Cell type-selective secretome profiling in vivo.
Nature chemical biology
Extracellular vesicle-mediated amyloid transfer to neural progenitor cells: implications for RAGE and HIV infection
Metabolomic changes in human adipose tissue derived products following non-enzymatic microfacturing.
European review for medical and pharmacological sciences
2018; 22 (10): 3249?60
Secreted polypeptides are a fundamental axis of intercellular and endocrine communication. However, a global understanding of the composition and dynamics of cellular secretomes in intact mammalian organisms has been lacking. Here, we introduce a proximity biotinylation strategy that enables labeling, detection and enrichment of secreted polypeptides in a cell type-selective manner in mice. We generate a proteomic atlas of hepatocyte, myocyte, pericyte and myeloid cell secretomes by direct purification of biotinylated secreted proteins from blood plasma. Our secretome dataset validates known cell type-protein pairs, reveals secreted polypeptides that distinguish between cell types and identifies new cellular sources for classical plasma proteins. Lastly, we uncover a dynamic and previously undescribed nutrient-dependent reprogramming of the hepatocyte secretome characterized by the increased unconventional secretion of the cytosolic enzyme betaine-homocysteine S-methyltransferase (BHMT). This secretome profiling strategy enables dynamic and cell type-specific dissection of the plasma proteome and the secreted polypeptides that mediate intercellular signaling.
View details for DOI 10.1038/s41589-020-00698-y
View details for PubMedID 33199915
Dual mechanism of type VII collagen transfer by bone marrow mesenchymal stem cell extracellular vesicles to recessive dystrophic epidermolysis bullosa fibroblasts.
2018; 155: 50?58
In this study, we evaluated the metabolomic profiling of cryopreserved Lipogems® tissue products and the initial lipoaspirates before microfracturing, to determine altered metabolites that could result from the non-enzymatic processing or the cryopreservation method.Human Lipoaspirate samples (n=10) were divided in two aliquots, of which one was non-processed and the other was processed by Lipogems® device. Non-processed lipoaspirates and Lipogems® processed tissues were stored at -80°C fresh frozen (N=3 per group) or in the presence of 0.5 M dimethyl sulfoxide (DMSO) (N=7 per group). A global non-targeted metabolic profile on these samples was performed.Differences were observed in carbohydrate and nucleotide metabolism. These alterations translated in long chain and polyunsaturated fatty acid levels and amino acid metabolites showed divergent trends. When Lipogems® and Lipoaspirate tissue products were cryopreserved with DMSO, amino acids tended to increase in Lipogems® product. However, in the absence of DMSO aminoacids and their catabolites, tended to decrease in Lipogems® fat tissue product.Microfractured human adipose tissue has been shown to provide a more effective source of adult stromal cells compared to the initial lipoaspirated tissue material. These could be, according to our findings, due to the changes in the metabolic profile of lipoaspirate tissues products.
View details for DOI 10.26355/eurrev_201805_15088
View details for PubMedID 29863273
Inflammasome Proteins in Serum and Serum-Derived Extracellular Vesicles as Biomarkers of Stroke.
Frontiers in molecular neuroscience
2018; 11: 309
Recessive dystrophic epidermolysis bullosa (RDEB) is a severe blistering disease resulting from a lack of type VII collagen production. Recent clinical trials have shown efficacy of bone marrow-derived mesenchymal stem cells (BM-MSCs) in the treatment of epidermolysis bullosa, including improved basement membrane restructuring and cutaneous wound healing. The mechanism as to how type VII collagen is transferred from donor stem cell to recipient RDEB cells has not been defined. Here, we submit the model that BM-MSC-derived extracellular vesicles serve at least two roles: 1) to help transport type VII collagen within the extracellular space; and 2) to feed RDEB fibroblasts with messenger RNA that codes for type VII collagen, resulting in COL7A1 translation and synthesis of type VII collagen alpha chain proteins by RDEB fibroblasts. Utilizing a chemoselective ligation detection method, we found RDEB cells that were treated simultaneously with BM-MSC EVs and an l-methionine analog, l-homopropargylglycine (HPG), synthesized collagen VII alpha chain protein that contained the alkyne group of HPG to react (i.e. undergo the Click-iT® reaction) with azide-modified Alexa 594, suggesting de novo synthesis of type VII collagen by RDEB fibroblasts. Thus, our results support a model in which BM-MSC EVs help increase type VII collagen levels available to recipient cells by 1) donating BM-MSC type VII collagen protein and 2) inducing RDEB fibroblasts to make their own type VII collagen protein. These findings allow us to hypothesize that the secretome of BM-MSCs could have therapeutic value in the treatment of RDEB-related skin disorders.
View details for DOI 10.1016/j.biochi.2018.04.007
View details for PubMedID 29653141
Extracellular vesicles of the blood-brain barrier: Role in the HIV-1 associated amyloid beta pathology.
Molecular and cellular neurosciences
2017; 79: 12?22
The inflammasome is a key contributor to the inflammatory innate immune response after stroke. We have previously shown that inflammasome proteins are released in extracellular vesicles (EV) after brain and spinal cord injury. In addition, we have shown that inflammasome proteins offer great promise as biomarkers of central nervous system (CNS) injury following brain trauma. In the present study, we used a Simple Plex Assay (Protein Simple), a novel multi-analyte automated microfluidic immunoassay platform, to analyze serum and serum-derived EV samples from stroke patients and control subjects for inflammasome protein levels of caspase-1, apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), Interleukins (IL)-1?, and (IL)-18. Receiver operator characteristic (ROC) curves with associated confidence intervals obtained from the analysis of serum samples revealed that the area under the curve (AUC) for ASC was 0.99 with a confidence interval between 0.9914 and 1.004, whereas the AUC for caspase-1, IL-1?, and IL-18 were 0.75, 0.61, and 0.67, respectively. Thus, these data indicate that ASC is a potential biomarker of stroke and highlight the role of the inflammasome in the inflammatory response after brain ischemia.
View details for DOI 10.3389/fnmol.2018.00309
View details for PubMedID 30233311
View details for PubMedCentralID PMC6131639
Comprehensive Metabolomics Study To Assess Longitudinal Biochemical Changes and Potential Early Biomarkers in Nonobese Diabetic Mice That Progress to Diabetes.
Journal of proteome research
2017; 16 (10): 3873?90
HIV-infected brains are characterized by increased amyloid beta (A?) deposition. It is believed that the blood-brain barrier (BBB) is critical for A? homeostasis and contributes to A? accumulation in the brain. Extracellular vesicles (ECV), like exosomes, recently gained a lot of attention as potentially playing a significant role in A? pathology. In addition, HIV-1 hijacks the exosomal pathway for budding and release. Therefore, we investigated the involvement of BBB-derived ECV in the HIV-1-induced A? pathology in the brain. Our results indicate that HIV-1 increases ECV release from brain endothelial cells as well as elevates their A? cargo when compared to controls. Interestingly, brain endothelial cell-derived ECV transferred A? to astrocytes and pericytes. Infusion of brain endothelial ECV carrying fluorescent A? into the internal carotid artery of mice resulted in A? fluorescence associated with brain microvessels and in the brain parenchyma. These results suggest that ECV carrying A? can be successfully transferred across the BBB into the brain. Based on these observations, we conclude that HIV-1 facilitates the shedding of brain endothelial ECV carrying A?; a process that may increase A? exposure of cells of neurovascular unit, and contribute to amyloid deposition in HIV-infected brain.
View details for DOI 10.1016/j.mcn.2016.12.006
View details for PubMedID 28040512
View details for PubMedCentralID PMC5315639
Bone Marrow Mesenchymal Stem Cell-Derived CD63+ Exosomes Transport Wnt3a Exteriorly and Enhance Dermal Fibroblast Proliferation, Migration, and Angiogenesis In Vitro.
Stem cells and development
2017; 26 (19): 1384?98
A global nontargeted longitudinal metabolomics study was carried out in male and female NOD mice to characterize the time-profile of the changes in the metabolic signature caused by onset of type 1 diabetes (T1D) and identify possible early biomarkers in T1D progressors. Metabolomics profiling of samples collected at five different time-points identified 676 and 706 biochemicals in blood and feces, respectively. Several metabolites were expressed at significantly different levels in progressors at all time-points, and their proportion increased strongly following onset of hyperglycemia. At the last time-point, when all progressors were diabetic, a large percentage of metabolites had significantly different levels: 57.8% in blood and 27.8% in feces. Metabolic pathways most strongly affected included the carbohydrate, lipid, branched-chain amino acid, and oxidative ones. Several biochemicals showed considerable (>4×) change. Maltose, 3-hydroxybutyric acid, and kojibiose increased, while 1,5-anhydroglucitol decreased more than 10-fold. At the earliest time-point (6-week), differences between the metabolic signatures of progressors and nonprogressors were relatively modest. Nevertheless, several compounds had significantly different levels and show promise as possible early T1D biomarkers. They include fatty acid phosphocholine derivatives from the phosphatidylcholine subpathway (elevated in both blood and feces) as well as serotonin, ribose, and arabinose (increased) in blood plus 13-HODE, tocopherol (increased), diaminopimelate, valerate, hydroxymethylpyrimidine, and dulcitol (decreased) in feces. A combined metabolic signature based on these compounds might serve as an early predictor of T1D-progressors.
View details for DOI 10.1021/acs.jproteome.7b00512
View details for PubMedID 28799767
Plasma-derived exosome characterization reveals a distinct microRNA signature in long duration Type 1 diabetes.
2017; 7 (1): 5998
Wnts are secreted glycoproteins that regulate stem cell self-renewal, differentiation, and cell-to-cell communication during embryonic development and in adult tissues. Bone marrow mesenchymal stem cells (BM-MSCs) have been shown to stimulate dermis repair and regeneration; however, it is unclear how BM-MSCs may modulate downstream Wnt signaling. While recent reports implicate that Wnt ligands and Wnt messenger RNAs (such as Wnt4) exist within the interior compartment of exosomes, it has been debated whether or not Wnts exist on the exterior surface of exosomes to travel in the extracellular space. To help answer this question, we utilized flow cytometry of magnetic beads coated with anti-CD63 antibodies and found, for the first time, that Wnt3a protein is detectable exteriorly on CD63+ exosomes derived from BM-MSCs over-secreting Wnt3a into serum-free conditioned media (Wnt3a CM). Our data suggest that CD63+ exosomes significantly help transport exterior Wnt3a signal to recipient cells to promote fibroblast and endothelial functions. During purification of exosomes, we unexpectedly found that use of ultracentrifugation alone significantly decreased the ability to detect exteriorly bound Wnt3a on CD63+ exosomes, however, polyethylene glycol (PEG)-mediated exosome-enrichment before exosome-purification (with ultracentrifugation into a sucrose cushion) resulted in exosomes more likely to retain exterior Wnt3a detectability and downstream Wnt/beta-catenin activity. Our findings indicate the important role that purification methods may have on stem cell-derived Wnt-exosome activity in downstream assays. The ability for BM-MSC Wnt3a CM and exosomes to stimulate dermal fibroblast proliferation and migration, and endothelial angiogenesis in vitro, was significantly decreased after CD63+-exosome depletion or knockdown of Wnt coreceptor LRP6 in recipient cells, suggesting both are required for optimal Wnt-exosome activity in our system. Thus, BM-MSC-derived CD63+ exosomes are a significant carrier of exterior Wnt3a within high Wnt environments, resulting in downstream fibroblast proliferation, migration, and angiogenesis in vitro.
View details for DOI 10.1089/scd.2017.0087
View details for PubMedID 28679315
Metabolomics Study of the Effects of Inflammation, Hypoxia, and High Glucose on Isolated Human Pancreatic Islets.
Journal of proteome research
2017; 16 (6): 2294?2306
Type 1 diabetes mellitus (T1DM) results from an autoimmune attack against the insulin-producing ß cells which leads to chronic hyperglycemia. Exosomes are lipid vesicles derived from cellular multivesicular bodies that are enriched in specific miRNAs, potentially providing a disease-specific diagnostic signature. To assess the value of exosome miRNAs as biomarkers for T1DM, miRNA expression in plasma-derived exosomes was measured. Nanoparticle tracking analysis and transmission electron microscopy confirmed the presence of plasma-derived exosomes (EXOs) isolated by differential centrifugation. Total RNA extracted from plasma-derived EXOs of 12 T1DM and 12 control subjects was hybridized onto Nanostring human v2 miRNA microarray array and expression data were analyzed on nSolver analysis software. We found 7 different miRNAs (1 up-regulated and 6 down-regulated), that were differentially expressed in T1DM. The selected candidate miRNAs were validated by qRT-PCR analysis of cohorts of 24 T1DM and 24 control subjects. Most of the deregulated miRNAs are involved in progression of T1DM. These findings highlight the potential of EXOs miRNA profiling in the diagnosis as well as new insights into the molecular mechanisms involved in T1DM.
View details for DOI 10.1038/s41598-017-05787-y
View details for PubMedID 28729721
View details for PubMedCentralID PMC5519761
Exosomes as biomarkers and therapeutic tools for type 1 diabetes mellitus.
European review for medical and pharmacological sciences
2017; 21 (12): 2940?56
The transplantation of human pancreatic islets is a therapeutic possibility for a subset of type 1 diabetic patients who experience severe hypoglycemia. Pre- and post-transplantation loss in islet viability and function, however, is a major efficacy-limiting impediment. To investigate the effects of inflammation and hypoxia, the main obstacles hampering the survival and function of isolated, cultured, and transplanted islets, we conducted a comprehensive metabolomics evaluation of human islets in parallel with dynamic glucose-stimulated insulin release (GSIR) perifusion studies for functional evaluation. Metabolomics profiling of media and cell samples identified a total of 241 and 361 biochemicals, respectively. Metabolites that were altered in highly significant manner in both included, for example, kynurenine, kynurenate, citrulline, and mannitol/sorbitol under inflammation (all elevated) plus lactate (elevated) and N-formylmethionine (depressed) for hypoxia. Dynamic GSIR experiments, which capture both first- and second-phase insulin release, found severely depressed insulin-secretion under hypoxia, whereas elevated baseline and stimulated insulin-secretion was measured for islet exposed to the inflammatory cytokine cocktail (IL-1?, IFN-?, and TNF-?). Because of the uniquely large changes observed in kynurenine and kynurenate, they might serve as potential biomarkers of islet inflammation, and indoleamine-2,3-dioxygenase on the corresponding pathway could be a worthwhile therapeutic target to dampen inflammatory effects.
View details for DOI 10.1021/acs.jproteome.7b00160
View details for PubMedID 28452488
View details for PubMedCentralID PMC5557342
Combination high-dose omega-3 fatty acids and high-dose cholecalciferol in new onset type 1 diabetes: a potential role in preservation of beta-cell mass.
European review for medical and pharmacological sciences
2016; 20 (15): 3313?18
Early diagnosis of diabetes mellitus can significantly improve therapeutic strategies and overall health span. Identifying biomarkers as a tool for determining the risk of developing diabetes as well as a monitoring strategy for progression of the disease state would be useful in predicting potential complications while simultaneously improving our ability to prevent and treat diabetes. Extracellular vesicles (EV) have recently emerged as prominent mediators of intercellular communication and as a potential source for the discovery of novel biomarkers. A deeper understanding of the cargo molecules present in EVs obtained from type 1 diabetes mellitus (T1D) patients may aid in the identification of novel diagnostic and prognostic biomarkers, and can potentially lead to the discovery of new therapeutic targets.
View details for PubMedID 28682421
Diet and Inflammation: Possible Effects on Immunity, Chronic Diseases, and Life Span.
Journal of the American College of Nutrition
2015; 34 Suppl 1: 10?13
Several studies have evaluated the role of inflammation in type 1 diabetes (T1D). The safety profile and anti-inflammatory properties of high dose omega-3 fatty acids combined with Vitamin D supplementation make this therapy a possible candidate for T1D intervention trials. Herein, we describe the case of a 14-year-old boy with new onset T1D treated with high dose Omega-3 and vitamin D3. By 12 months, peak C-peptide increased to 0.55 nmol/L (1.66 ng/mL) corresponding to a 20% increment from baseline and AUC C-peptide was slightly higher compared to 9 months (0.33 vs. 0.30 nmol/L/min) although remaining slightly lower than baseline. Combination high-dose Omega-3 fatty acids and high-dose vitamin D3 therapy was well tolerated and may have beneficial effects on beta-cell function. Randomized controlled trials could be of assistance to determine whether this therapy may result in the preservation of beta-cell function in patients with new onset T1D.
View details for PubMedID 27467009
Hierarchical paracrine interaction of breast cancer associated fibroblasts with cancer cells via hMAPK-microRNAs to drive ER-negative breast cancer phenotype.
Cancer biology & therapy
2015; 16 (11): 1671?81
Chronic inflammation negatively impacts all physiological functions, causing an array of degenerative conditions including diabetes; cancer; cardiovascular, osteo-articular, and neurodegenerative diseases; autoimmunity disorders; and aging. In particular, there is a growing knowledge of the role that gene transcription factors play in the inflammatory process. Obesity, metabolic syndrome, and diabetes represent multifactorial conditions resulting from improper balances of hormones and gene expression. In addition, these conditions have a strong inflammatory component that can potentially be impacted by the diet. It can reduce pro-inflammatory eicosanoids that can alter hormonal signaling cascades to the modulation of the innate immune system and gene transcription factors. Working knowledge of the impact of how nutrients, especially dietary fatty acids and polyphenols, can impact these various molecular targets makes it possible to develop a general outline of an anti-inflammatory diet that offers a unique, nonpharmacological approach in treating obesity, metabolic syndrome, and diabetes. Several important bioactive dietary components can exert their effect through selected inflammatory pathways that can affect metabolic and genetic changes. In fact, dietary components that can modulate glucose and insulin levels, as well as any other mediator that can activate nuclear factor-kB, can also trigger inflammation through common pathway master switches.
View details for DOI 10.1080/07315724.2015.1080101
View details for PubMedID 26400428
Therapeutic potential of human adipose-derived stem cells (ADSCs) from cancer patients: a pilot study.
2014; 9 (11): e113288
Multiple juxtacrine and paracrine interactions occur between cancer cells and non-cancer cells of the tumor microenvironment (TME) that direct tumor progression. Cancer Associated Fibroblasts (CAFs) are an integral component of the TME, and the majority of breast tumor stroma is comprised of CAFs. Heterotypic interactions between cancer cells and non-cancer cells of the TME occur via soluble agents, including cytokines, hormones, growth factors, and secreted microRNAs. We previously identified a microRNA signature indicative of hyperactive MAPK signaling (hMAPK-miRNA signature) that significantly associated with reduced recurrence-free and overall survival. Here we report that the hMAPK-miRNA signature associates with a high metric of stromal cell infiltrate, and we investigate the role of microRNAs, particularly hMAPK-microRNAs, secreted by CAFs on estrogen receptor (ER) expression in breast cancer cells. ER-positive MCF-7/ltE2- cells were treated with conditioned media (CM) from CAFs derived from breast cancers of different PAM50 subtypes (CAFBAS, CAFHER2, and CAFLA). CAF CM isolated specifically from ER-negative primary breast tumors led to ER repression in vitro. Nanoparticle tracking analysis and transmission electron microscopy confirmed the presence of CAF-secreted exosomes in CM and the uptake of these exosomes by the ER+ MCF-7/ltE2- cells. Differentially expressed microRNAs in CAF CM as well as in MCF-7/ltE2- cells treated with this CM were identified. Knockdown of miR-221/222 in CAFBAS resulted in knockdown of miR221/222 levels in the conditioned media and the CM from CAFBAS; miR221/222 knockdown rescued ER repression in ER-positive cell lines treated with CAFBAS-CM. Collectively, our results demonstrate that CAF-secreted microRNAs are directly involved in ER-repression, and may contribute to the MAPK-induced ER repression in breast cancer cells.
View details for DOI 10.1080/15384047.2015.1071742
View details for PubMedID 26186233
View details for PubMedCentralID PMC4846097
Mesenchymal stem cells from adipose tissue (ADSCs) are an important source of cells for regenerative medicine. The therapeutic effect of culture-expanded adipose derived stem cells has been shown; however, optimal xeno-free culture conditions remain to be determined. Cancer patients, specifically those undergoing invasive surgery, constitute a subgroup of patients who could benefit from autologous stem cell transplantation. Although regenerative potential of their ADSCs could be affected by the disease and/or treatment, we are not aware of any study that has evaluated the therapeutic potential of ADSCs isolated from cancer patients in reference to that of ADSCs derived from healthy subjects. Here we report that ADSCs isolated from subabdominal adipose tissue of patients with urological neoplasms yielded similar growth kinetics, presented equivalent mesenchymal surface markers and showed similar differentiation potential into distinct mesodermal cell lineages: adipocytes, chondroblasts and osteoblasts than ADSCs isolated from adipose tissue of age-matched non-oncogenic participants, all under xeno-free growth culture conditions. Molecular karyotyping of patient expanded ADSCs genomes showed no disease-related alterations indicating their safety. In addition, vesicles <100 nm identified as exosomes (EXOs) which may be at least partly responsible for the attributed therapeutic paracrine effects of the ADSCs were effectively isolated from ADSCs and showed equivalent miRNA content regardless they were derived from cancer patients or non-oncogenic participants indicating that the repair capabilities of xeno-free expanded ADSCs are not compromised by patient condition and therefore their xeno-free culture expanded ADSCs should be suitable for autologous stem cell transplantation in a clinical setting.
View details for DOI 10.1371/journal.pone.0113288
View details for PubMedID 25412325
View details for PubMedCentralID PMC4239050