Instructor, Cardiothoracic Surgery
Interleukin-8 (IL8) is highly expressed by injured arteries in a variety of diseases and is a chemoattractant for neutrophils which express IL8 receptors IL8RA and RB (IL8RA/B) on their membranes. Neutrophils interact with the damaged endothelium and initiate an inflammatory cascade at the site of injury. We have generated a novel translational targeted cell therapy for acute vascular injury using adenoviral vectors to overexpress IL8RA/B and green fluorescent protein (GFP) on the surface of endothelial cells (ECs) derived from human induced pluripotent stem cells (HiPS-IL8RA/B-ECs). We hypothesize that HiPS-IL8RA/B-ECs transfused intravenously into rats with balloon injury of the carotid artery will target to the injured site and compete with neutrophils, thus inhibiting inflammation and neointima formation. Young adult male Sprague-Dawley rats underwent balloon injury of the right carotid artery and received intravenous transfusion of saline vehicle, 1.5 × 10(6) HiPS-ECs, 1.5 × 10(6) HiPS-Null-ECs, or 1.5 × 10(6) HiPS-IL8RA/B-ECs immediately after endoluminal injury. Tissue distribution of HiPS-IL8RA/B-ECs was analyzed by a novel GFP DNA qPCR method. Cytokine and chemokine expression and leukocyte infiltration were measured in injured and uninjured arteries at 24 h postinjury by ELISA and immunohistochemistry, respectively. Neointimal, medial areas, and reendothelialization were measured 14 days postinjury. HiPS-IL8RA/B-ECs homed to injured arteries, inhibited inflammatory mediator expression and inflammatory cell infiltration, accelerated reendothelialization, and attenuated neointima formation after endoluminal injury while control HiPS-ECs and HiPS-Null-ECs did not. HiPS-IL8RA/B-ECs transfused into rats with endoluminal carotid artery injury target to the injured artery and provide a novel strategy to treat vascular injury.
View details for DOI 10.1152/ajpheart.00587.2015
View details for PubMedID 26801304
The objective of this study was to enhance the angiogenic capacity of endothelial cells (ECs) using nanoscale signaling cues from aligned nanofibrillar scaffolds in the setting of tissue ischemia. Thread-like nanofibrillar scaffolds with porous structure were fabricated from aligned-braided membranes generated under shear from liquid crystal collagen solution. Human ECs showed greater outgrowth from aligned scaffolds than from nonpatterned scaffolds. Integrin α1 was in part responsible for the enhanced cellular outgrowth on aligned nanofibrillar scaffolds, as the effect was abrogated by integrin α1 inhibition. To test the efficacy of EC-seeded aligned nanofibrillar scaffolds in improving neovascularization in vivo, the ischemic limbs of mice were treated with EC-seeded aligned nanofibrillar scaffold; EC-seeded nonpatterned scaffold; ECs in saline; aligned nanofibrillar scaffold alone; or no treatment. After 14 days, laser Doppler blood spectroscopy demonstrated significant improvement in blood perfusion recovery when treated with EC-seeded aligned nanofibrillar scaffolds, in comparison to ECs in saline or no treatment. In ischemic hindlimbs treated with scaffolds seeded with human ECs derived from induced pluripotent stem cells (iPSC-ECs), single-walled carbon nanotube (SWNT) fluorophores were systemically delivered to quantify microvascular density after 28 days. Near infrared-II (NIR-II, 1000-1700 nm) imaging of SWNT fluorophores demonstrated that iPSC-EC-seeded aligned scaffolds group showed significantly higher microvascular density than the saline or cells groups. These data suggest that treatment with EC-seeded aligned nanofibrillar scaffolds improved blood perfusion and arteriogenesis, when compared to treatment with cells alone or scaffold alone, and have important implications in the design of therapeutic cell delivery strategies.
View details for DOI 10.1021/acsnano.5b00545
View details for PubMedID 26061869
We developed an aligned bi-layered vascular graft derived from human induced pluripotent stem cells (iPSCs) that recapitulates the cellular composition, orientation, and anti-inflammatory function of blood vessels.The luminal layer consisted of longitudinal-aligned nanofibrillar collagen containing primary endothelial cells (ECs) or iPSC-derived ECs (iPSC-ECs). The outer layer contained circumferentially oriented nanofibrillar collagen with primary smooth muscle cells (SMCs) or iPSC-derived SMCs(iPSC-SMCs).On the aligned scaffolds, cells organized F-actin assembly within 8º from the direction of nanofibrils. When compared to randomly-oriented scaffolds, EC-seeded aligned scaffolds had significant reduced inflammatory response, based on adhesivity to monocytes.This study highlights the importance of anisotropic scaffolds in directing cell form and function, and has therapeutic significance as physiologically relevant blood vessels.
View details for DOI 10.2217/rme.15.45
View details for Web of Science ID 000364574000005
View details for PubMedID 26440211
It is generally agreed that engineered cardiovascular tissues require cellular interactions with the local milieu. Within the microenvironment, the extracellular matrix (ECM) is an important support structure that provides dynamic signaling cues in part through its chemical, physical, and mechanical properties. In response to ECM factors, cells activate biochemical and mechanotransduction pathways that modulate their survival, growth, migration, differentiation, and function. This Review describes the role of ECM chemical composition, spatial patterning, and mechanical stimulation in the specification of cardiovascular lineages, with a focus on stem cell differentiation, direct transdifferentiation, and endothelial-to-mesenchymal transition. The translational application of ECMs will be discussed in the context of cardiovascular tissue engineering and regenerative medicine.
View details for DOI 10.1002/adhm.201300620
View details for PubMedID 24443420
Real-time vascular imaging that provides both anatomic and hemodynamic information could greatly facilitate the diagnosis of vascular diseases and provide accurate assessment of therapeutic effects. Here, we have developed a novel fluorescence-based all-optical method, named near-infrared II (NIR-II) fluorescence imaging, to image murine hindlimb vasculature and blood flow in an experimental model of peripheral arterial disease, by exploiting fluorescence in the NIR-II region (1000-1400 nm) of photon wavelengths.Because of the reduced photon scattering of NIR-II fluorescence compared with traditional NIR fluorescence imaging and thus much deeper penetration depth into the body, we demonstrated that the mouse hindlimb vasculature could be imaged with higher spatial resolution than in vivo microscopic computed tomography. Furthermore, imaging during 26 days revealed a significant increase in hindlimb microvascular density in response to experimentally induced ischemia within the first 8 days of the surgery (P<0.005), which was confirmed by histological analysis of microvascular density. Moreover, the tissue perfusion in the ischemic hindlimb could be quantitatively measured by the dynamic NIR-II method, revealing the temporal kinetics of blood flow recovery that resembled microbead-based blood flowmetry and laser Doppler blood spectroscopy.The penetration depth of millimeters, high spatial resolution, and fast acquisition rate of NIR-II imaging make it a useful imaging tool for murine models of vascular disease.
View details for DOI 10.1161/CIRCIMAGING.113.000305
View details for PubMedID 24657826
Therapeutic delivery of cardiomyocytes derived from human pluripotent stem cells (hPSC-CMs) represents a novel clinical approach to regenerate the injured myocardium. However, poor survival and contractility of these cells are a significant bottleneck to their clinical use. To better understand the role of cell-cell communication in enhancing the phenotype and contractile properties of hPSC-CMs, we developed a three-dimensional (3D) hydrogel composed of hPSC-CMs, human pluripotent stem cell-derived endothelial cells (hPSC-ECs), and/or human amniotic mesenchymal stem cells (hAMSCs). The objective of this study was to examine the role of multi-cellular interactions among hPSC-ECs and hAMSCs on the survival and long-term contractile phenotype of hPSC-CMs in a 3D hydrogel. Quantification of spontaneous contractility of hPSC-CMs in tri-culture demonstrated a 6-fold increase in the area of contractile motion after 6 weeks with characteristic rhythmic contraction frequency, when compared to hPSC-CMs alone (P < 0.05). This finding was supported by a statistically significant increase in cardiac troponin T protein expression in the tri-culture hydrogel construct at 6 weeks, when compared to hPSC-CMs alone (P < 0.001). The sustained hPSC-CM survival and contractility in tri-culture was associated with a significant upregulation in the gene expression of L-type Ca(2+) ion channel, Cav1.2, and the inward-rectifier potassium channel, Kir2.1 (P < 0.05), suggesting a role of ion channels in mediating these processes. These findings demonstrate that multi-cellular interactions modulate hPSC-CM phenotype, function, and survival, and they will have important implications in engineering cardiac tissues for treatment of cardiovascular diseases.
View details for PubMedID 25628783
Initial steps in establishing an optimal strategy for functional bioengineered tissues is generation of three-dimensional constructs containing cells with the appropriate organization and phenotype. To effectively utilize rhesus monkey decellularized kidney scaffolds, these studies evaluated two key parameters: (1) residual scaffold components after decellularization including proteomics analysis, and (2) the use of undifferentiated human embryonic stem cells (hESCs) for recellularization in order to explore cellular differentiation in a tissue-specific manner. Sections of kidney and lung were selected for a comparative evaluation because of their similar pattern of organogenesis. Proteomics analysis revealed the presence of growth factors and antimicrobial proteins as well as stress proteins and complement components. Immunohistochemistry of recellularized kidney scaffolds showed the generation of Cytokeratin+ epithelial tubule phenotypes throughout the scaffold that demonstrated a statistically significant increase in expression of kidney-associated genes compared to baseline hESC gene expression. Recellularization of lung scaffolds showed that cells lined the alveolar spaces and demonstrated statistically significant upregulation of key lung-associated genes. However, overall expression of kidney and lung-associated markers was not statistically different when the kidney and lung recellularized scaffolds were compared. These results suggest that decellularized scaffolds have an intrinsic spatial ability to influence hESC differentiation by physically shaping cells into tissue-appropriate structures and phenotypes, and that additional approaches may be needed to ensure consistent recellularization throughout the matrix.
View details for DOI 10.1371/journal.pone.0064134
View details for PubMedID 23717553
New therapies for severely damaged kidneys are needed due to limited regenerative capacity and organ donor shortages. The goal of this study was to repopulate decellularized kidney sections in vitro and to determine the impact of donor age on recellularization. This was addressed by generating decellularized kidney scaffolds from fetal, juvenile, and adult rhesus monkey kidney sections using a procedure that removes cellular components while preserving the structural and functional properties of the native extracellular matrix (ECM). Kidney scaffolds were recellularized using explants from different age groups (fetal, juvenile, adult) and fetal renal cell fractions. Results showed vimentin+ cytokeratin+ calbindin+ cell infiltration and organization around the scaffold ECM. The extent of cellular repopulation was greatest with scaffolds from the youngest donors, and with seeding of mixed fetal renal aggregates that formed tubular structures within the kidney scaffolds. These findings suggest that decellularized kidney sections from different age groups can be effectively repopulated with donor cells and the age of the donor is a critical factor in repopulation efficiency.
View details for DOI 10.1089/ten.tea.2010.0714
View details for Web of Science ID 000298077900001
View details for PubMedID 21902603
The goal of this study was the production of a decellularized kidney scaffold with structural, mechanical, and physiological properties necessary for engineering basic renal structures in vitro. Fetal, infant, juvenile, and adult rhesus monkey kidney sections were treated with either 1% (v/v) sodium dodecyl sulfate or Triton X-100 followed by quantitative and qualitative analysis. Comparison of decellularization agents and incubation temperatures demonstrated sodium dodecyl sulfate at 4 degrees C to be most effective in preserving the native architecture. Hematoxylin and eosin staining confirmed the removal of cellular material, and immunohistochemistry demonstrated preservation of native expression patterns of extracellular matrix proteins, including heparan sulfate proteoglycan, fibronectin, collagen types I and IV, and laminin. Biomechanical testing revealed a decrease in the compressive modulus of decellularized compared to fresh kidneys. Layering of fetal kidney explants on age-matched decellularized kidney scaffolds demonstrated the capacity of the scaffold to support Pax2+/vimentin+ cell attachment and migration to recellularize the scaffold. These findings demonstrate that decellularized kidney sections retain critical structural and functional properties necessary for use as a three-dimensional scaffold and promote cellular repopulation. Further, this study provides the initial steps in developing new regenerative medicine strategies for renal tissue engineering and repair.
View details for DOI 10.1089/ten.tea.2009.0602
View details for Web of Science ID 000279455500010
View details for PubMedID 20156112