-molecule therapeutics, the hydroxylase inhibitor dimethyloxalylglycine and the iron chelator deferoxamine, for attenuating diabetes-associated deficits in cutaneous wound healing by enhancing HIF-1α activation.HIF-1α stabilization, phosphorylation, and transactivation were measured in murine fibroblasts cultured under normoxic or hypoxic and low-glucose or high-glucose conditions following treatment with deferoxamine or dimethyloxalylglycine. In addition, diabetic wound healing and neovascularization were evaluated in db/db mice treated with topical solutions of either deferoxamine or dimethyloxalylglycine, and the efficacy of these molecules was also compared in aged mice.The authors show that deferoxamine stabilizes HIF-1α expression and improves HIF-1α transactivity in hypoxic and hyperglycemic states in vitro, whereas the effects of dimethyloxalylglycine are significantly blunted under hyperglycemic hypoxic conditions. In vivo, both dimethyloxalylglycine and deferoxamine enhance wound healing and vascularity in aged mice, but only deferoxamine universally augmented wound healing and neovascularization in the setting of both advanced age and diabetes.This first direct comparison of deferoxamine and dimethyloxalylglycine in the treatment of impaired wound healing suggests significant therapeutic potential for topical deferoxamine treatment in ischemic and diabetic disease.

    View details for DOI 10.1097/PRS.0000000000003072

    View details for PubMedID 28234841

    View details for PubMedCentralID PMC5327844

  • Pharmacological rescue of diabetic skeletal stem cell niches. Science translational medicine Tevlin, R., Seo, E. Y., Marecic, O., McArdle, A., Tong, X., Zimdahl, B., Malkovskiy, A., Sinha, R., Gulati, G., Li, X., Wearda, T., Morganti, R., Lopez, M., Ransom, R. C., Duldulao, C. R., Rodrigues, M., Nguyen, A., Januszyk, M., Maan, Z., Paik, K., Yapa, K., Rajadas, J., Wan, D. C., Gurtner, G. C., Snyder, M., Beachy, P. A., Yang, F., Goodman, S. B., Weissman, I. L., Chan, C. K., Longaker, M. T. 2017; 9 (372)

    Abstract

    Diabetes mellitus (DM) is a metabolic disease frequently associated with impaired bone healing. Despite its increasing prevalence worldwide, the molecular etiology of DM-linked skeletal complications remains poorly defined. Using advanced stem cell characterization techniques, we analyzed intrinsic and extrinsic determinants of mouse skeletal stem cell (mSSC) function to identify specific mSSC niche-related abnormalities that could impair skeletal repair in diabetic (Db) mice. We discovered that high serum concentrations of tumor necrosis factor-α directly repressed the expression of Indian hedgehog (Ihh) in mSSCs and in their downstream skeletogenic progenitors in Db mice. When hedgehog signaling was inhibited during fracture repair, injury-induced mSSC expansion was suppressed, resulting in impaired healing. We reversed this deficiency by precise delivery of purified Ihh to the fracture site via a specially formulated, slow-release hydrogel. In the presence of exogenous Ihh, the injury-induced expansion and osteogenic potential of mSSCs were restored, culminating in the rescue of Db bone healing. Our results present a feasible strategy for precise treatment of molecular aberrations in stem and progenitor cell populations to correct skeletal manifestations of systemic disease.

    View details for DOI 10.1126/scitranslmed.aag2809

    View details for PubMedID 28077677

  • The Role of Focal Adhesion Kinase in Keratinocyte Fibrogenic Gene Expression. International journal of molecular sciences Januszyk, M., Kwon, S. H., Wong, V. W., Padmanabhan, J., Maan, Z. N., Whittam, A. J., Major, M. R., Gurtner, G. C. 2017; 18 (9)

    Abstract

    Abnormal skin scarring causes functional impairment, psychological stress, and high socioeconomic cost. Evidence shows that altered mechanotransduction pathways have been linked to both inflammation and fibrosis, and that focal adhesion kinase (FAK) is a key mediator of these processes. We investigated the importance of keratinocyte FAK at the single cell level in key fibrogenic pathways critical for scar formation. Keratinocytes were isolated from wildtype and keratinocyte-specific FAK-deleted mice, cultured, and sorted into single cells. Keratinocytes were evaluated using a microfluidic-based platform for high-resolution transcriptional analysis. Partitive clustering, gene enrichment analysis, and network modeling were applied to characterize the significance of FAK on regulating keratinocyte subpopulations and fibrogenic pathways important for scar formation. Considerable transcriptional heterogeneity was observed within the keratinocyte populations. FAK-deleted keratinocytes demonstrated increased expression of genes integral to mechanotransduction and extracellular matrix production, including Igtbl, Mmpla, and Col4a1. Transcriptional activities upon FAK deletion were not identical across all single keratinocytes, resulting in higher frequency of a minor subpopulation characterized by a matrix-remodeling profile compared to wildtype keratinocyte population. The importance of keratinocyte FAK signaling gene expression was revealed. A minor subpopulation of keratinocytes characterized by a matrix-modulating profile may be a keratinocyte subset important for mechanotransduction and scar formation.

    View details for PubMedID 28880199

    View details for PubMedCentralID PMC5618564

  • Delivery of monocyte lineage cells in a biomimetic scaffold enhances tissue repair. JCI insight Hu, M. S., Walmsley, G. G., Barnes, L. A., Weiskopf, K., Rennert, R. C., Duscher, D., Januszyk, M., Maan, Z. N., Hong, W. X., Cheung, A. T., Leavitt, T., Marshall, C. D., Ransom, R. C., Malhotra, S., Moore, A. L., Rajadas, J., Lorenz, H. P., Weissman, I. L., Gurtner, G. C., Longaker, M. T. 2017; 2 (19)

    Abstract

    The monocyte lineage is essential to normal wound healing. Macrophage inhibition or knockout in mice results in impaired wound healing through reduced neovascularization, granulation tissue formation, and reepithelialization. Numerous studies have either depleted macrophages or reduced their activity in the context of wound healing. Here, we demonstrate that by increasing the number of macrophages or monocytes in the wound site above physiologic levels via pullulan-collagen composite dermal hydrogel scaffold delivery, the rate of wound healing can be significantly accelerated in both wild-type and diabetic mice, with no adverse effect on the quality of repair. Macrophages transplanted onto wounds differentiate into M1 and M2 phenotypes of different proportions at various time points, ultimately increasing angiogenesis. Given that monocytes can be readily isolated from peripheral blood without in vitro manipulation, these findings hold promise for translational medicine aimed at accelerating wound healing across a broad spectrum of diseases.

    View details for PubMedID 28978794

  • Systematic Reviews in Craniofacial Trauma-Strengths and Weaknesses. Annals of plastic surgery Hunter, C., Januszyk, M., Wan, D. C., Momeni, A. 2016; 77 (3): 363-368

    Abstract

    Despite substantial advances in the management of craniofacial trauma, numerous clinical questions remain. These are increasingly being answered using systematic reviews (SRs). However, caution is warranted as their validity and role in influencing clinical practice has been called into question.A PubMed search was performed in October 2014 to identify SRs published up to and including September 2014 in 35 scientific journals. Two authors independently reviewed the literature and extracted data from included studies. Discrepancies were resolved by consensus. Assessment of multiple systematic reviews (AMSTAR) was used to determine the quality of SRs.The initial search retrieved 3080 articles of which 3051 articles were excluded after screening title and abstract. After full-text review of the remaining 29 articles, 3 additional articles were excluded, thus, leaving 26 SRs for final analysis. Regression analysis demonstrated that the overall number of published SRs increased significantly throughout the period analyzed (P = 0.022). The median AMSTAR score of all SRs was 4.5, consistent with a "poor-to-fair" quality. The interobserver agreement was high, as evidenced by a mean κ of 0.91. Although there appeared to be a trend toward an increase in AMSTAR score by year over the period analyzed, this failed to reach statistical significance in terms of median (P = 0.36) or absolute (P = 0.26) counts.A tremendous opportunity exists for improvements in the quality of SRs focusing on craniofacial trauma. In addition to familiarizing authors with quality criteria for SRs, adoption of strict reporting criteria by scientific journals may result in long-term improvements in the quality of reporting.

    View details for DOI 10.1097/SAP.0000000000000633

    View details for PubMedID 26418794

  • Microfluidic single-cell transcriptional analysis rationally identifies novel surface marker profiles to enhance cell-based therapies NATURE COMMUNICATIONS Rennert, R. C., Januszyk, M., Sorkin, M., Rodrigues, M., Maan, Z. N., Duscher, D., Whittam, A. J., Kosaraju, R., Chung, M. T., Paik, K., Li, A. Y., Findlay, M., Glotzbach, J. P., Butte, A. J., Gurtner, G. C. 2016; 7

    Abstract

    Current progenitor cell therapies have only modest efficacy, which has limited their clinical adoption. This may be the result of a cellular heterogeneity that decreases the number of functional progenitors delivered to diseased tissue, and prevents correction of underlying pathologic cell population disruptions. Here, we develop a high-resolution method of identifying phenotypically distinct progenitor cell subpopulations via single-cell transcriptional analysis and advanced bioinformatics. When combined with high-throughput cell surface marker screening, this approach facilitates the rational selection of surface markers for prospective isolation of cell subpopulations with desired transcriptional profiles. We establish the usefulness of this platform in costly and highly morbid diabetic wounds by identifying a subpopulation of progenitor cells that is dysfunctional in the diabetic state, and normalizes diabetic wound healing rates following allogeneic application. We believe this work presents a logical framework for the development of targeted cell therapies that can be customized to any clinical application.

    View details for DOI 10.1038/ncomms11945

    View details for Web of Science ID 000379085200001

    View details for PubMedID 27324848

  • Concomitant Liposuction Reduces Complications of Vertical Medial Thigh Lift in Massive Weight Loss Patients PLASTIC AND RECONSTRUCTIVE SURGERY Schmidt, M., Pollhammer, M. S., Januszyk, M., Duscher, D., Huemer, G. M. 2016; 137 (6): 1748–57

    Abstract

    Medial thigh lift procedures in the massive weight loss population have been associated with significant complication rates. Liposuction-assisted medial thighplasty has recently been introduced as a technical advancement to improve outcomes. To date, no study is available directly comparing the traditional approach and this new technique. Here, the authors evaluate outcomes and complications of both techniques in a retrospective cohort study.Outcomes of 59 patients undergoing vertical medial thighplasty at the authors' institution between 2008 and 2014 were assessed retrospectively. Evaluated parameters include age, sex, body mass indices, method of weight loss, comorbidities, and complications (e.g., seroma, infection, wound dehiscence, hematoma, and surgical revision). Appropriate statistical analysis was performed.There were 29 patients in the excision-only group and 30 patients in the liposuction-assisted group (all women; average age, 41.5 years). The overall complication rate was significantly reduced in the liposuction-assisted group (13 percent versus 59 percent; p < 0.001). The incidence of individual complications such as seroma formation (zero patients versus 10 patients; p < 0.001) and wound infection (one patient versus eight patients; p = 0.01) was significantly less in the liposuction-assisted group. In addition, we observed a significantly shorter hospital stay (6.0 days versus 7.8 days), reduced number of follow-up visits (2.0 versus 4.4), and reduced time to drain removal (1.8 days versus 4.1 days; p < 0.001) in the liposuction-assisted group.Liposuction-assisted medial thighplasty led to a significant reduction of complications and faster recovery in the massive weight loss patient population. As a consequence, the excision-only vertical thigh lift has been completely abandoned in the authors' clinical practice.Therapeutic, III.

    View details for PubMedID 27219231

  • Multiple Subsets of Brain Tumor Initiating Cells Coexist in Glioblastoma STEM CELLS Rennert, R. C., Achrol, A. S., Januszyk, M., Kahn, S. A., Liu, T. T., Liu, Y., Sahoo, D., Rodrigues, M., Maan, Z. N., Wong, V. W., Cheshier, S. H., Chang, S. D., Steinberg, G. K., Harsh, G. R., Gurtner, G. C. 2016; 34 (6): 1702-1707

    Abstract

    Brain tumor-initiating cells (BTICs) are self-renewing multipotent cells critical for tumor maintenance and growth. Using single-cell microfluidic profiling, we identified multiple subpopulations of BTICs co-existing in human glioblastoma, characterized by distinct surface marker expression and single-cell molecular profiles relating to distinct bulk tissue molecular subtypes. These data suggest BTIC subpopulation heterogeneity as an underlying source of intra-tumoral bulk tissue molecular heterogeneity, and will support future studies into BTIC subpopulation-specific therapies. This article is protected by copyright. All rights reserved.

    View details for DOI 10.1002/stem.2359

    View details for Web of Science ID 000378089500025

    View details for PubMedID 26991945

  • Microsurgical ear replantation is venous repair necessary? A systematic review MICROSURGERY Momeni, A., Liu, X., Januszyk, M., Wan, D. C., Buncke, G. M., Buntic, R. F., Parrett, B. M. 2016; 36 (4): 345-350

    Abstract

    A common postoperative observation after microsurgical ear replantation has been venous congestion necessitating alternate modes of decongestion, frequently in conjunction with blood transfusion. A comprehensive literature search was performed to assess the relationship between mode of vascular reconstruction and postoperative outcome as well as postoperative transfusion requirement after microsurgical ear replantation.The search was limited to cases of microsurgical ear replantation following complete amputation. Only articles published in English and indexed in PubMed were included.The initial search retrieved 285 articles, which was narrowed down to 40 articles reporting on 60 cases that matched the aforementioned criteria. Reconstruction of the arterial and venous limb (Group 1) was performed in 63.3% of patients and artery-only anastomosis (Group 2) was performed in 31.7%. Among measurable outcomes, only the duration of surgery was significantly different between groups (2.6 hours longer in Group 1 than Group 2; P = 0.0042).In light of contemporary data demonstrating successful artery-only ear replantation, replantation should not be abandoned when unable to establish venous outflow microsurgically. © 2015 Wiley Periodicals, Inc. Microsurgery 36:345-350, 2016.

    View details for DOI 10.1002/micr.22411

    View details for Web of Science ID 000377114900013

    View details for PubMedID 25847853

  • High-Resolution Microfluidic Single-Cell Transcriptional Profiling Reveals Clinically Relevant Subtypes among Human Stem Cell Populations Commonly Utilized in Cell-Based Therapies FRONTIERS IN NEUROLOGY Rennert, R. C., Schaefer, R., Bliss, T., Januszyk, M., Sorkin, M., Achrol, A. S., Rodrigues, M., Maan, Z. N., Kluba, T., Steinberg, G. K., Gurtner, G. C. 2016; 7

    Abstract

    Stem cell therapies can promote neural repair and regeneration, yet controversy regarding optimal cell source and mechanism of action has slowed clinical translation, potentially due to undefined cellular heterogeneity. Single-cell resolution is needed to identify clinically relevant subpopulations with the highest therapeutic relevance. We combine single-cell microfluidic analysis with advanced computational modeling to study for the first time two common sources for cell-based therapies, human NSCs and MSCs. This methodology has the potential to logically inform cell source decisions for any clinical application.

    View details for DOI 10.3389/fneur.2016.00041

    View details for Web of Science ID 000372534400001

    View details for PubMedCentralID PMC4801858

  • Extracellular superoxide dismutase deficiency impairs wound healing in advanced age by reducing neovascularization and fibroblast function EXPERIMENTAL DERMATOLOGY Fujiwara, T., Duscher, D., Rustad, K. C., Kosaraju, R., Rodrigues, M., Whittam, A. J., Januszyk, M., Maan, Z. N., Gurtner, G. C. 2016; 25 (3): 206-211

    Abstract

    Advanced age is characterized by impairments in wound healing, and evidence is accumulating that this may be due in part to a concomitant increase in oxidative stress. Extended exposure to reactive oxygen species (ROS) is thought to lead to cellular dysfunction and organismal death via the destructive oxidation of intra-cellular proteins, lipids and nucleic acids. Extracellular superoxide dismutase (ecSOD/SOD3) is a prime antioxidant enzyme in the extracellular space that eliminates ROS. Here, we demonstrate that reduced SOD3 levels contribute to healing impairments in aged mice. These impairments include delayed wound closure, reduced neovascularization, impaired fibroblast proliferation and increased neutrophil recruitment. We further establish that SOD3 KO and aged fibroblasts both display reduced production of TGF-β1, leading to decreased differentiation of fibroblasts into myofibroblasts. Taken together, these results suggest that wound healing impairments in ageing are associated with increased levels of ROS, decreased SOD3 expression and impaired extracellular oxidative stress regulation. Our results identify SOD3 as a possible target to correct age-related cellular dysfunction in wound healing.

    View details for DOI 10.1111/exd.12909

    View details for Web of Science ID 000373072800285

  • Extracellular superoxide dismutase deficiency impairs wound healing in advanced age by reducing neovascularization and fibroblast function. Experimental dermatology Fujiwara, T., Duscher, D., Rustad, K. C., Kosaraju, R., Rodrigues, M., Whittam, A. J., Januszyk, M., Maan, Z. N., Gurtner, G. C. 2016; 25 (3): 206-211

    Abstract

    Advanced age is characterized by impairments in wound healing, and evidence is accumulating that this may be due in part to a concomitant increase in oxidative stress. Extended exposure to reactive oxygen species (ROS) is thought to lead to cellular dysfunction and organismal death via the destructive oxidation of intra-cellular proteins, lipids and nucleic acids. Extracellular superoxide dismutase (ecSOD/SOD3) is a prime antioxidant enzyme in the extracellular space that eliminates ROS. Here, we demonstrate that reduced SOD3 levels contribute to healing impairments in aged mice. These impairments include delayed wound closure, reduced neovascularization, impaired fibroblast proliferation and increased neutrophil recruitment. We further establish that SOD3 KO and aged fibroblasts both display reduced production of TGF-β1, leading to decreased differentiation of fibroblasts into myofibroblasts. Taken together, these results suggest that wound healing impairments in ageing are associated with increased levels of ROS, decreased SOD3 expression and impaired extracellular oxidative stress regulation. Our results identify SOD3 as a possible target to correct age-related cellular dysfunction in wound healing.

    View details for DOI 10.1111/exd.12909

    View details for PubMedID 26663425

  • Adipose-Derived Stem Cell-Seeded Hydrogels Increase Endogenous Progenitor Cell Recruitment and Neovascularization in Wounds TISSUE ENGINEERING PART A Kosaraju, R., Rennert, R. C., Maan, Z. N., Duscher, D., Barrera, J., Whittam, A. J., Januszyk, M., Rajadas, J., Rodrigues, M., Gurtner, G. C. 2016; 22 (3-4): 295-305

    Abstract

    Adipose-derived mesenchymal stem cells (ASCs) are appealing for cell-based wound therapies because of their accessibility and ease of harvest, but their utility is limited by poor cell survival within the harsh wound microenvironment. In prior work, our laboratory has demonstrated that seeding ASCs within a soft pullulan-collagen hydrogel enhances ASC survival and improves wound healing. To more fully understand the mechanism of this therapy, we examined whether ASC-seeded hydrogels were able to modulate the recruitment and/or functionality of endogenous progenitor cells. Employing a parabiosis model and fluorescence-activated cell sorting analysis, we demonstrate that application of ASC-seeded hydrogels to wounds, when compared with injected ASCs or a noncell control, increased the recruitment of provascular circulating bone marrow-derived mesenchymal progenitor cells (BM-MPCs). BM-MPCs comprised 23.0% of recruited circulating progenitor cells in wounds treated with ASC-seeded hydrogels versus 8.4% and 2.1% in those treated with controls, p < 0.05. Exploring the potential for functional modulation of BM-MPCs, we demonstrate a statistically significant increase in BM-MPC migration, proliferation, and tubulization when exposed to hydrogel-seeded ASC-conditioned medium versus control ASC-conditioned medium (73.8% vs. 51.4% scratch assay closure; 9.1% vs. 1.4% proliferation rate; 10.2 vs. 5.5 tubules/HPF; p < 0.05 for all assays). BM-MPC expression of genes related to cell stemness and angiogenesis was also significantly increased following exposure to hydrogel-seeded ASC-conditioned medium (p < 0.05). These data suggest that ASC-seeded hydrogels improve both progenitor cell recruitment and functionality to effect greater neovascularization.

    View details for DOI 10.1089/ten.tea.2015.0277

    View details for Web of Science ID 000369987900012

    View details for PubMedID 26871860

    View details for PubMedCentralID PMC4779321

  • Challenges and Opportunities in Drug Delivery for Wound Healing. Advances in wound care Whittam, A. J., Maan, Z. N., Duscher, D., Wong, V. W., Barrera, J. A., Januszyk, M., Gurtner, G. C. 2016; 5 (2): 79-88

    Abstract

    Significance: Chronic wounds remain a significant public health problem. Alterations in normal physiological processes caused by aging or diabetes lead to impaired tissue repair and the development of chronic and nonhealing wounds. Understanding the unique features of the wound environment will be required to develop new therapeutics that impact these disabling conditions. New drug-delivery systems (DDSs) may enhance current and future therapies for this challenging clinical problem. Recent Advances: Historically, physical barriers and biological degradation limited the efficacy of DDSs in wound healing. In aiming at improving and optimizing drug delivery, recent data suggest that combinations of delivery mechanisms, such as hydrogels, small molecules, RNA interference (RNAi), as well as growth factor and stem cell-based therapies (biologics), could offer exciting new opportunities for improving tissue repair. Critical Issues: The lack of effective therapeutic approaches to combat the significant disability associated with chronic wounds has become an area of increasing clinical concern. However, the unique challenges of the wound environment have limited the development of effective therapeutic options for clinical use. Future Directions: New platforms presented in this review may provide clinicians and scientists with an improved understanding of the alternatives for drug delivery in wound care, which may facilitate the development of new therapeutic approaches for patients.

    View details for PubMedID 26862465

  • Rapid identification of slow healing wounds. Wound repair and regeneration Jung, K., Covington, S., Sen, C. K., Januszyk, M., Kirsner, R. S., Gurtner, G. C., Shah, N. H. 2016; 24 (1): 181-188

    Abstract

    Chronic nonhealing wounds have a prevalence of 2% in the United States, and cost an estimated $50 billion annually. Accurate stratification of wounds for risk of slow healing may help guide treatment and referral decisions. We have applied modern machine learning methods and feature engineering to develop a predictive model for delayed wound healing that uses information collected during routine care in outpatient wound care centers. Patient and wound data was collected at 68 outpatient wound care centers operated by Healogics Inc. in 26 states between 2009 and 2013. The dataset included basic demographic information on 59,953 patients, as well as both quantitative and categorical information on 180,696 wounds. Wounds were split into training and test sets by randomly assigning patients to training and test sets. Wounds were considered delayed with respect to healing time if they took more than 15 weeks to heal after presentation at a wound care center. Eleven percent of wounds in this dataset met this criterion. Prognostic models were developed on training data available in the first week of care to predict delayed healing wounds. A held out subset of the training set was used for model selection, and the final model was evaluated on the test set to evaluate discriminative power and calibration. The model achieved an area under the curve of 0.842 (95% confidence interval 0.834-0.847) for the delayed healing outcome and a Brier reliability score of 0.00018. Early, accurate prediction of delayed healing wounds can improve patient care by allowing clinicians to increase the aggressiveness of intervention in patients most at risk.

    View details for DOI 10.1111/wrr.12384

    View details for PubMedID 26606167

    View details for PubMedCentralID PMC4820011

  • Rapid identification of slow healing wounds WOUND REPAIR AND REGENERATION Jung, K., Covington, S., Sen, C. K., Januszyk, M., Kirsner, R. S., Gurtner, G. C., Shah, N. H. 2016; 24 (1): 181-188

    Abstract

    Chronic nonhealing wounds have a prevalence of 2% in the United States, and cost an estimated $50 billion annually. Accurate stratification of wounds for risk of slow healing may help guide treatment and referral decisions. We have applied modern machine learning methods and feature engineering to develop a predictive model for delayed wound healing that uses information collected during routine care in outpatient wound care centers. Patient and wound data was collected at 68 outpatient wound care centers operated by Healogics Inc. in 26 states between 2009 and 2013. The dataset included basic demographic information on 59,953 patients, as well as both quantitative and categorical information on 180,696 wounds. Wounds were split into training and test sets by randomly assigning patients to training and test sets. Wounds were considered delayed with respect to healing time if they took more than 15 weeks to heal after presentation at a wound care center. Eleven percent of wounds in this dataset met this criterion. Prognostic models were developed on training data available in the first week of care to predict delayed healing wounds. A held out subset of the training set was used for model selection, and the final model was evaluated on the test set to evaluate discriminative power and calibration. The model achieved an area under the curve of 0.842 (95% confidence interval 0.834-0.847) for the delayed healing outcome and a Brier reliability score of 0.00018. Early, accurate prediction of delayed healing wounds can improve patient care by allowing clinicians to increase the aggressiveness of intervention in patients most at risk.

    View details for DOI 10.1111/wrr.12384

    View details for Web of Science ID 000372925500018

    View details for PubMedCentralID PMC4820011

  • High-Resolution Microfluidic Single-Cell Transcriptional Profiling Reveals Clinically Relevant Subtypes among Human Stem Cell Populations Commonly Utilized in Cell-Based Therapies. Frontiers in neurology Rennert, R. C., Schäfer, R., Bliss, T., Januszyk, M., Sorkin, M., Achrol, A. S., Rodrigues, M., Maan, Z. N., Kluba, T., Steinberg, G. K., Gurtner, G. C. 2016; 7: 41-?

    Abstract

    Stem cell therapies can promote neural repair and regeneration, yet controversy regarding optimal cell source and mechanism of action has slowed clinical translation, potentially due to undefined cellular heterogeneity. Single-cell resolution is needed to identify clinically relevant subpopulations with the highest therapeutic relevance. We combine single-cell microfluidic analysis with advanced computational modeling to study for the first time two common sources for cell-based therapies, human NSCs and MSCs. This methodology has the potential to logically inform cell source decisions for any clinical application.

    View details for DOI 10.3389/fneur.2016.00041

    View details for PubMedID 27047447

  • Stem Cells in Wound Healing: The Future of Regenerative Medicine? A Mini-Review. Gerontology Duscher, D., Barrera, J., Wong, V. W., Maan, Z. N., Whittam, A. J., Januszyk, M., Gurtner, G. C. 2016; 62 (2): 216-225

    Abstract

    The increased risk of disease and decreased capacity to respond to tissue insult in the setting of aging results from complex changes in homeostatic mechanisms, including the regulation of oxidative stress and cellular heterogeneity. In aged skin, the healing capacity is markedly diminished resulting in a high risk for chronic wounds. Stem cell-based therapies have the potential to enhance cutaneous regeneration, largely through trophic and paracrine activity. Candidate cell populations for therapeutic application include adult mesenchymal stem cells, embryonic stem cells and induced pluripotent stem cells. Autologous cell-based approaches are ideal to minimize immune rejection but may be limited by the declining cellular function associated with aging. One strategy to overcome age-related impairments in various stem cell populations is to identify and enrich with functionally superior stem cell subsets via single cell transcriptomics. Another approach is to optimize cell delivery to the harsh environment of aged wounds via scaffold-based cell applications to enhance engraftment and paracrine activity of therapeutic stem cells. In this review, we shed light on challenges and recent advances surrounding stem cell therapies for wound healing and discuss limitations for their clinical adoption.

    View details for DOI 10.1159/000381877

    View details for PubMedID 26045256

  • Fibroblast-Specific Deletion of Hypoxia Inducible Factor-1 Critically Impairs Murine Cutaneous Neovascularization and Wound Healing PLASTIC AND RECONSTRUCTIVE SURGERY Duscher, D., Maan, Z. N., Whittam, A. J., Sorkin, M., Hu, M. S., Walmsley, G. G., Baker, H., Fischer, L. H., Januszyk, M., Wong, V. W., Gurtner, G. C. 2015; 136 (5): 1004-1013

    Abstract

    Diabetes and aging are known risk factors for impaired neovascularization in response to ischemic insult, resulting in chronic wounds, and poor outcomes following myocardial infarction and cerebrovascular injury. Hypoxia-inducible factor (HIF)-1α, has been identified as a critical regulator of the response to ischemic injury and is dysfunctional in diabetic and elderly patients. To better understand the role of this master hypoxia regulator within cutaneous tissue, the authors generated and evaluated a fibroblast-specific HIF-1α knockout mouse model.The authors generated floxed HIF-1 mice (HIF-1) by introducing loxP sites around exon 1 of the HIF-1 allele in C57BL/6J mice. Fibroblast-restricted HIF-1α knockout (FbKO) mice were generated by breeding our HIF-1 with tamoxifen-inducible Col1a2-Cre mice (Col1a2-CreER). HIF-1α knockout was evaluated on a DNA, RNA, and protein level. Knockout and wild-type mice were subjected to ischemic flap and wound healing models, and CD31 immunohistochemistry was performed to assess vascularity of healed wounds.Quantitative real-time polymerase chain reaction of FbKO skin demonstrated significantly reduced Hif1 and Vegfa expression compared with wild-type. This finding was confirmed at the protein level (p < 0.05). HIF-1α knockout mice showed significantly impaired revascularization of ischemic tissue and wound closure and vascularity (p < 0.05).Loss of HIF-1α from fibroblasts results in delayed wound healing, reduced wound vascularity, and significant impairment in the ischemic neovascular response. These findings provide new insight into the importance of cell-specific responses to hypoxia during cutaneous neovascularization.

    View details for Web of Science ID 000364092800001

    View details for PubMedID 26505703

  • Impairment in Fracture Healing in a Mouse Model of Type 2 Diabetes Is Driven by Skeletal Stem Cell Niche Dysregulation Tevlin, R., Seo, E., Mc Ardle, A., Tong, X., Januszyk, M., Yang, F., Gurtner, G. C., Chan, C. F., Weissman, I. L., Longaker, M. T. ELSEVIER SCIENCE INC. 2015: S115
  • Delivery of Macrophages in a Biomimetic Scaffold Accelerates Diabetic Wound Healing Through Enhanced Angiogenesis Walmsley, G. G., Hu, M. S., Duscher, D., Januszyk, M., Maan, Z. N., Senarath-Yapa, K., Tevlin, R., Zielins, E. R., Gurtner, G. C., Longaker, M. T. ELSEVIER SCIENCE INC. 2015: S113–S114
  • Microfluidic single cell transcriptional analysis reveals subpopulations of adipose derived stromal cells with enhanced angiogenic potential Zielins, E. R., Januszyk, M., Luan, A., Brett, E. A., Paik, K., Walmsley, G. G., Gurtner, G. C., Longaker, M. T., Wan, D. C. ELSEVIER SCIENCE INC. 2015: E26
  • Melanoma Progression Depends on CXCL12 Expression by Host Endothelium Maan, Z. N., Hu, M. S., Whittam, A., Fischer, L. H., Duscher, D., Walmsley, G. G., Januszyk, M., Whitmore, A. J., Longaker, M. T., Gurtner, G. C. ELSEVIER SCIENCE INC. 2015: S116
  • Using the wisdom of the crowds to find critical errors in biomedical ontologies: a study of SNOMED CT. Journal of the American Medical Informatics Association Mortensen, J. M., Minty, E. P., Januszyk, M., Sweeney, T. E., Rector, A. L., Noy, N. F., Musen, M. A. 2015; 22 (3): 640-648

    Abstract

    The verification of biomedical ontologies is an arduous process that typically involves peer review by subject-matter experts. This work evaluated the ability of crowdsourcing methods to detect errors in SNOMED CT (Systematized Nomenclature of Medicine Clinical Terms) and to address the challenges of scalable ontology verification.We developed a methodology to crowdsource ontology verification that uses micro-tasking combined with a Bayesian classifier. We then conducted a prospective study in which both the crowd and domain experts verified a subset of SNOMED CT comprising 200 taxonomic relationships.The crowd identified errors as well as any single expert at about one-quarter of the cost. The inter-rater agreement (κ) between the crowd and the experts was 0.58; the inter-rater agreement between experts themselves was 0.59, suggesting that the crowd is nearly indistinguishable from any one expert. Furthermore, the crowd identified 39 previously undiscovered, critical errors in SNOMED CT (eg, 'septic shock is a soft-tissue infection').The results show that the crowd can indeed identify errors in SNOMED CT that experts also find, and the results suggest that our method will likely perform well on similar ontologies. The crowd may be particularly useful in situations where an expert is unavailable, budget is limited, or an ontology is too large for manual error checking. Finally, our results suggest that the online anonymous crowd could successfully complete other domain-specific tasks.We have demonstrated that the crowd can address the challenges of scalable ontology verification, completing not only intuitive, common-sense tasks, but also expert-level, knowledge-intensive tasks.

    View details for DOI 10.1136/amiajnl-2014-002901

    View details for PubMedID 25342179

  • Skin fibrosis. Identification and isolation of a dermal lineage with intrinsic fibrogenic potential. Science Rinkevich, Y., Walmsley, G. G., Hu, M. S., Maan, Z. N., Newman, A. M., Drukker, M., Januszyk, M., Krampitz, G. W., Gurtner, G. C., Lorenz, H. P., Weissman, I. L., Longaker, M. T. 2015; 348 (6232)

    Abstract

    Dermal fibroblasts represent a heterogeneous population of cells with diverse features that remain largely undefined. We reveal the presence of at least two fibroblast lineages in murine dorsal skin. Lineage tracing and transplantation assays demonstrate that a single fibroblast lineage is responsible for the bulk of connective tissue deposition during embryonic development, cutaneous wound healing, radiation fibrosis, and cancer stroma formation. Lineage-specific cell ablation leads to diminished connective tissue deposition in wounds and reduces melanoma growth. Using flow cytometry, we identify CD26/DPP4 as a surface marker that allows isolation of this lineage. Small molecule-based inhibition of CD26/DPP4 enzymatic activity during wound healing results in diminished cutaneous scarring. Identification and isolation of these lineages hold promise for translational medicine aimed at in vivo modulation of fibrogenic behavior.

    View details for DOI 10.1126/science.aaa2151

    View details for PubMedID 25883361

    View details for PubMedCentralID PMC5088503

  • Studies in Fat Grafting: Part IV. Adipose-Derived Stromal Cell Gene Expression in Cell-Assisted Lipotransfer PLASTIC AND RECONSTRUCTIVE SURGERY Garza, R. M., Rennert, R. C., Paik, K. J., Atashroo, D., Chung, M. T., Duscher, D., Januszyk, M., Gurtner, G. C., Longaker, M. T., Wan, D. C. 2015; 135 (4): 1045-1055

    Abstract

    Fat graft volume retention remains highly unpredictable, but addition of adipose-derived stromal cells to fat grafts has been shown to improve retention. The present study aimed to investigate the mechanisms involved in adipose-derived stromal cell enhancement of fat grafting.Adipose-derived stromal cells isolated from human lipoaspirate were labeled with green fluorescent protein and luciferase. Fat grafts enhanced with adipose-derived stromal cells were injected into the scalp and bioluminescent imaging was performed to follow retention of adipose-derived stromal cells within the fat graft. Fat grafts were also explanted at days 1, 5, and 10 after grafting for adipose-derived stromal cell extraction and single-cell gene analysis. Finally, CD31 immunohistochemical staining was performed on fat grafts enriched with adipose-derived stromal cells.Bioluminescent imaging demonstrated significant reduction in luciferase-positive adipose-derived stromal cells within fat grafts at 5 days after grafting. A similar reduction in viable green fluorescent protein-positive adipose-derived stromal cells retrieved from explanted grafts was also noted. Single-cell analysis revealed expression of multiple genes/markers related to cell survival and angiogenesis, including BMPR2, CD90, CD105, FGF2, CD248, TGFß1, and VEGFA. Genes involved in adipogenesis were not expressed by adipose-derived stromal cells. Finally, CD31 staining revealed significantly higher vascular density in fat grafts explanted at day 10 after grafting.Although adipose-derived stromal cell survival in the hypoxic graft environment decreases significantly over time, these cells provide multiple angiogenic growth factors. Therefore, improved fat graft volume retention with adipose-derived stromal cell enrichment may be attributable to improved graft vascularization.

    View details for DOI 10.1097/PRS.0000000000001104

    View details for Web of Science ID 000351910200043

    View details for PubMedID 25502860

    View details for PubMedCentralID PMC4376612

  • Cell recruitment by amnion chorion grafts promotes neovascularization JOURNAL OF SURGICAL RESEARCH Maan, Z. N., Rennert, R. C., Koob, T. J., Januszyk, M., Li, W. W., Gurtner, G. C. 2015; 193 (2): 953-962

    Abstract

    Nonhealing wounds are a significant health burden. Stem and progenitor cells can accelerate wound repair and regeneration. Human amniotic membrane has demonstrated efficacy in promoting wound healing, though the underlying mechanisms remain unknown. A dehydrated human amnion chorion membrane (dHACM) was tested for its ability to recruit hematopoietic progenitor cells to a surgically implanted graft in a murine model of cutaneous ischemia.dHACM was subcutaneously implanted under elevated skin (ischemic stimulus) in either wild-type mice or mice surgically parabiosed to green fluorescent protein (GFP) + reporter mice. A control acellular dermal matrix, elevated skin without an implant, and normal unwounded skin were used as controls. Wound tissue was harvested and processed for histology and flow cytometric analysis.Implanted dHACMs recruited significantly more progenitor cells compared with controls (*P < 0.05) and displayed in vivo SDF-1 expression with incorporation of CD34 + progenitor cells within the matrix. Parabiosis modeling confirmed the circulatory origin of recruited cells, which coexpressed progenitor cell markers and were localized to foci of neovascularization within implanted matrices.In summary, dHACM effectively recruits circulating progenitor cells, likely because of stromal derived factor 1 (SDF-1) expression. The recruited cells express markers of "stemness" and localize to sites of neovascularization, providing a partial mechanism for the clinical efficacy of human amniotic membrane in the treatment of chronic wounds.

    View details for DOI 10.1016/j.jss.2014.08.045

    View details for Web of Science ID 000346244300056

    View details for PubMedID 25266600

  • Exercise induces stromal cell-derived factor-1a-mediated release of endothelial progenitor cells with increased vasculogenic function. Plastic and reconstructive surgery Chang, E., Paterno, J., Duscher, D., Maan, Z. N., Chen, J. S., Januszyk, M., Rodrigues, M., Rennert, R. C., Bishop, S., Whitmore, A. J., Whittam, A. J., Longaker, M. T., Gurtner, G. C. 2015; 135 (2): 340e-50e

    Abstract

    Endothelial progenitor cells have been shown to traffic to and incorporate into ischemic tissues, where they participate in new blood vessel formation, a process termed vasculogenesis. Previous investigation has demonstrated that endothelial progenitor cells appear to mobilize from bone marrow to the peripheral circulation after exercise. In this study, the authors investigate potential etiologic factors driving this mobilization and investigate whether the mobilized endothelial progenitor cells are the same as those present at baseline.Healthy volunteers (n = 5) performed a monitored 30-minute run to maintain a heart rate greater than 140 beats/min. Venous blood samples were collected before, 10 minutes after, and 24 hours after exercise. Endothelial progenitor cells were isolated and evaluated.Plasma levels of stromal cell-derived factor-1α significantly increased nearly two-fold immediately after exercise, with a nearly four-fold increase in circulating endothelial progenitor cells 24 hours later. The endothelial progenitor cells isolated following exercise demonstrated increased colony formation, proliferation, differentiation, and secretion of angiogenic cytokines. Postexercise endothelial progenitor cells also exhibited a more robust response to hypoxic stimulation.Exercise appears to mobilize endothelial progenitor cells and augment their function by means of stromal cell-derived factor 1α-dependent signaling. The population of endothelial progenitor cells mobilized following exercise is primed for vasculogenesis with increased capacity for proliferation, differentiation, secretion of cytokines, and responsiveness to hypoxia. Given the evidence demonstrating positive regenerative effects of exercise, this may be one possible mechanism for its benefits.

    View details for DOI 10.1097/PRS.0000000000000917

    View details for PubMedID 25626819

  • Transdermal deferoxamine prevents pressure-induced diabetic ulcers. Proceedings of the National Academy of Sciences of the United States of America Duscher, D., Neofytou, E., Wong, V. W., Maan, Z. N., Rennert, R. C., Inayathullah, M., Januszyk, M., Rodrigues, M., Malkovskiy, A. V., Whitmore, A. J., Walmsley, G. G., Galvez, M. G., Whittam, A. J., Brownlee, M., Rajadas, J., Gurtner, G. C. 2015; 112 (1): 94-99

    Abstract

    There is a high mortality in patients with diabetes and severe pressure ulcers. For example, chronic pressure sores of the heels often lead to limb loss in diabetic patients. A major factor underlying this is reduced neovascularization caused by impaired activity of the transcription factor hypoxia inducible factor-1 alpha (HIF-1α). In diabetes, HIF-1α function is compromised by a high glucose-induced and reactive oxygen species-mediated modification of its coactivator p300, leading to impaired HIF-1α transactivation. We examined whether local enhancement of HIF-1α activity would improve diabetic wound healing and minimize the severity of diabetic ulcers. To improve HIF-1α activity we designed a transdermal drug delivery system (TDDS) containing the FDA-approved small molecule deferoxamine (DFO), an iron chelator that increases HIF-1α transactivation in diabetes by preventing iron-catalyzed reactive oxygen stress. Applying this TDDS to a pressure-induced ulcer model in diabetic mice, we found that transdermal delivery of DFO significantly improved wound healing. Unexpectedly, prophylactic application of this transdermal delivery system also prevented diabetic ulcer formation. DFO-treated wounds demonstrated increased collagen density, improved neovascularization, and reduction of free radical formation, leading to decreased cell death. These findings suggest that transdermal delivery of DFO provides a targeted means to both prevent ulcer formation and accelerate diabetic wound healing with the potential for rapid clinical translation.

    View details for DOI 10.1073/pnas.1413445112

    View details for PubMedID 25535360

  • Reduced Regenerative Capacity of Aged Adipose Derived Stem Cells is Caused by Alterations of Cell Subpopulation Dynamics Duscher, D., Rennert, R. C., Januszyk, M., Maan, Z. N., Whittam, A. J., Hu, M. S., Walmsley, G. G., Atashroo, D., Longaker, M. T., Gurtner, G. C. ELSEVIER SCIENCE INC. 2014: S136
  • Noncontact, low-frequency ultrasound therapy enhances neovascularization and wound healing in diabetic mice. Plastic and reconstructive surgery Maan, Z. N., Januszyk, M., Rennert, R. C., Duscher, D., Rodrigues, M., Fujiwara, T., Ho, N., Whitmore, A., Hu, M. S., Longaker, M. T., Gurtner, G. C. 2014; 134 (3): 402e-11e

    Abstract

    Chronic wounds are a major source of morbidity for patients and represent a significant health burden. Implementing noninvasive techniques that accelerate healing of these wounds would provide great benefit. Ultrasound appears to be an effective modality for the treatment of chronic wounds in humans. MIST Therapy is a noncontact, low-frequency ultrasound treatment delivered through a saline mist. A variety of mechanisms have been proposed to explain the efficacy of ultrasound therapy, but the underlying molecular and cellular pathways impacted by this technique remain unclear. The in vivo effect of noncontact, low-frequency ultrasound was therefore examined in a humanized excisional wound model.The treatment group received noncontact, low-frequency ultrasound therapy three times per week, whereas the control group received a standard dressing change. Wounds were photographed at regular intervals to calculate healing kinetics. Wound tissue was harvested and processed for histology, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay.The MIST group demonstrated significantly accelerated wound healing, with 17.3 days to wound closure compared with 24 days in the controls (p < 0.05). This improvement became evident by day 9, with healing evidenced by significantly decreased mean wound area relative to original size (68 percent versus 80 percent; p < 0.01). Expression of markers of neovascularization (stromal cell-derived factor 1, vascular endothelial growth factor, and CD31) was also increased in the wound beds of noncontact, low-frequency ultrasound-treated mice compared with controls.Noncontact, low-frequency ultrasound treatment improves neovascularization and wound closure rates in excisional wounds for diabetic mice, likely because of the stimulated release of angiogenic factors.

    View details for DOI 10.1097/PRS.0000000000000467

    View details for PubMedID 25158717

  • A Randomized Controlled Trial of the embrace Advanced Scar Therapy Device to Reduce Incisional Scar Formation. Plastic and reconstructive surgery Longaker, M. T., Rohrich, R. J., Greenberg, L., Furnas, H., Wald, R., Bansal, V., Seify, H., Tran, A., Weston, J., Korman, J. M., Chan, R., Kaufman, D., Dev, V. R., Mele, J. A., Januszyk, M., Cowley, C., McLaughlin, P., Beasley, B., Gurtner, G. C. 2014; 134 (3): 536-546

    Abstract

    Scarring represents a significant biomedical burden in clinical medicine. Mechanomodulation has been linked to scarring through inflammation, but until now a systematic approach to attenuate mechanical force and reduce scarring has not been possible.The authors conducted a 12-month, prospective, open-label, randomized, multicenter clinical trial to evaluate abdominoplasty scar appearance following postoperative treatment with the embrace Advanced Scar Therapy device to reduce mechanical forces on healing surgical incisions. Incisions from 65 healthy adult subjects were randomized to receive embrace treatment on one half of an abdominoplasty incision and control treatment (surgeon's optimal care methods) on the other half. The primary endpoint for this study was the difference between assessments of scar appearance for the treated and control sides using the visual analogue scale scar score.Final 12-month study photographs were obtained from 36 subjects who completed at least 5 weeks of dressing application. The mean visual analogue scale score for embrace-treated scars (2.90) was significantly improved compared with control-treated scars (3.29) at 12 months (difference, 0.39; 95 percent confidence interval, 0.14 to 0.66; p = 0.027). Both subjects and investigators found that embrace-treated scars demonstrated significant improvements in overall appearance at 12 months using the Patient and Observer Scar Assessment Scale evaluation (p = 0.02 and p < 0.001, respectively). No serious adverse events were reported.These results demonstrate that the embrace device significantly reduces scarring following abdominoplasty surgery. To the authors' knowledge, this represents the first level I evidence for postoperative scar reduction.Therapeutic, II.

    View details for DOI 10.1097/PRS.0000000000000417

    View details for PubMedID 24804638

  • Diabetes irreversibly depletes bone marrow-derived mesenchymal progenitor cell subpopulations. Diabetes Januszyk, M., Sorkin, M., Glotzbach, J. P., Vial, I. N., Maan, Z. N., Rennert, R. C., Duscher, D., Thangarajah, H., Longaker, M. T., Butte, A. J., Gurtner, G. C. 2014; 63 (9): 3047-3056

    Abstract

    Diabetic vascular pathology is largely attributable to impairments in tissue recovery from hypoxia. Circulating progenitor cells have been postulated to play a role in ischemic recovery and deficiencies in these cells have been well described in diabetic patients. Here, we examine bone marrow-derived mesenchymal progenitor cells (BM-MPCs) that have previously been shown to be important for new blood vessel formation, and demonstrate significant deficits in the context of diabetes. Further, we determine that this dysfunction is attributable to intrinsic defects in diabetic BM-MPCs that are not correctable by restoring glucose homeostasis. We identify two transcriptionally distinct subpopulations that are selectively depleted by both type 1 and type 2 diabetes, and these subpopulations have pro-vasculogenic expression profiles, suggesting that they are vascular progenitor cells. These results suggest that the clinically observed deficits in progenitor cells may be attributable to selective and irreversible depletion of progenitor cell subsets in patients with diabetes.

    View details for DOI 10.2337/db13-1366

    View details for PubMedID 24740572

  • Clonal analysis reveals nerve-dependent and independent roles on mammalian hind limb tissue maintenance and regeneration PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rinkevich, Y., Montoro, D. T., Muhonen, E., Walmsley, G. G., Lo, D., Hasegawa, M., Januszyk, M., Connolly, A. J., Weissman, I. L., Longaker, M. T. 2014; 111 (27): 9846-9851

    Abstract

    The requirement and influence of the peripheral nervous system on tissue replacement in mammalian appendages remain largely undefined. To explore this question, we have performed genetic lineage tracing and clonal analysis of individual cells of mouse hind limb tissues devoid of nerve supply during regeneration of the digit tip, normal maintenance, and cutaneous wound healing. We show that cellular turnover, replacement, and cellular differentiation from presumed tissue stem/progenitor cells within hind limb tissues remain largely intact independent of nerve and nerve-derived factors. However, regenerated digit tips in the absence of nerves displayed patterning defects in bone and nail matrix. These nerve-dependent phenotypes mimic clinical observations of patients with nerve damage resulting from spinal cord injury and are of significant interest for translational medicine aimed at understanding the effects of nerves on etiologies of human injury.

    View details for DOI 10.1073/pnas.1410097111

    View details for Web of Science ID 000338514800040

    View details for PubMedCentralID PMC4103362

  • Clonal analysis reveals nerve-dependent and independent roles on mammalian hind limb tissue maintenance and regeneration. Proceedings of the National Academy of Sciences of the United States of America Rinkevich, Y., Montoro, D. T., Muhonen, E., Walmsley, G. G., Lo, D., Hasegawa, M., Januszyk, M., Connolly, A. J., Weissman, I. L., Longaker, M. T. 2014; 111 (27): 9846-9851

    Abstract

    The requirement and influence of the peripheral nervous system on tissue replacement in mammalian appendages remain largely undefined. To explore this question, we have performed genetic lineage tracing and clonal analysis of individual cells of mouse hind limb tissues devoid of nerve supply during regeneration of the digit tip, normal maintenance, and cutaneous wound healing. We show that cellular turnover, replacement, and cellular differentiation from presumed tissue stem/progenitor cells within hind limb tissues remain largely intact independent of nerve and nerve-derived factors. However, regenerated digit tips in the absence of nerves displayed patterning defects in bone and nail matrix. These nerve-dependent phenotypes mimic clinical observations of patients with nerve damage resulting from spinal cord injury and are of significant interest for translational medicine aimed at understanding the effects of nerves on etiologies of human injury.

    View details for DOI 10.1073/pnas.1410097111

    View details for PubMedID 24958860

  • Gene expression in fetal murine keratinocytes and fibroblasts JOURNAL OF SURGICAL RESEARCH Hu, M. S., Januszyk, M., Hong, W. X., Walmsley, G. G., Zielins, E. R., Atashroo, D. A., Maan, Z. N., McArdle, A., Takanishi, D. M., Gurtner, G. C., Longaker, M. T., Lorenz, H. P. 2014; 190 (1): 344-357

    Abstract

    Early fetuses heal wounds without the formation of a scar. Many studies have attempted to explain this remarkable phenomenon. However, the exact mechanism remains unknown. Herein, we examine the predominant cell types of the epidermis and dermis-the keratinocyte and fibroblast-during different stages of fetal development to better understand the changes that lead to scarring wound repair versus regeneration.Keratinocytes and fibroblasts were harvested and cultured from the dorsal skin of time-dated BALB/c fetuses. Total RNA was isolated and microarray analysis was performed using chips with 42,000 genes. Significance analysis of microarrays was used to select genes with >2-fold expression differences with a false discovery rate <2. Enrichment analysis was performed on significant genes to identify differentially expressed pathways.By comparing the gene expression profile of keratinocytes from E16 versus E18 fetuses, we identified 24 genes that were downregulated at E16. Analysis of E16 and E18 fibroblasts revealed 522 differentially expressed genes. Enrichment analysis showed the top 20 signaling pathways that were downregulated in E16 keratinocytes and upregulated or downregulated in E16 fibroblasts.Our data reveal 546 differentially expressed genes in keratinocytes and fibroblasts between the scarless and scarring transition. In addition, a total of 60 signaling pathways have been identified to be either upregulated or downregulated in these cell types. The genes and pathways recognized by our study may prove to be essential targets that may discriminate between fetal wound regeneration and adult wound repair.

    View details for DOI 10.1016/j.jss.2014.02.030

    View details for Web of Science ID 000338444700051

  • Mechanotransduction and fibrosis JOURNAL OF BIOMECHANICS Duscher, D., Maan, Z. N., Wong, V. W., Rennert, R. C., Januszyk, M., Rodrigues, M., Hu, M., Whitmore, A. J., Whittam, A. J., Longaker, M. T., Gurtner, G. C. 2014; 47 (9): 1997-2005

    Abstract

    Scarring and tissue fibrosis represent a significant source of morbidity in the United States. Despite considerable research focused on elucidating the mechanisms underlying cutaneous scar formation, effective clinical therapies are still in the early stages of development. A thorough understanding of the various signaling pathways involved is essential to formulate strategies to combat fibrosis and scarring. While initial efforts focused primarily on the biochemical mechanisms involved in scar formation, more recent research has revealed a central role for mechanical forces in modulating these pathways. Mechanotransduction, which refers to the mechanisms by which mechanical forces are converted to biochemical stimuli, has been closely linked to inflammation and fibrosis and is believed to play a critical role in scarring. This review provides an overview of our current understanding of the mechanisms underlying scar formation, with an emphasis on the relationship between mechanotransduction pathways and their therapeutic implications.

    View details for DOI 10.1016/j.jbiomech.2014.03.031

    View details for Web of Science ID 000338621900009

  • Transcriptional profiling of rapamycin-treated fibroblasts from hypertrophic and keloid scars. Annals of plastic surgery Wong, V. W., You, F., Januszyk, M., Gurtner, G. C., Kuang, A. A. 2014; 72 (6): 711-719

    Abstract

    Excess scar formation after cutaneous injury can result in hypertrophic scar (HTS) or keloid formation. Modern strategies to treat pathologic scarring represent nontargeted approaches that produce suboptimal results. Mammalian target of rapamycin (mTOR), a central mediator of inflammation, has been proposed as a novel target to block fibroproliferation. To examine its mechanism of action, we performed genomewide microarray on human fibroblasts (from normal skin, HTS, and keloid scars) treated with the mTOR inhibitor, rapamycin. Hypertrophic scar and keloid fibroblasts demonstrated overexpression of collagen I and III that was effectively abrogated with rapamycin. Blockade of mTOR specifically impaired fibroblast expression of the collagen biosynthesis genes PLOD, PCOLCE, and P4HA, targets significantly overexpressed in HTS and keloid scars. These data suggest that pathologic scarring can be abrogated via modulation of mTOR pathways in procollagen and collagen processing.

    View details for DOI 10.1097/SAP.0b013e31826956f6

    View details for PubMedID 24835866

  • In Vivo clonal analysis reveals lineage-restricted progenitor characteristics in Mammalian kidney development, maintenance, and regeneration. Cell reports Rinkevich, Y., Montoro, D. T., Contreras-Trujillo, H., Harari-Steinberg, O., Newman, A. M., Tsai, J. M., Lim, X., Van-Amerongen, R., Bowman, A., Januszyk, M., Pleniceanu, O., Nusse, R., Longaker, M. T., Weissman, I. L., Dekel, B. 2014; 7 (4): 1270-1283

    Abstract

    The mechanism and magnitude by which the mammalian kidney generates and maintains its proximal tubules, distal tubules, and collecting ducts remain controversial. Here, we use long-term in vivo genetic lineage tracing and clonal analysis of individual cells from kidneys undergoing development, maintenance, and regeneration. We show that the adult mammalian kidney undergoes continuous tubulogenesis via expansions of fate-restricted clones. Kidneys recovering from damage undergo tubulogenesis through expansions of clones with segment-specific borders, and renal spheres developing in vitro from individual cells maintain distinct, segment-specific fates. Analysis of mice derived by transfer of color-marked embryonic stem cells (ESCs) into uncolored blastocysts demonstrates that nephrons are polyclonal, developing from expansions of singly fated clones. Finally, we show that adult renal clones are derived from Wnt-responsive precursors, and their tracing in vivo generates tubules that are segment specific. Collectively, these analyses demonstrate that fate-restricted precursors functioning as unipotent progenitors continuously maintain and self-preserve the mouse kidney throughout life.

    View details for DOI 10.1016/j.celrep.2014.04.018

    View details for PubMedID 24835991

    View details for PubMedCentralID PMC4425291

  • Tracking the elusive fibrocyte: identification and characterization of collagen-producing hematopoietic lineage cells during murine wound healing. Stem cells Suga, H., Rennert, R. C., Rodrigues, M., Sorkin, M., Glotzbach, J. P., Januszyk, M., Fujiwara, T., Longaker, M. T., Gurtner, G. C. 2014; 32 (5): 1347-1360

    Abstract

    Fibrocytes are a unique population of circulating cells reported to exhibit characteristics of both hematopoietic and mesenchymal cells, and play an important role in wound healing. However, putative fibrocytes have been found to lose expression of hematopoietic surface markers such as CD45 during differentiation, making it difficult to track these cells in vivo with conventional methodologies. In this study, to distinguish hematopoietic and nonhematopoietic cells without surface markers, we took advantage of the gene vav 1, which is expressed solely on hematopoietic cells but not on other cell types, and established a novel transgenic mouse, in which hematopoietic cells are irreversibly labeled with green fluorescent protein and nonhematopoietic cells with red fluorescent protein. Use of single-cell transcriptional analysis in this mouse model revealed two discrete types of collagen I (Col I) expressing cells of hematopoietic lineage recruited into excisional skin wounds. We confirmed this finding on a protein level, with one subset of these Col I synthesizing cells being CD45+ and CD11b+, consistent with the traditional definition of a fibrocyte, while another was CD45- and Cd11b-, representing a previously unidentified population. Both cell types were found to initially peak, then reduce posthealing, consistent with a disappearance from the wound site and not a loss of identifying surface marker expression. Taken together, we have unambiguously identified two cells of hematopoietic origin that are recruited to the wound site and deposit collagen, definitively confirming the existence and natural time course of fibrocytes in cutaneous healing. Stem Cells 2014;32:1347-1360.

    View details for DOI 10.1002/stem.1648

    View details for PubMedID 24446236

  • Mechanical offloading of incisional wounds is associated with transcriptional downregulation of inflammatory pathways in a large animal model ORGANOGENESIS Januszyk, M., Wong, V. W., Bhatt, K. A., Vial, I. N., Paterno, J., Longaker, M. T., Gurtner, G. C. 2014; 10 (2): 186-193

    Abstract

    Cutaneous scarring is a major source of morbidity and current therapies to mitigate scar formation remain ineffective. Although wound fibrosis and inflammation are highly linked, only recently have mechanical forces been implicated in these pathways. Our group has developed a topical polymer device that significantly reduces post-injury scar formation via the manipulation of mechanical forces. Here we extend these studies to examine the genomewide transcriptional effects of mechanomodulation during scar formation using a validated large animal model, the red Duroc pig. We demonstrate that mechanical loading of incisional wounds upregulates expression of genes associated with inflammatory and fibrotic pathways, and that device-mediated offloading of these wounds reverses these effects. Validation studies are needed to clarify the clinical significance of these findings.

    View details for DOI 10.4161/org.28818

    View details for Web of Science ID 000341807300005

    View details for PubMedID 24739276

    View details for PubMedCentralID PMC4154952

  • Abstract 15: Characterization of the Endothelial Progenitor Cell from Adult Tissue using Vav/Cre RFP-GFP Murine Model and Single Cell Microfluidics. Plastic and reconstructive surgery Rodrigues, M., Rennert, R. C., Bishop, S., Januszyk, M., Maan, Z., Sorkin, M., Duscher, D., Gurtner, G. C. 2014; 133 (3): 25-?

    View details for DOI 10.1097/01.prs.0000445018.79483.78

    View details for PubMedID 25942126

  • Abstract 8: SDF-1 Regulates Adipose Niche Homeostasis and Adipose Derived Stromal Cell Function. Plastic and reconstructive surgery Maan, Z. N., Rennert, R. C., Duscher, D., Januszyk, M., Paik, K., Chung, M. T., Paik, K., Fujiwara, T., Rodrigues, M., Ho, N., Baker, H., Perez, M., Hu, M., Sorkin, M., Longaker, M. T., Gurtner, G. C. 2014; 133 (3): 15-16

    View details for DOI 10.1097/01.prs.0000444941.23852.ce

    View details for PubMedID 25942119

  • Abstract 33: Stress Offloading through Mechanomodulation is Associated with Down-Regulation of Inflammatory Pathways in a Large Animal Model. Plastic and reconstructive surgery Januszyk, M., Wong, V. W., Bhatt, K., Vial, I. N., Dauskardt, R., Longaker, M. T., Gurtner, G. C. 2014; 133 (3): 44-?

    View details for DOI 10.1097/01.prs.0000445066.09978.3b

    View details for PubMedID 25942144

  • Reduced BMPR2 expression induces GM-CSF translation and macrophage recruitment in humans and mice to exacerbate pulmonary hypertension. journal of experimental medicine Sawada, H., Saito, T., Nickel, N. P., Alastalo, T., Glotzbach, J. P., Chan, R., Haghighat, L., Fuchs, G., Januszyk, M., Cao, A., Lai, Y., Perez, V. d., Kim, Y., Wang, L., Chen, P., Spiekerkoetter, E., Mitani, Y., Gurtner, G. C., Sarnow, P., Rabinovitch, M. 2014; 211 (2): 263-280

    Abstract

    Idiopathic pulmonary arterial hypertension (PAH [IPAH]) is an insidious and potentially fatal disease linked to a mutation or reduced expression of bone morphogenetic protein receptor 2 (BMPR2). Because intravascular inflammatory cells are recruited in IPAH pathogenesis, we hypothesized that reduced BMPR2 enhances production of the potent chemokine granulocyte macrophage colony-stimulating factor (GM-CSF) in response to an inflammatory perturbation. When human pulmonary artery (PA) endothelial cells deficient in BMPR2 were stimulated with tumor necrosis factor (TNF), a twofold increase in GM-CSF was observed and related to enhanced messenger RNA (mRNA) translation. The mechanism was associated with disruption of stress granule formation. Specifically, loss of BMPR2 induced prolonged phospho-p38 mitogen-activated protein kinase (MAPK) in response to TNF, and this increased GADD34-PP1 phosphatase activity, dephosphorylating eukaryotic translation initiation factor (eIF2α), and derepressing GM-CSF mRNA translation. Lungs from IPAH patients versus unused donor controls revealed heightened PA expression of GM-CSF co-distributing with increased TNF and expanded populations of hematopoietic and endothelial GM-CSF receptor α (GM-CSFRα)-positive cells. Moreover, a 3-wk infusion of GM-CSF in mice increased hypoxia-induced PAH, in association with increased perivascular macrophages and muscularized distal arteries, whereas blockade of GM-CSF repressed these features. Thus, reduced BMPR2 can subvert a stress granule response, heighten GM-CSF mRNA translation, increase inflammatory cell recruitment, and exacerbate PAH.

    View details for DOI 10.1084/jem.20111741

    View details for PubMedID 24446489

  • The embrace Device Significantly Decreases Scarring following Scar Revision Surgery in a Randomized Controlled Trial. Plastic and reconstructive surgery Lim, A. F., Weintraub, J., Kaplan, E. N., Januszyk, M., Cowley, C., McLaughlin, P., Beasley, B., Gurtner, G. C., Longaker, M. T. 2014; 133 (2): 398-405

    Abstract

    Mechanically offloading or shielding an incision significantly reduces scarring in both animal and first-in-human studies. Whether or not this strategy would be effective following scar revision surgery was previously unknown. In this article, the authors report that the embrace device, which uses principles of mechanomodulation, significantly improves aesthetic outcomes following scar revision surgery.A prospective, open-label, randomized, single-center study was conducted to evaluate the appearance of scars following revision and embrace treatment. Revision surgery was performed on 12 patients, each acting as his or her own control, and outcomes were assessed at 6 months. A visual analogue scale was used to evaluate each scar, rated by four independent surgeons who were not involved in the study.Evaluation of 6-month scar images by four independent surgeons using the visual analogue scale demonstrated a highly significant improvement in scar appearance following embrace treatment (p < 0.005).The embrace device represents a powerful new technology for significantly improving scar appearance following revision surgery.Therapeutic, II.

    View details for DOI 10.1097/01.prs.0000436526.64046.d0

    View details for PubMedID 24105084

  • Aging disrupts cell subpopulation dynamics and diminishes the function of mesenchymal stem cells. Scientific reports Duscher, D., Rennert, R. C., Januszyk, M., Anghel, E., Maan, Z. N., Whittam, A. J., Perez, M. G., Kosaraju, R., Hu, M. S., Walmsley, G. G., Atashroo, D., Khong, S., Butte, A. J., Gurtner, G. C. 2014; 4: 7144-?

    Abstract

    Advanced age is associated with an increased risk of vascular morbidity, attributable in part to impairments in new blood vessel formation. Mesenchymal stem cells (MSCs) have previously been shown to play an important role in neovascularization and deficiencies in these cells have been described in aged patients. Here we utilize single cell transcriptional analysis to determine the effect of aging on MSC population dynamics. We identify an age-related depletion of a subpopulation of MSCs characterized by a pro-vascular transcriptional profile. Supporting this finding, we demonstrate that aged MSCs are also significantly compromised in their ability to support vascular network formation in vitro and in vivo. Finally, aged MSCs are unable to rescue age-associated impairments in cutaneous wound healing. Taken together, these data suggest that age-related changes in MSC population dynamics result in impaired therapeutic potential of aged progenitor cells. These findings have critical implications for therapeutic cell source decisions (autologous versus allogeneic) and indicate the necessity of strategies to improve functionality of aged MSCs.

    View details for DOI 10.1038/srep07144

    View details for PubMedID 25413454

  • Understanding regulatory pathways of neovascularization in diabetes EXPERT REVIEW OF ENDOCRINOLOGY & METABOLISM Maan, Z. N., Rodrigues, M., Rennert, R. C., Whitmore, A., Duscher, D., Januszyk, M., Hu, M., Whittam, A. J., Davis, C. R., Gurtner, G. C. 2014; 9 (5): 487–501
  • Mechanotransduction and fibrosis. Journal of biomechanics Duscher, D., Maan, Z. N., Wong, V. W., Rennert, R. C., Januszyk, M., Rodrigues, M., Hu, M., Whitmore, A. J., Whittam, A. J., Longaker, M. T., Gurtner, G. C. 2014

    Abstract

    Scarring and tissue fibrosis represent a significant source of morbidity in the United States. Despite considerable research focused on elucidating the mechanisms underlying cutaneous scar formation, effective clinical therapies are still in the early stages of development. A thorough understanding of the various signaling pathways involved is essential to formulate strategies to combat fibrosis and scarring. While initial efforts focused primarily on the biochemical mechanisms involved in scar formation, more recent research has revealed a central role for mechanical forces in modulating these pathways. Mechanotransduction, which refers to the mechanisms by which mechanical forces are converted to biochemical stimuli, has been closely linked to inflammation and fibrosis and is believed to play a critical role in scarring. This review provides an overview of our current understanding of the mechanisms underlying scar formation, with an emphasis on the relationship between mechanotransduction pathways and their therapeutic implications.

    View details for PubMedID 24709567

  • Gene expression in fetal murine keratinocytes and fibroblasts. The Journal of surgical research Hu, M. S., Januszyk, M., Hong, W. X., Walmsley, G. G., Zielins, E. R., Atashroo, D. A., Maan, Z. N., McArdle, A., Takanishi, D. M., Gurtner, G. C., Longaker, M. T., Lorenz, H. P. 2014

    Abstract

    Early fetuses heal wounds without the formation of a scar. Many studies have attempted to explain this remarkable phenomenon. However, the exact mechanism remains unknown. Herein, we examine the predominant cell types of the epidermis and dermis-the keratinocyte and fibroblast-during different stages of fetal development to better understand the changes that lead to scarring wound repair versus regeneration.Keratinocytes and fibroblasts were harvested and cultured from the dorsal skin of time-dated BALB/c fetuses. Total RNA was isolated and microarray analysis was performed using chips with 42,000 genes. Significance analysis of microarrays was used to select genes with >2-fold expression differences with a false discovery rate <2. Enrichment analysis was performed on significant genes to identify differentially expressed pathways.By comparing the gene expression profile of keratinocytes from E16 versus E18 fetuses, we identified 24 genes that were downregulated at E16. Analysis of E16 and E18 fibroblasts revealed 522 differentially expressed genes. Enrichment analysis showed the top 20 signaling pathways that were downregulated in E16 keratinocytes and upregulated or downregulated in E16 fibroblasts.Our data reveal 546 differentially expressed genes in keratinocytes and fibroblasts between the scarless and scarring transition. In addition, a total of 60 signaling pathways have been identified to be either upregulated or downregulated in these cell types. The genes and pathways recognized by our study may prove to be essential targets that may discriminate between fetal wound regeneration and adult wound repair.

    View details for PubMedID 24726057

  • Understanding regulatory pathways of neovascularization in diabetes. Expert review of endocrinology & metabolism Maan, Z. N., Rodrigues, M., Rennert, R. C., Whitmore, A., Duscher, D., Januszyk, M., Hu, M., Whittam, A. J., Davis, C. R., Gurtner, G. C. 2014; 9 (5): 487–501

    Abstract

    Diabetes mellitus and its associated comorbidities represent a significant health burden worldwide. Vascular dysfunction is the major contributory factor in the development of these comorbidities, which include impaired wound healing, cardiovascular disease and proliferative diabetic retinopathy. While the etiology of abnormal neovascularization in diabetes is complex and paradoxical, the dysregulation of the varied processes contributing to the vascular response are due in large part to the effects of hyperglycemia. In this review, we explore the mechanisms by which hyperglycemia disrupts chemokine expression and function, including the critical hypoxia inducible factor-1 axis. We place particular emphasis on the therapeutic potential of strategies addressing these pathways; as such targeted approaches may one day help alleviate the healthcare burden of diabetic sequelae.

    View details for PubMedID 30736211

  • High-Throughput Single-Cell Analysis for Wound Healing Applications. Advances in wound care Januszyk, M., Gurtner, G. C. 2013; 2 (9): 457-469

    Abstract

    Wound repair is a complex biological process that integrates multiple physiologic pathways to restore skin homeostasis after a wide array of gross and anatomical insults. As such, a scientific examination of the wound typically requires broad sampling of numerous factors and is commonly achieved through DNA microarray analysis.In the last several years, it has become increasingly evident that the granularity afforded by such traditional population-based assays may be insufficient to capture the complex relationships in heterogeneous processes such as those associated with wound healing and stem cell biology.Several emerging technologies have recently become available that permit high-throughput single-cell gene expression analysis in a manner which provides novel insights into the relationships of complex tissue. The most prominent among these employs microfluidic-based devices to achieve a high-resolution analysis of tissue samples.The intrinsically heterogeneous nature of injured tissue, in conjunction with its temporal dynamics, makes wound repair and tissue regeneration an attractive target for high-throughput single-cell analysis. Given the staggering costs associated with chronic and non-healing wounds, the development of predictive and diagnostic tools using this technology would likely be attractive to healthcare providers.

    View details for PubMedID 24527358

  • Tacrolimus fails to regulate collagen expression in dermal fibroblasts. journal of surgical research Wong, V. W., You, F., Januszyk, M., Kuang, A. A. 2013; 184 (1): 678-690

    Abstract

    BACKGROUND: The purpose of this study was to investigate the effects of tacrolimus on human fibroblasts derived from unwounded skin, hypertrophic scars (HTS), and keloids. We hypothesized that tacrolimus, a potent anti-inflammatory and immunosuppressant drug known to attenuate solid organ transplant fibrosis, would block collagen expression in human dermal fibroblasts. METHODS: We performed genomewide microarray analysis on human dermal fibroblasts treated with tacrolimus in vitro. We used principal component analysis and hierarchical clustering to identify targets regulated by tacrolimus. We performed quantitative polymerase chain reaction to validate the effect of tacrolimus on collagen 1 and 3 expression. RESULTS: We identified 62, 136, and 185 gene probes on microarray analysis that were significantly regulated (P < 0.05) by tacrolimus in normal, HTS, and keloid fibroblasts, respectively. Collagen pathways were not blocked after tacrolimus exposure in any of the fibroblast groups; we validated these findings using quantitative polymerase chain reaction for collagen 1 and 3. Microarray gene expression of NME/NM23 nucleoside diphosphate kinase 1 and heterogeneous nuclear ribonucleoprotein H3-2H9 were significantly downregulated (P < 0.05) by tacrolimus in both HTS and keloid fibroblast populations but not normal fibroblasts. CONCLUSIONS: Tacrolimus does not modulate the expression of collagen 1 or 3 in human dermal fibroblasts in vitro. Microarray gene expression of NME/NM23 nucleoside diphosphate kinase 1 and heterogeneous nuclear ribonucleoprotein H3-2H9 are blocked by tacrolimus in pathologic fibroblasts but not normal fibroblasts, and may represent novel genes underlying HTS and keloid pathogenesis. Tacrolimus-based anti-fibrotics might prove more effective if non-fibroblast populations such as inflammatory cells and keratinocytes are targeted.

    View details for DOI 10.1016/j.jss.2013.04.006

    View details for PubMedID 23647800

  • Molecular analysis and differentiation capacity of adipose-derived stem cells from lymphedema tissue. Plastic and reconstructive surgery Levi, B., Glotzbach, J. P., Sorkin, M., Hyun, J., Januszyk, M., Wan, D. C., Li, S., Nelson, E. R., Longaker, M. T., Gurtner, G. C. 2013; 132 (3): 580-589

    Abstract

    Many breast cancer patients are plagued by the disabling complication of upper limb lymphedema after axillary surgery. Conservative treatments using massage and compression therapy do not offer a lasting relief, as they fail to address the chronic transformation of edema into excess adipose tissue. Liposuction to address the adipose nature of the lymphedema has provided an opportunity for a detailed analysis of the stromal fraction of lymphedema-associated fat to clarify the molecular mechanisms for this adipogenic transformation.Adipose-derived stem cells were harvested from human lipoaspirate of the upper extremity from age-matched patients with lymphedema (n = 3) or subcutaneous adipose tissue from control patients undergoing cosmetic procedures (n = 3). Immediately after harvest, adipose-derived stem cells were analyzed using single-cell transcriptional profiling techniques. Osteogenic, adipogenic, and vasculogenic gene expression and differentiation were assessed by quantitative real-time polymerase chain reaction and standard in vitro differentiation assays.Differential transcriptional clusters of adipose-derived stem cells were found between lymphedema and subcutaneous fat. Interestingly, lymphedema-associated stem cells had a much higher adipogenic gene expression and enhanced ability to undergo adipogenic differentiation. Conversely, they had lower vasculogenic gene expression and diminished capability to form tubules in vitro, whereas the osteogenic differentiation capacity was not significantly altered.Adipose-derived stem cells from extremities affected by lymphedema appear to exhibit transcriptional profiles similar to those of abdominal adipose-derived stem cells; however, their adipogenic differentiation potential is strongly increased and their vasculogenic capacity is compromised. These results suggest that the underlying pathophysiology of lymphedema drives adipose-derived stem cells toward adipogenic differentiation.

    View details for DOI 10.1097/PRS.0b013e31829ace13

    View details for PubMedID 23985633

  • Micro-Computed Tomography Evaluation of Human Fat Grafts in Nude Mice TISSUE ENGINEERING PART C-METHODS Chung, M. T., Hyun, J. S., Lo, D. D., Montoro, D. T., Hasegawa, M., Levi, B., Januszyk, M., Longaker, M. T., Wan, D. C. 2013; 19 (3): 227-232

    Abstract

    Although autologous fat grafting has revolutionized the field of soft tissue reconstruction and augmentation, long-term maintenance of fat grafts is unpredictable. Recent studies have reported survival rates of fat grafts to vary anywhere between 10% and 80% over time. The present study evaluated the long-term viability of human fat grafts in a murine model using a novel imaging technique allowing for in vivo volumetric analysis.Human fat grafts were prepared from lipoaspirate samples using the Coleman technique. Fat was injected subcutaneously into the scalp of 10 adult Crl:NU-Foxn1(nu) CD-1 male mice. Micro-computed tomography (CT) was performed immediately following injection and then weekly thereafter. Fat volume was rendered by reconstructing a three-dimensional (3D) surface through cubic-spline interpolation. Specimens were also harvested at various time points and sections were prepared and stained with hematoxylin and eosin (H&E), for macrophages using CD68 and for the cannabinoid receptor 1 (CB1). Finally, samples were explanted at 8- and 12-week time points to validate calculated micro-CT volumes.Weekly CT scanning demonstrated progressive volume loss over the time course. However, volumetric analysis at the 8- and 12-week time points stabilized, showing an average of 62.2% and 60.9% survival, respectively. Gross analysis showed the fat graft to be healthy and vascularized. H&E analysis and staining for CD68 showed minimal inflammatory reaction with viable adipocytes. Immunohistochemical staining with anti-human CB1 antibodies confirmed human origin of the adipocytes.Studies assessing the fate of autologous fat grafts in animals have focused on nonimaging modalities, including histological and biochemical analyses, which require euthanasia of the animals. In this study, we have demonstrated the ability to employ micro-CT for 3D reconstruction and volumetric analysis of human fat grafts in a mouse model. Importantly, this model provides a platform for subsequent study of fat manipulation and soft tissue engineering.

    View details for DOI 10.1089/ten.tec.2012.0371

    View details for Web of Science ID 000314179900006

    View details for PubMedID 22916732

    View details for PubMedCentralID PMC3557441

  • Cellular response to a novel fetal acellular collagen matrix: implications for tissue regeneration. International journal of biomaterials Rennert, R. C., Sorkin, M., Garg, R. K., Januszyk, M., Gurtner, G. C. 2013; 2013: 527957-?

    Abstract

    Introduction. PriMatrix (TEI Biosciences Inc., Boston, MA, USA) is a novel acellular collagen matrix derived from fetal bovine dermis that is designed for use in partial- and full-thickness wounds. This study analyzes the cellular response to PriMatrix in vivo, as well as the ability of this matrix to facilitate normal tissue regeneration. Methods. Five by five mm squares of rehydrated PriMatrix were implanted in a subcutaneous fashion on the dorsum of wild-type mice. Implant site tissue was harvested for histology, immunohistochemistry (IHC), and flow cytometric analyses at multiple time points until day 28. Results. PriMatrix implants were found to go through a biological progression initiated by a transient infiltrate of inflammatory cells, followed by mesenchymal cell recruitment and vascular development. IHC analysis revealed that the majority of the implanted fetal dermal collagen fibers persisted through day 28 but underwent remodeling and cellular repopulation to form tissue with a density and morphology consistent with healthy dermis. Conclusions. PriMatrix implants undergo progressive in vivo remodeling, facilitating the regeneration of histologically normal tissue through a mild inflammatory and progenitor cell response. Regeneration of normal tissue is especially important in a wound environment, and these findings warrant further investigation of PriMatrix in this setting.

    View details for DOI 10.1155/2013/527957

    View details for PubMedID 23970899

    View details for PubMedCentralID PMC3736474

  • Focal adhesion kinase links mechanical force to skin fibrosis via inflammatory signaling. Nature medicine Wong, V. W., Rustad, K. C., Akaishi, S., Sorkin, M., Glotzbach, J. P., Januszyk, M., Nelson, E. R., Levi, K., Paterno, J., Vial, I. N., Kuang, A. A., Longaker, M. T., Gurtner, G. C. 2012; 18 (1): 148-152

    Abstract

    Exuberant fibroproliferation is a common complication after injury for reasons that are not well understood. One key component of wound repair that is often overlooked is mechanical force, which regulates cell-matrix interactions through intracellular focal adhesion components, including focal adhesion kinase (FAK). Here we report that FAK is activated after cutaneous injury and that this process is potentiated by mechanical loading. Fibroblast-specific FAK knockout mice have substantially less inflammation and fibrosis than control mice in a model of hypertrophic scar formation. We show that FAK acts through extracellular-related kinase (ERK) to mechanically trigger the secretion of monocyte chemoattractant protein-1 (MCP-1, also known as CCL2), a potent chemokine that is linked to human fibrotic disorders. Similarly, MCP-1 knockout mice form minimal scars, indicating that inflammatory chemokine pathways are a major mechanism by which FAK mechanotransduction induces fibrosis. Small-molecule inhibition of FAK blocks these effects in human cells and reduces scar formation in vivo through attenuated MCP-1 signaling and inflammatory cell recruitment. These findings collectively indicate that physical force regulates fibrosis through inflammatory FAK-ERK-MCP-1 pathways and that molecular strategies targeting FAK can effectively uncouple mechanical force from pathologic scar formation.

    View details for DOI 10.1038/nm.2574

    View details for PubMedID 22157678

  • CD105 Protein Depletion Enhances Human Adipose-derived Stromal Cell Osteogenesis through Reduction of Transforming Growth Factor beta 1 (TGF-beta 1) Signaling JOURNAL OF BIOLOGICAL CHEMISTRY Levi, B., Wan, D. C., Glotzbach, J. P., Hyun, J., Januszyk, M., Montoro, D., Sorkin, M., James, A. W., Nelson, E. R., Li, S., Quarto, N., Lee, M., Gurtner, G. C., Longaker, M. T. 2011; 286 (45): 39497-39509

    Abstract

    Clinically available sources of bone for repair and reconstruction are limited by the accessibility of autologous grafts, infectious risks of cadaveric materials, and durability of synthetic substitutes. Cell-based approaches for skeletal regeneration can potentially fill this need, and adipose tissue represents a promising source for development of such therapies. Here, we enriched for an osteogenic subpopulation of cells derived from human subcutaneous adipose tissue utilizing microfluidic-based single cell transcriptional analysis and fluorescence-activated cell sorting (FACS). Statistical analysis of single cell transcriptional profiles demonstrated that low expression of endoglin (CD105) correlated with a subgroup of adipose-derived cells with increased osteogenic gene expression. FACS-sorted CD105(low) cells demonstrated significantly enhanced in vitro osteogenic differentiation and in vivo bone regeneration when compared with either CD105(high) or unsorted cells. Evaluation of the endoglin pathway suggested that enhanced osteogenesis among CD105(low) adipose-derived cells is likely due to identification of a subpopulation with lower TGF-β1/Smad2 signaling. These findings thus highlight a potential avenue to promote osteogenesis in adipose-derived mesenchymal cells for skeletal regeneration.

    View details for DOI 10.1074/jbc.M111.256529

    View details for PubMedID 21949130

  • An Information Theoretic, Microfluidic-Based Single Cell Analysis Permits Identification of Subpopulations among Putatively Homogeneous Stem Cells PLOS ONE Glotzbach, J. P., Januszyk, M., Vial, I. N., Wong, V. W., Gelbard, A., Kalisky, T., Thangarajah, H., Longaker, M. T., Quake, S. R., Chu, G., Gurtner, G. C. 2011; 6 (6)

    Abstract

    An incomplete understanding of the nature of heterogeneity within stem cell populations remains a major impediment to the development of clinically effective cell-based therapies. Transcriptional events within a single cell are inherently stochastic and can produce tremendous variability, even among genetically identical cells. It remains unclear how mammalian cellular systems overcome this intrinsic noisiness of gene expression to produce consequential variations in function, and what impact this has on the biologic and clinical relevance of highly 'purified' cell subgroups. To address these questions, we have developed a novel method combining microfluidic-based single cell analysis and information theory to characterize and predict transcriptional programs across hundreds of individual cells. Using this technique, we demonstrate that multiple subpopulations exist within a well-studied and putatively homogeneous stem cell population, murine long-term hematopoietic stem cells (LT-HSCs). These subgroups are defined by nonrandom patterns that are distinguishable from noise and are consistent with known functional properties of these cells. We anticipate that this analytic framework can also be applied to other cell types to elucidate the relationship between transcriptional and phenotypic variation.

    View details for DOI 10.1371/journal.pone.0021211

    View details for Web of Science ID 000292033700046

    View details for PubMedID 21731674

    View details for PubMedCentralID PMC3120839

  • Engineered Pullulan-Collagen Composite Dermal Hydrogels Improve Early Cutaneous Wound Healing TISSUE ENGINEERING PART A Wong, V. W., Rustad, K. C., Galvez, M. G., Neofyotou, E., Glotzbach, J. P., Januszyk, M., Major, M. R., Sorkin, M., Longaker, M. T., Rajadas, J., Gurtner, G. C. 2011; 17 (5-6): 631-644

    Abstract

    New strategies for skin regeneration are needed to address the significant medical burden caused by cutaneous wounds and disease. In this study, pullulan-collagen composite hydrogel matrices were fabricated using a salt-induced phase inversion technique, resulting in a structured yet soft scaffold for skin engineering. Salt crystallization induced interconnected pore formation, and modification of collagen concentration permitted regulation of scaffold pore size. Hydrogel architecture recapitulated the reticular distribution of human dermal matrix while maintaining flexible properties essential for skin applications. In vitro, collagen hydrogel scaffolds retained their open porous architecture and viably sustained human fibroblasts and murine mesenchymal stem cells and endothelial cells. In vivo, hydrogel-treated murine excisional wounds demonstrated improved wound closure, which was associated with increased recruitment of stromal cells and formation of vascularized granulation tissue. In conclusion, salt-induced phase inversion techniques can be used to create modifiable pullulan-collagen composite dermal scaffolds that augment early wound healing. These novel biomatrices can potentially serve as a structured delivery template for cells and biomolecules in regenerative skin applications.

    View details for DOI 10.1089/ten.tea.2010.0298

    View details for Web of Science ID 000287801600005

    View details for PubMedID 20919949

  • Statistics in Medicine PLASTIC AND RECONSTRUCTIVE SURGERY Januszyk, M., Gurtner, G. C. 2011; 127 (1): 437-444

    Abstract

    The scope of biomedical research has expanded rapidly during the past several decades, and statistical analysis has become increasingly necessary to understand the meaning of large and diverse quantities of raw data. As such, a familiarity with this lexicon is essential for critical appraisal of medical literature. This article attempts to provide a practical overview of medical statistics, with an emphasis on the selection, application, and interpretation of specific tests. This includes a brief review of statistical theory and its nomenclature, particularly with regard to the classification of variables. A discussion of descriptive methods for data presentation is then provided, followed by an overview of statistical inference and significance analysis, and detailed treatment of specific statistical tests and guidelines for their interpretation.

    View details for DOI 10.1097/PRS.0b013e3181f95dd2

    View details for Web of Science ID 000285992100060

    View details for PubMedID 21200241

  • Anatomical and Technical Tips for Use of the Superficial Inferior Epigastric Artery (SIEA) Flap in Breast Reconstructive Surgery JOURNAL OF RECONSTRUCTIVE MICROSURGERY Dorafshar, A. H., Januszyk, M., Song, D. H. 2010; 26 (6): 381-389

    Abstract

    Techniques for autologous breast reconstruction have evolved to minimize donor-site morbidity and reduce flap-specific complications. When available, the superficial inferior epigastric artery (SIEA) flap represents the optimal method to achieve the former. However, many microsurgeons have been reluctant to adopt this procedure due to technical challenges inherent to the surgery, as well as concerns with the intrinsic capacity of the superficial vessel system to adequately support this flap. This article sets forth a simple approach to the SIEA flap harvest and demonstrates that favorable results may be achieved even for small caliber vessels. A total of 46 patients underwent 53 SIEA breast reconstructions over a 6-year period using a modified approach for pedicle dissection and arterial inclusion criteria solely on the basis of presence of a palpable pulse. Average pedicle length harvested for all SIEA flaps was 6.07 cm; and mean arterial (0.96 mm) and venous (2.27 mm) diameters represent the lowest published values. Three flaps (5.7%) demonstrated fat necrosis or partial flap necrosis, with one (1.9%) complete flap loss. These results compare favorably with those of previous SIEA series employing diameter-based selection criteria, suggesting that the presence of a palpable arterial pulse may be sufficient to permit successful utilization of this flap.

    View details for DOI 10.1055/s-0030-1249604

    View details for Web of Science ID 000279057300005

    View details for PubMedID 20301060

  • HIF-1 alpha dysfunction in diabetes CELL CYCLE Thangarajah, H., Vial, I. N., Grogan, R. H., Yao, D., Shi, Y., Januszyk, M., Galiano, R. D., Chang, E. I., Galvez, M. G., Glotzbach, J. P., Wong, V. W., Brownlee, M., Gurtner, G. C. 2010; 9 (1): 75-79

    Abstract

    Diabetic wounds are a significant public health burden, with slow or nonhealing diabetic foot ulcers representing the leading cause of non-traumatic lower limb amputation in developed countries. These wounds heal poorly as a result of compromised blood vessel formation in response to ischemia. We have recently shown that this impairment in neovascularization results from a high glucose-induced defect in transactivation of hypoxia-inducible factor-1alpha (HIF-1alpha), the transcription factor regulating vascular endothelial growth factor (VEGF) expression. HIF-1 dysfunction is the end result of reactive oxygen species-induced modification of its coactivator p300 by the glycolytic metabolite methylglyoxal. Use of the iron chelator-antioxidant deferoxamine (DFO) reversed these effects and normalized healing of humanized diabetic wounds in mice. Here, we present additional data demonstrating that HIF-1alpha activity, not stability, is impaired in the high glucose environment. We demonstrate that high glucose-induced impairments in HIF-1alpha transactivation persist even in the setting of constitutive HIF-1alpha protein overexpression. Further, we show that high glucose-induced hydroxylation of the C-terminal transactivation domain of HIF-1alpha (the primary pathway regulating HIF-1alpha/p300 binding) does not alter HIF-1alpha activity. We extend our study of DFO's therapeutic efficacy in the treatment of impaired wound healing by demonstrating improvements in tissue viability in diabetic mice with DFO-induced increases in VEGF expression and vascular proliferation. Since DFO has been in clinical use for decades, the potential of this drug to treat a variety of ischemic conditions in humans can be evaluated relatively quickly.

    View details for Web of Science ID 000273236800025

    View details for PubMedID 20016290

  • The molecular basis for impaired hypoxia-induced VEGF expression in diabetic tissues PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Thangarajah, H., Yao, D., Chang, E. I., Shi, Y., Jazayeri, L., Vial, I. N., Galiano, R. D., Du, X., Grogan, R., Galvez, M. G., Januszyk, M., Brownlee, M., Gurtner, G. C. 2009; 106 (32): 13505-13510

    Abstract

    Diabetes is associated with poor outcomes following acute vascular occlusive events. This results in part from a failure to form adequate compensatory microvasculature in response to ischemia. Since vascular endothelial growth factor (VEGF) is an essential mediator of neovascularization, we examined whether hypoxic up-regulation of VEGF was impaired in diabetes. Both fibroblasts isolated from type 2 diabetic patients, and normal fibroblasts exposed chronically to high glucose, were defective in their capacity to up-regulate VEGF in response to hypoxia. In vivo, diabetic animals demonstrated an impaired ability to increase VEGF production in response to soft tissue ischemia. This resulted from a high glucose-induced decrease in transactivation by the transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha), which mediates hypoxia-stimulated VEGF expression. Decreased HIF-1alpha functional activity was specifically caused by impaired HIF-1alpha binding to the coactivator p300. We identify covalent modification of p300 by the dicarbonyl metabolite methylglyoxal as being responsible for this decreased association. Administration of deferoxamine abrogated methylglyoxal conjugation, normalizing both HIF-1alpha/p300 interaction and transactivation by HIF-1alpha. In diabetic mice, deferoxamine promoted neovascularization and enhanced wound healing. These findings define molecular defects that underlie impaired VEGF production in diabetic tissues and offer a promising direction for therapeutic intervention.

    View details for DOI 10.1073/pnas.0906670106

    View details for Web of Science ID 000268877300065

    View details for PubMedID 19666581

    View details for PubMedCentralID PMC2726398

  • IFATS Collection: Adipose Stromal Cells Adopt a Proangiogenic Phenotype Under the Influence of Hypoxia STEM CELLS Thangarajah, H., Vial, I. N., Chang, E., El-Ftesi, S., Januszyk, M., Chang, E. I., Paterno, J., Neofytou, E., Longaker, M. T., Gurtner, G. C. 2009; 27 (1): 266-274

    Abstract

    Evolving evidence suggests a possible role for adipose stromal cells (ASCs) in adult neovascularization, although the specific cues that stimulate their angiogenic behavior are poorly understood. We evaluated the effect of hypoxia, a central mediator of new blood vessel development within ischemic tissue, on proneovascular ASC functions. Murine ASCs were exposed to normoxia (21% oxygen) or hypoxia (5%, 1% oxygen) for varying lengths of time. Vascular endothelial growth factor (VEGF) secretion by ASCs increased as an inverse function of oxygen tension, with progressively higher VEGF expression at 21%, 5%, and 1% oxygen, respectively. Greater VEGF levels were also associated with longer periods in culture. ASCs were able to migrate towards stromal cell-derived factor (SDF)-1, a chemokine expressed by ischemic tissue, with hypoxia augmenting ASC expression of the SDF-1 receptor (CXCR4) and potentiating ASC migration. In vivo, ASCs demonstrated the capacity to proliferate in response to a hypoxic insult remote from their resident niche, and this was supported by in vitro studies showing increasing ASC proliferation with greater degrees of hypoxia. Hypoxia did not significantly alter the expression of endothelial surface markers by ASCs. However, these cells did assume an endothelial phenotype as evidenced by their ability to tubularize when seeded with differentiated endothelial cells on Matrigel. Taken together, these data suggest that ASCs upregulate their proneovascular activity in response to hypoxia, and may harbor the capacity to home to ischemic tissue and function cooperatively with existing vasculature to promote angiogenesis.

    View details for DOI 10.1634/stemcells.2008-0276

    View details for Web of Science ID 000263032400030

    View details for PubMedID 18974212