The impact of hypoxia preconditioning on mesenchymal stem cells performance in hypertensive kidney disease.

BACKGROUND: Autologous mesenchymal stem cells (MSCs) have emerged as a therapeutic option for many diseases. Hypertensive kidney disease (HKD) might impair MSCs' reparative ability by altering the biomolecular properties, but the characteristics of this impairment are unclear. In our previous pre-clinical studies, we found hypoxic preconditioning (HPC) enhanced angiogenesis and suppressed senescence gene expression. Thus, we hypothesize that HPC would improve human MSCs by enhancing their functionality and angiogenesis, creating an anti-inflammatory and anti-senescence environment. METHODS: MSC samples (n = 12 each) were collected from the abdominal fat of healthy kidney donors (HC), hypertensive patients (HTN), and patients with hypertensive kidney disease (HKD). MSCs were harvested and cultured in Normoxic (20% O2) or Hypoxic (1% O2) conditions. MSC functionality was measured by proliferation assays and cytokine released in conditioned media. Senescence was evaluated by senescence-associated beta-galactosidase (SA-beta-gal) activity. Additionally, transcriptome analysis using RNA-sequencing and quantitative PCR (qPCR) were performed. RESULTS: At baseline, normoxic HTN-MSCs had higher proliferation capacity compared to HC. However, HPC augmented proliferation in HC. HPC did not affect the release of pro-angiogenic protein VEGF, but increased EGF in HC-MSC, and decreased HGF in HC and HKD MSCs. Under HPC, SA-ß-gal activity tended to decrease, particularly in HC group. HPC upregulated mostly the pro-angiogenic and inflammatory genes in HC and HKD and a few senescence genes in HKD. CONCLUSIONS: HPC has a more favorable functional effect on HC- than on HKD-MSC, reflected in increased proliferation and EGF release, and modest decrease in senescence, whereas it has little effect on HTN or HKD MSCs.

Obesity Blunts the Effect of Mesenchymal Stem Cell-Derived Extracellular Vesicles.

Introduction: Mesenchymal stem/stromal cell-derived extracellular vesicles (MSC-EVs) are paracrine vectors with therapeutic functions comparable to their parent cells. However, it remains unclear if donor obesity affects their therapeutic functions. We tested the hypothesis that the curative effect of human adipose tissue-derived MSC-EVs (A-MSC-EVs) is blunted by obesity. Methods: MSC-EVs were isolated by ultracentrifugation from mesenchymal stem/stromal cells (MSCs) collected from abdominal subcutaneous fat of obese and lean human subjects (obese and lean-MSC-EVs, respectively) and injected into the aorta of mice 2 weeks after renal artery stenosis (RAS) induction. Magnetic resonance imaging studies were conducted 2 weeks after MSC-EVs delivery to determine renal function. The effect of MSC-EVs on tissue injury was assessed by histology and gene expression of inflammatory factors, including interleukin (IL)-1ß, IL-6, monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor alpha (TNF-α). Oxidative damage, macrophage infiltration, plasma renin, and hypoxia inducible factor-1α (HIF-1α) were also assessed. Results: Tracking showed that MSC-EVs localized in the kidney tissue, including glomeruli and tubules. All MSC-EVs decreased systolic blood pressure (SBP) and plasma renin and improved the poststenotic kidney (STK) volume, but obese-MSC-EVs were less effective than lean-MSC-EVs in improving medullary hypoxia, fibrosis, and tubular injury. Lean-MSC-EVs decreased inflammation, whereas obesity attenuated this effect. Only lean-MSC-EVs decreased STK cortical HIF-1α expression. Conclusion: Obesity attenuates the antihypoxia, antifibrosis, antiinflammation, and tubular repair functions of human MSC-EVs in chronic ischemic kidney disease. These observations may have implications for the self-repair potency of obese subjects and for the use of autologous MSC-EVs in regenerative medicine.

Obesity blunts amelioration of cardiac hypertrophy and fibrosis by human mesenchymal stem/stromal cell-derived extracellular vesicles.

Renovascular hypertension (RVH) can induce cardiac damage that is reversible using adipose tissue-derived mesenchymal stromal/stem cells (A-MSCs). However, A-MSCs isolated from patients with obesity are less effective than lean-A-MSC in blunting hypertensive cardiomyopathy in mice with RVH. We tested the hypothesis that this impairment extends to their obese A-MSC-extracellular vesicles (EVs) progeny. MSCs were harvested from the subcutaneous fat of obese and lean human subjects, and their EVs were collected and injected into the aorta of mice 2 wk after renal artery stenosis or sham surgery. Cardiac left ventricular (LV) function was studied with MRI 2 wk later, and myocardial tissue ex vivo. Blood pressure, LV myocardial wall thickness, mass, and fibrosis that were elevated in RVH mice were suppressed only by lean EVs. Hence, human A-MSC-derived lean EVs are more effective than obese EVs in blunting hypertensive cardiac injury in RVH mice. These observations highlight impaired paracrine repair potency of endogenous MSCs in patients with obesity.NEW & NOTEWORTHY Injection of A-MSC-derived EVs harvested from patients who are lean can resolve myocardial injury in mice with experimental renovascular hypertension more effectively than A-MSC-derived EVs from patients with obesity. These observations underscore and might have important ramifications for the self-healing capacity of patients with obesity and for the use of autologous EVs as a regenerative tool.

Obesity and dyslipidemia are associated with partially reversible modifications to DNA hydroxymethylation of apoptosis- and senescence-related genes in swine adipose-derived mesenchymal stem/stromal cells.

BACKGROUND: Obesity dysregulates key biological processes underlying the functional homeostasis, fate decisions, and reparative potential of mesenchymal stem/stromal cells (MSCs). Mechanisms directing obesity-induced phenotypic alterations in MSCs remain unclear, but emerging drivers include dynamic modification of epigenetic marks, like 5-hydroxymethylcytosine (5hmC). We hypothesized that obesity and cardiovascular risk factors induce functionally relevant, locus-specific changes in 5hmC of swine adipose-derived MSCs and evaluated their reversibility using an epigenetic modulator, vitamin-C. METHODS: Female domestic pigs were fed a 16-week Lean or Obese diet (n = 6 each). MSCs were harvested from subcutaneous adipose tissue, and 5hmC profiles were examined through hydroxymethylated DNA immunoprecipitation sequencing (hMeDIP-seq) followed by an integrative (hMeDIP and mRNA sequencing) gene set enrichment analysis. For clinical context, we compared 5hmC profiles of adipose tissue-derived human MSCs harvested from patients with obesity and healthy controls. RESULTS: hMeDIP-seq revealed 467 hyper- (fold change ≥ 1.4; p-value ≤ 0.05) and 591 hypo- (fold change ≤ 0.7; p-value ≤ 0.05) hydroxymethylated loci in swine Obese- versus Lean-MSCs. Integrative hMeDIP-seq/mRNA-seq analysis identified overlapping dysregulated gene sets and discrete differentially hydroxymethylated loci with functions related to apoptosis, cell proliferation, and senescence. These 5hmC changes were associated with increased senescence in cultured MSCs (p16/CDKN2A immunoreactivity, senescence-associated ß-galactosidase [SA-ß-Gal] staining), were partly reversed in swine Obese-MSCs treated with vitamin-C, and shared common pathways with 5hmC changes in human Obese-MSCs. CONCLUSIONS: Obesity and dyslipidemia are associated with dysregulated DNA hydroxymethylation of apoptosis- and senescence-related genes in swine and human MSCs, potentially affecting cell vitality and regenerative functions. Vitamin-C may mediate reprogramming of this altered epigenomic landscape, providing a potential strategy to improve the success of autologous MSC transplantation in obese patients.

Autologous Extracellular Vesicles Attenuate Cardiac Injury in Experimental Atherosclerotic Renovascular Disease More Effectively Than Their Parent Mesenchymal Stem/Stromal Cells.

Atherosclerotic renovascular disease (RVD) leads to hypertension, chronic kidney disease (CKD), and heart disease. Intrarenal delivery of mesenchymal stem cells (MSCs) and MSC-derived extracellular vesicles (EVs) attenuate renal injury and suppress release of inflammatory cytokines in porcine RVD. We hypothesized that this strategy would also be useful for cardioprotection. Pigs with renovascular hypertension and metabolic syndrome were studied 4 weeks after treatment with a single intrarenal infusion of autologous MSCs, EVs, or vehicle. Cardiac structure and function were assessed in vivo, and myocardial remodeling and expression of the pro-fibrotic factor growth factor receptor-bound protein-2 (Grb2) were measured ex-vivo. Inflammatory cytokine levels were measured in the systemic circulation and myocardial tissue. Blood pressure was elevated in all RVD groups, but serum creatinine increased in RVD and decreased in both RVD + MSCs and RVD + EVs. RVD-induced diastolic dysfunction (lower E/A ratio) was normalized in both MSCs- and EVs- treated pigs. Intrarenal delivery of MSCs and EVs also attenuated RVD-induced myocardial fibrosis, collagen deposition, and Grb2 expression, yet EVs restored capillary density and inflammation more effectively than MSCs. These observations suggest that autologous EVs attenuate cardiac injury in experimental RVD more effectively than their parent MSCs.

Effect of Hypoxia Preconditioning on the Regenerative Capacity of Adipose Tissue Derived Mesenchymal Stem Cells in a Model of Renal Artery Stenosis.

Atherosclerotic renal artery stenosis (ARAS) is associated with irreversible parenchymal renal disease and regenerative stem cell therapies may improve renal outcomes. Hypoxia preconditioning (HPC) may improve the regenerative functions of adipose tissue-derived mesenchymal stem cells (AMSC) by affecting DNA 5-hydroxymethylcytosine (5hmC) marks in angiogenic genes. Here, we investigated using a porcine ARAS model, whether growth of ARAS AMSCs in hypoxia (Hx) versus normoxia (Nx) would enhance renal tissue repair, and comprehensively analyze how HPC modifies DNA hydroxymethylation compared to untreated ARAS and healthy/normal pigs (n=5 each). ARAS pigs exhibited elevated serum cholesterol, serum creatinine and renal artery stenosis, with a concomitant decrease in renal blood flow (RBF) and increased blood pressure (BP) compared to healthy pigs. Renal artery injection of either autologous Nx or Hx AMSCs improved diastolic BP, reduced kidney tissue fibrosis, and inflammation (CD3+ T-cells) in ARAS pigs. In addition, renal medullary hypoxia significantly lowered with Nx but not Hx AMSC treatment. Mechanistically, levels of epigenetic 5hmC marks (which reflect gene activation) estimated using DNA immunoprecipitation technique were elevated in profibrotic and inflammatory genes in ARAS compared with normal AMSCs. HPC significantly reduced 5hmC levels in cholesterol biosynthesis and oxidative stress response pathways in ARAS AMSCs. Thus, autologous AMSCs improve key renovascular parameters and inflammation in ARAS pigs, with HPC mitigating pathological molecular effects on inflammatory and profibrotic genes which may play a role in augmenting regenerative capacity of AMSCs.

Effects of Elamipretide on Autophagy in Renal Cells of Pigs with Metabolic Syndrome.

Autophagy eliminates excessive nutrients and maintains homeostasis. Obesity and metabolic syndrome (MetS) dysregulate autophagy, possibly partly due to mitochondria injury and inflammation. Elamipretide (ELAM) improves mitochondrial function. We hypothesized that MetS blunts kidney autophagy, which ELAM would restore. Domestic pigs were fed a control or MetS-inducing diet for 16 weeks. During the 4 last weeks, MetS pigs received subcutaneous injections of ELAM (0.1 mg/kg/day, MetS + ELAM) or vehicle (MetS), and kidneys were then harvested to measure protein expression of autophagy mediators and apoptosis. Systemic and renal venous levels of inflammatory cytokines were measured to calculate renal release. The function of isolated mitochondria was assessed by oxidative stress, energy production, and pro-apoptotic activity. MetS slightly downregulated renal expression of autophagy mediators including p62, ATG5-12, mTOR, and AMPK vs. control. Increased mitochondrial H2O2 production accompanied decreased ATP production, elevated apoptosis, and renal fibrosis. In MetS + ELAM, mito-protection restored autophagic protein expression, improved mitochondrial energetics, and blunted renal cytokine release and fibrosis. In vitro, mitoprotection restored mitochondrial membrane potential and reduced oxidative stress in injured proximal tubular epithelial cells. Our study suggests that swine MetS mildly affects renal autophagy, possibly secondary to mitochondrial damage, and may contribute to kidney structural damage in MetS.

Microvascular remodeling and altered angiogenic signaling in human kidneys distal to occlusive atherosclerotic renal artery stenosis.

BACKGROUND: Renal artery stenosis (RAS) is an important cause of chronic kidney disease and secondary hypertension. In animal models, renal ischemia leads to downregulation of growth factor expression and loss of intrarenal microcirculation. However, little is known about the sequelae of large-vessel occlusive disease on the microcirculation within human kidneys. METHOD: This study included five patients who underwent nephrectomy due to renovascular occlusion and seven nonstenotic discarded donor kidneys (four deceased donors). Micro-computed tomography was performed to assess microvascular spatial densities and tortuosity, an index of microvascular immaturity. Renal protein expression, gene expression and histology were studied in vitro using immunoblotting, polymerase chain reaction and staining. RESULTS: RAS demonstrated a loss of medium-sized vessels (0.2-0.3 mm) compared with donor kidneys (P = 0.037) and increased microvascular tortuosity. RAS kidneys had greater protein expression of angiopoietin-1, hypoxia-inducible factor-1α and thrombospondin-1 but lower protein expression of vascular endothelial growth factor (VEGF) than donor kidneys. Renal fibrosis, loss of peritubular capillaries (PTCs) and pericyte detachment were greater in RAS, yet they had more newly formed PTCs than donor kidneys. Therefore, our study quantified significant microvascular remodeling in the poststenotic human kidney. RAS induced renal microvascular loss, vascular remodeling and fibrosis. Despite downregulated VEGF, stenotic kidneys upregulated compensatory angiogenic pathways related to angiopoietin-1. CONCLUSIONS: These observations underscore the nature of human RAS as a microvascular disease distal to main vessel stenosis and support therapeutic strategies directly targeting the poststenotic kidney microcirculation in patients with RAS.

Mesenchymal Stem/Stromal Cell-Derived Extracellular Vesicles Elicit Better Preservation of the Intra-Renal Microvasculature Than Renal Revascularization in Pigs with Renovascular Disease.

BACKGROUND: Percutaneous transluminal renal angioplasty (PTRA) confers clinical and mortality benefits in select 'high-risk' patients with renovascular disease (RVD). Intra-renal-delivered extracellular vesicles (EVs) released from mesenchymal stem/stromal cells (MSCs) protect the kidney in experimental RVD, but have not been compared side-by-side to clinically applied interventions, such as PTRA. We hypothesized that MSC-derived EVs can comparably protect the post-stenotic kidney via direct tissue effects. METHODS: Five groups of pigs (n = 6 each) were studied after 16 weeks of RVD, RVD treated 4 weeks earlier with either PTRA or MSC-derived EVs, and normal controls. Single-kidney renal blood flow (RBF) and glomerular filtration rate (GFR) were assessed in vivo with multi-detector CT, and renal microvascular architecture (3D micro CT) and injury pathways ex vivo. RESULTS: Despite sustained hypertension, EVs conferred greater improvement of intra-renal microvascular and peritubular capillary density compared to PTRA, associated with attenuation of renal inflammation, oxidative stress, and tubulo-interstitial fibrosis. Nevertheless, stenotic kidney RBF and GFR similarly rose in both PTRA- and EV-treated pigs compared RVD + Sham. mRNA sequencing reveled that EVs were enriched with pro-angiogenic, anti-inflammatory, and antioxidants genes. CONCLUSION: MSC-derived EVs elicit a better preservation of the stenotic kidney microvasculature and greater attenuation of renal injury and fibrosis compared to PTRA, possibly partly attributed to their cargo of vasculo-protective genes. Yet, both strategies similarly improve renal hemodynamics and function. These observations shed light on diverse mechanisms implicated in improvement of post-stenotic kidney function and position EVs as a promising therapeutic intervention in RVD.

Comparable in vitro Function of Human Liver-Derived and Adipose Tissue-Derived Mesenchymal Stromal Cells: Implications for Cell-Based Therapy.

Mesenchymal stem/stromal cells (MSCs) have been investigated extensively for their immunotherapeutic and regenerative properties, which may differ by cell source. In MSCs harvested from donors matched for sex, age, and body mass index, we compared the proliferative and migration functions of liver-derived MSCs (L-MSCs) and adipose tissue-derived MSCs (A-MSCs) (n = 6 donors each). Cellular senescence was evaluated by senescence-associated beta-galactosidase enzyme activity and expression of senescence-associated secretory phenotype (SASP) factors using real-time quantitative polymerase chain and by western blot assay. The pro-angiogenic and reparative potency of MSCs was compared by co-culturing MSCs with injured human umbilical vein endothelial cells (HUVEC). The proliferation and migration properties were similar in L-MSCs and A-MSCs. Although cell cycle arrest and SASP genes were similarly expressed in both MSCs, tumor necrosis factor alpha gene and protein expression were significantly downregulated in L-MSCs. In co-cultured injured HUVEC, A-MSCs restored significantly more tubes and tube connections than L-MSCs. Therefore, despite many functional similarities between L-MSCs and A-MSCs, L-MSCs have enhanced immunomodulatory properties, while A-MSCs appear to have better pro-angiogenic and vascular reparative potency. Availability of a broad range of cellular options might enable selecting cell-based therapy appropriate for the specific underlying disease.

Hypoxic preconditioning induces epigenetic changes and modifies swine mesenchymal stem cell angiogenesis and senescence in experimental atherosclerotic renal artery stenosis.

BACKGROUND: Atherosclerotic renal artery stenosis (ARAS) is a risk factor for ischemic and hypertensive kidney disease (HKD) for which autologous mesenchymal stem cell (MSC) appears to be a promising therapy. However, MSCs from ARAS patients exhibit impaired function, senescence, and DNA damage, possibly due to epigenetic mechanisms. Hypoxia preconditioning (HPC) exerts beneficial effects on cellular proliferation, differentiation, and gene and protein expression. We hypothesized that HPC could influence MSC function and senescence in ARAS by epigenetic mechanisms and modulating gene expression of chromatin-modifying enzymes. METHODS: Adipose-derived MSC harvested from healthy control (N = 8) and ARAS (N = 8) pigs were cultured under normoxia (20%O2) or hypoxia (1%O2) conditions. MSC function was assessed by migration, proliferation, and cytokine release in conditioned media. MSC senescence was evaluated by SA-ß-gal activity. Specific pro-angiogenic and senescence genes were assessed by reverse transcription polymerase chain reaction (RT-PCR). Dot blotting was used to measure global genome 5-hydroxymethylcytosine (5hmC) levels on DNA and Western blotting of modified histone 3 (H3) proteins to quantify tri-methylated lysine-4 (H3K4me3), lysine-9 (H3K9me3), and lysine-27 (H3K27me3) residues. RESULTS: Specific pro-angiogenic genes in ARAS assessed by RT-PCR were lower at baseline but increased under HPC, while pro-senescence genes were higher in ARAS at baseline as compared healthy MSCs. ARAS MSCs under basal conditions, displayed higher H3K4me3, H3K27me3, and 5hmC levels compared to healthy MSCs. During HPC, global 5hmC levels were decreased while no appreciable changes occurred in histone H3 tri-methylation. ARAS MSCs cultured under HPC had higher migratory and proliferative capacity as well as increased vascular endothelial growth factor and epidermal growth factor expression compared to normoxia, and SA-ß-gal activity decreased in both animal groups. CONCLUSIONS: These data demonstrate that swine ARAS MSCs have decreased angiogenesis and increased senescence compared to healthy MSCs and that HPC mitigates MSC dysfunction, senescence, and DNA hydroxymethylation in ARAS MSC. Thus, HPC for MSCs may be considered for their optimization to improve autologous cell therapy in patients with nephropathies.

Diabetic Kidney Disease Alters the Transcriptome and Function of Human Adipose-Derived Mesenchymal Stromal Cells but Maintains Immunomodulatory and Paracrine Activities Important for Renal Repair.

Mesenchymal stem/stromal cells (MSCs) facilitate repair in experimental diabetic kidney disease (DKD). However, the hyperglycemic and uremic milieu may diminish regenerative capacity of patient-derived therapy. We hypothesized that DKD reduces human MSC paracrine function. Adipose-derived MSC from 38 participants with DKD and 16 control subjects were assessed for cell surface markers, trilineage differentiation, RNA sequencing (RNA-seq), in vitro function (coculture or conditioned medium experiments with T cells and human kidney cells [HK-2]), secretome profile, and cellular senescence abundance. The direction of association between MSC function and patient characteristics were also tested. RNA-seq analysis identified 353 differentially expressed genes and downregulation of several immunomodulatory genes/pathways in DKD-MSC versus Control-MSC. DKD-MSC phenotype, differentiation, and tube formation capacity were preserved, but migration was reduced. DKD-MSC with and without interferon-γ priming inhibited T-cell proliferation greater than Control-MSC. DKD-MSC medium contained higher levels of anti-inflammatory cytokines (indoleamine 2,3-deoxygenase 1 and prostaglandin-E2) and prorepair factors (hepatocyte growth factor and stromal cell-derived factor 1) but lower IL-6 versus control-MSC medium. DKD-MSC medium protected high glucose plus transforming growth factor-ß-exposed HK-2 cells by reducing apoptotic, fibrotic, and inflammatory marker expression. Few DKD-MSC functions were affected by patient characteristics, including age, sex, BMI, hemoglobin A1c, kidney function, and urine albumin excretion. However, senescence-associated ß-galactosidase activity was lower in DKD-MSC from participants on metformin therapy. Therefore, while DKD altered the transcriptome and migratory function of culture-expanded MSCs, DKD-MSC functionality, trophic factor secretion, and immunomodulatory activities contributing to repair remained intact. These observations support testing of patient-derived MSC therapy and may inform preconditioning regimens in DKD clinical trials.

Renovascular Hypertension Induces Myocardial Mitochondrial Damage, Contributing to Cardiac Injury and Dysfunction in Pigs With Metabolic Syndrome.

BACKGROUND: Renovascular hypertension (RVH) often manifest with metabolic syndrome (MetS) as well. Coexisting MetS and hypertension increases cardiovascular morbidity and mortality, but the mechanisms underlying cardiac injury remain unknown. We hypothesized that superimposition of MetS induces myocardial mitochondrial damage, leading to cardiac injury and dysfunction in swine RVH. METHODS: Pigs were studied after 16 weeks of diet-induced MetS with or without RVH (unilateral renal artery stenosis), and Lean controls (n = 6 each). Systolic and diastolic cardiac function were assessed by multidetector CT, and cardiac mitochondrial morphology (electron microscopy) and myocardial function in tissue and isolated mitochondria. RESULTS: Body weight was similarly higher in MetS groups vs. Lean. RVH groups achieved significant stenosis and developed hypertension. Mitochondrial matrix density and adenosine triphosphate production were lower and H2O2 production higher in RVH groups vs. Lean and MetS. Lean + RVH (but not MetS + RVH) activated mitophagy, which was associated with decreased myocardial expression of mitophagy-related microRNAs. MetS groups exhibited higher numbers of intermitochondrial junctions, which could have prevented membrane depolarization/activation of mitophagy in MetS + RVH. Cardiac fibrosis, hypertrophy (increased left ventricular muscle mass), and diastolic function (decreased E/A ratio) were greater in MetS + RVH vs. Lean + RVH. CONCLUSIONS: MetS+RVH induces myocardial mitochondrial damage and dysfunction. MetS + RVH failed to activate mitophagy, resulting in greater cardiac remodeling, fibrosis, and diastolic dysfunction. Mitochondrial injury and impaired mitophagy may constitute important mechanisms and therapeutic targets to ameliorate cardiac damage and dysfunction in patients with coexisting MetS and RVH.

Global epigenetic alterations of mesenchymal stem cells in obesity: the role of vitamin C reprogramming.

Obesity promotes dysfunction and impairs the reparative capacity of mesenchymal stem/stromal cells (MSCs), and alters their transcription, protein content, and paracrine function. Whether these adverse effects are mediated by chromatin-modifying epigenetic changes remains unclear. We tested the hypothesis that obesity imposes global DNA hydroxymethylation and histone tri-methylation alterations in obese swine abdominal adipose tissue-derived MSCs compared to lean pig MSCs. MSCs from female lean (n = 7) and high-fat-diet fed obese (n = 7) domestic pigs were assessed using global epigenetic assays, before and after in-vitro co-incubation with the epigenetic modulator vitamin-C (VIT-C) (50 µg/ml). Dot blotting was used to measure across the whole genome 5-hydroxyemthycytosine (5hmC) residues, and Western blotting to quantify in genomic histone-3 protein tri-methylated lysine-4 (H3K4me3), lysine-9 (H3K9me3), and lysine-27 (H3K27me3) residues. MSC migration and proliferation were studied in-vitro. Obese MSCs displayed reduced global 5hmC and H3K4m3 levels, but comparable H3K9me3 and H3K27me3, compared to lean MSCs. Global 5hmC, H3K4me3, and HK9me3 marks correlated with MSC migration and reduced proliferation, as well as clinical and metabolic characteristics of obesity. Co-incubation of obese MSCs with VIT-C enhanced 5hmC marks, and reduced their global levels of H3K9me3 and H3K27me3. Contrarily, VIT-C did not affect 5hmC, and decreased H3K4me3 in lean MSCs. Obesity induces global genomic epigenetic alterations in swine MSCs, involving primarily genomic transcriptional repression, which are associated with MSC function and clinical features of obesity. Some of these alterations might be reversible using the epigenetic modulator VIT-C, suggesting epigenetic modifications as therapeutic targets in obesity.

Metabolic syndrome increases senescence-associated micro-RNAs in extracellular vesicles derived from swine and human mesenchymal stem/stromal cells.

BACKGROUND: The metabolic syndrome (MetS) is a combination of cardiovascular risk-factors, including obesity, hypertension, hyperglycemia, and insulin resistance. MetS may induce senescence in mesenchymal stem/stromal cells (MSC) and impact their micro-RNA (miRNA) content. We hypothesized that MetS also alters senescence-associated (SA) miRNAs in MSC-derived extracellular vesicles (EVs), and interferes with their function. METHODS: EVs were collected from abdominal adipose tissue-derived MSCs from pigs with diet-induced MetS or Lean controls (n = 6 each), and from patients with MetS (n = 4) or age-matched Lean controls (n = 5). MiRNA sequencing was performed to identify dysregulated miRNAs in these EVs, and gene ontology to analyze their SA-genes targeted by dysregulated miRNAs. To test for EV function, MetS and Lean pig-EVs were co-incubated with renal tubular cells in-vitro or injected into pigs with renovascular disease (RVD, n = 6 each) in-vivo. SA-b-Galactosidase and trichrome staining evaluated cellular senescence and fibrosis, respectively. RESULTS: Both humans and pigs with MetS showed obesity, hypertension, and hyperglycemia/insulin resistance. In MetS pigs, several upregulated and downregulated miRNAs targeted 5768 genes in MSC-EVs, 68 of which were SA. In MetS patients, downregulated and upregulated miRNAs targeted 131 SA-genes, 57 of which overlapped with pig-EVs miRNA targets. In-vitro, MetS-MSC-EVs induced greater senescence in renal tubular cells than Lean-MSC-EVs. In-vivo, Lean-MSC-EVs attenuated renal senescence, fibrosis, and dysfunction more effectively than MetS-MSC-EVs. CONCLUSIONS: MetS upregulates SA-miRNAs in swine MSC-EVs, which is conserved in human subjects, and attenuates their ability to blunt cellular senescence and repair injured target organs. These alterations need to be considered when designing therapeutic regenerative approaches. Video abstract.

Metabolic Syndrome Impairs 3D Mitochondrial Structure, Dynamics, and Function in Swine Mesenchymal Stem Cells.

Transplantation of autologous mesenchymal stem cells (MSCs) is an effective therapy for several diseases. Mitochondria modulate several important aspects of MSC function, but might be damaged by comorbidities and cardiovascular risk factors. We hypothesized that metabolic syndrome (MetS) compromises 3D mitochondrial structure, dynamics, and function in swine adipose tissue-derived MSCs. Domestic pigs were fed a Lean or MetS diet (n = 6 each) for 16 weeks. MSCs were collected from subcutaneous abdominal fat and their mitochondria analyzed using state-of-the-art Serial Block Face Electron Microscopy and 3D reconstruction. Mitochondrial dynamics (fusion/fission) were assessed by mRNA sequencing and Western blotting, and bioenergetics by membrane potential (TMRE), cytochrome-c oxidase (COX)-IV activity, and Seahorse Analyzer. Expression of mitochondria-associated microRNAs (mitomiRs) was measured by quantitative polymerase chain reaction (qPCR). MetS pigs developed obesity, hypertension, insulin resistance, and hyperlipidemia. Mitochondrial density was similar between the groups, but 3D mitochondrial and matrix volumes were lower in MetS-MSCs versus Lean-MSCs. Mitochondrial fission was higher, but fusion lower in MetS-MSCs versus Lean-MSCs, as were membrane potential, COX-IV activity, and ATP production. Contrarily, expression of the mitomiRs miR15a, miR-137, and miR-181c, which target mitochondrial genes that support mitochondrial structure, energy pathways, and dynamics, was higher in MetS-MSCs compared to Lean-MSCs, suggesting a potential to modulate their expression. MetS damages MSC 3D mitochondrial structure, dynamics, and function, and may modulate genes encoding for mitochondrial proteins. These observations support development of mitoprotective strategies to preserve the regenerative potency of MSCs and their suitability for autologous transplantation in patients with MetS.

Human Obesity Induces Dysfunction and Early Senescence in Adipose Tissue-Derived Mesenchymal Stromal/Stem Cells.

BACKGROUND: Chronic inflammatory conditions like obesity may adversely impact the biological functions underlying the regenerative potential of mesenchymal stromal/stem cells (MSC). Obesity can impair MSC function by inducing cellular senescence, a growth-arrest program that transitions cells to a pro-inflammatory state. However, the effect of obesity on adipose tissue-derived MSC in human subjects remains unclear. We tested the hypothesis that obesity induces senescence and dysfunction in human MSC. METHODS: MSC were harvested from abdominal subcutaneous fat collected from obese and age-matched non-obese subjects (n = 40) during bariatric or kidney donation surgeries, respectively. MSC were characterized, their migration and proliferation assessed, and cellular senescence evaluated by gene expression of cell-cycle arrest and senescence-associated secretory phenotype markers. In vitro studies tested MSC effect on injured human umbilical vein endothelial cells (HUVEC) function. RESULTS: Mean age was 59 ± 8 years, 66% were females. Obese subjects had higher body-mass index (BMI) than non-obese. MSC from obese subjects exhibited lower proliferative capacities than non-obese-MSC, suggesting decreased function, whereas their migration remained unchanged. Senescent cell burden and phenotype, manifested as p16, p53, IL-6, and MCP-1 gene expression, were significantly upregulated in obese subjects' MSC. BMI correlated directly with expression of p16, p21, and IL-6. Furthermore, co-incubation with non-obese, but not with obese-MSC, restored VEGF expression and tube formation that were blunted in injured HUVEC. CONCLUSION: Human obesity triggers an early senescence program in adipose tissue-derived MSC. Thus, obesity-induced cellular injury may alter efficacy of this endogenous repair system and hamper the feasibility of autologous transplantation in obese individuals.

Experimental Renovascular Disease Induces Endothelial Cell Mitochondrial Damage and Impairs Endothelium-Dependent Relaxation of Renal Artery Segments.

BACKGROUND: Mitochondria modulate endothelial cell (EC) function, but may be damaged during renal disease. We hypothesized that the ischemic and metabolic constituents of swine renovascular disease (RVD) induce mitochondrial damage and impair the function of renal artery ECs. METHODS: Pigs were studied after 16 weeks of metabolic syndrome (MetS), renal artery stenosis (RAS), or MetS + RAS, and Lean pigs served as control (n = 6 each). Mitochondrial morphology, homeostasis, and function were measured in isolated primary stenotic-kidney artery ECs. EC functions were assessed in vitro, whereas vasoreactivity of renal artery segments was characterized in organ baths. RESULTS: Lean + RAS and MetS + RAS ECs showed increased mitochondrial area and decreased matrix density. Mitochondrial biogenesis was impaired in MetS and MetS + RAS compared with their respective controls. Mitochondrial membrane potential similarly decreased in MetS, Lean + RAS, and MetS + RAS groups, whereas production of reactive oxygen species increased in MetS vs. Lean, but further increased in both RAS groups. EC tube formation was impaired in MetS, RAS, and MetS + RAS vs. Lean, but EC proliferation and endothelial-dependent relaxation of renal artery segments were blunted in MetS vs. Lean, but further attenuated in Lean + RAS and MetS + RAS. CONCLUSIONS: MetS and RAS damage mitochondria in pig renal artery ECs, which may impair EC function. Coexisting MetS and RAS did not aggravate EC mitochondrial damage in the short time of our in vivo studies, suggesting that mitochondrial injury is associated with impaired renal artery EC function.

Senolytics decrease senescent cells in humans: Preliminary report from a clinical trial of Dasatinib plus Quercetin in individuals with diabetic kidney disease.

BACKGROUND: Senescent cells, which can release factors that cause inflammation and dysfunction, the senescence-associated secretory phenotype (SASP), accumulate with ageing and at etiological sites in multiple chronic diseases. Senolytics, including the combination of Dasatinib and Quercetin (D + Q), selectively eliminate senescent cells by transiently disabling pro-survival networks that defend them against their own apoptotic environment. In the first clinical trial of senolytics, D + Q improved physical function in patients with idiopathic pulmonary fibrosis (IPF), a fatal senescence-associated disease, but to date, no peer-reviewed study has directly demonstrated that senolytics decrease senescent cells in humans. METHODS: In an open label Phase 1 pilot study, we administered 3 days of oral D 100 mg and Q 1000 mg to subjects with diabetic kidney disease (N = 9; 68·7 ±â€¯3·1 years old; 2 female; BMI:33·9 ±â€¯2·3 kg/m2; eGFR:27·0 ±â€¯2·1 mL/min/1·73m2). Adipose tissue, skin biopsies, and blood were collected before and 11 days after completing senolytic treatment. Senescent cell and macrophage/Langerhans cell markers and circulating SASP factors were assayed. FINDINGS: D + Q reduced adipose tissue senescent cell burden within 11 days, with decreases in p16INK4A-and p21CIP1-expressing cells, cells with senescence-associated ß-galactosidase activity, and adipocyte progenitors with limited replicative potential. Adipose tissue macrophages, which are attracted, anchored, and activated by senescent cells, and crown-like structures were decreased. Skin epidermal p16INK4A+ and p21CIP1+ cells were reduced, as were circulating SASP factors, including IL-1α, IL-6, and MMPs-9 and -12. INTERPRETATION: "Hit-and-run" treatment with senolytics, which in the case of D + Q have elimination half-lives <11 h, significantly decreases senescent cell burden in humans. FUND: NIH and Foundations. ClinicalTrials.gov Identifier: NCT02848131. Senescence, Frailty, and Mesenchymal Stem Cell Functionality in Chronic Kidney Disease: Effect of Senolytic Agents.