Eirin, Alfonso

Abstract Mesenchymal stem/stromal cells (MSCs) release extracellular vesicles (EVs), which shuttle proteins to recipient cells, promoting cellular repair. We hypothesized that cardiovascular risk factors may alter the pattern of proteins packed within MSC-derived EVs. To test this, we compared the protein cargo of EVs to their parent MSCs in pigs with metabolic syndrome (MetS) and Lean controls. Porcine MSCs were harvested from abdominal fat after 16 weeks of Lean- or MetS-diet (n = 5 each), and their EVs isolated. Following liquid chromatography mass spectrometry proteomic analysis, proteins were classified based on cellular component, molecular function, and protein class. Five candidate proteins were validated by Western blot. Clustering analysis was performed to identify primary functional categories of proteins enriched in or excluded from EVs. Proteomics analysis identified 6,690 and 6,790 distinct proteins in Lean- and MetS-EVs, respectively. Differential expression analysis revealed that 146 proteins were upregulated and 273 downregulated in Lean-EVs versus Lean-MSCs, whereas 787 proteins were upregulated and 185 downregulated in MetS-EVs versus MetS-MSCs. Proteins enriched in both Lean- and MetS-EVs participate in vesicle-mediated transport and cell-to-cell communication. Proteins enriched exclusively in Lean-EVs modulate pathways related to the MSC reparative capacity, including cell proliferation, differentiation, and activation, as well as transforming growth factor-β signaling. Contrarily, proteins enriched only in MetS-EVs are linked to proinflammatory pathways, including acute inflammatory response, leukocyte transendothelial migration, and cytokine production. Coculture with MetS-EVs increased renal tubular cell inflammation. MetS alters the protein cargo of porcine MSC-derived EVs, selectively packaging specific proinflammatory signatures that may impair their ability to repair damaged tissues. Stem Cells Translational Medicine 2019;8:430–440

Hypertension, an important cause of chronic kidney disease, is characterized by peritubular capillary (PTC) loss. Circulating levels of endothelial microparticles (EMPs) reflect systemic endothelial injury. We hypothesized that systemic and urinary PTC-EMPs levels would reflect renal microvascular injury in hypertensive patients. We prospectively measured by flow cytometry renal vein, inferior vena cava, and urinary levels of EMPs in essential (n=14) and renovascular (RVH; n=24) hypertensive patients and compared them with peripheral blood and urinary levels in healthy volunteers (n=14). PTC-EMPs were identified as urinary exosomes positive for the PTC marker plasmalemmal-vesicle–associated protein. In 7 RVH patients, PTC and fibrosis were also quantified in renal biopsy, and in 18 RVH patients, PTC-EMPs were measured again 3 months after continued medical therapy with or without stenting (n=9 each). Renal vein and systemic PTC-EMPs levels were not different among the groups, whereas their urinary levels were elevated in both RVH and essential hypertension versus healthy volunteers (56.8%±12.7% and 62.8%±10.7% versus 34.0%±17.8%; both P ≤0.001). Urinary PTC-EMPs levels correlated directly with blood pressure and inversely with estimated glomerular filtration rate. Furthermore, in RVH, urinary PTC-EMPs levels correlated directly with stenotic kidney hypoxia, histological PTC count, and fibrosis and inversely with cortical perfusion. Three months after treatment, the change in urinary PTC-EMPs levels correlated inversely with a change in renal function ( r =−0.582; P =0.011). Therefore, urinary PTC-EMPs levels are increased in hypertensive patients and may reflect renal microcirculation injury, whereas systemic PTC-EMPs levels are unchanged. Urinary PTC-EMPs may be useful as novel biomarkers of intrarenal capillary loss.

Mesenchymal stem cells (MSCs) constitute an important repair system, but may be impaired by exposure to cardiovascular risk factors. Consequently, adipose tissue-derived MSCs from pigs with the metabolic syndrome (MetS) show decreased vitality. A growing number of microRNAs (miRNAs) are recognized as key modulators of senescence, but their role in regulating senescence in MSC in MetS is unclear. We tested the hypothesis that MetS upregulates in MSC expression of miRNAs that can serve as post-transcriptional regulators of senescence-associated (SA) genes. MSCs were collected from swine abdominal adipose tissue after 16 weeks of Lean or Obese diet ( n = 6 each). Next-generation miRNA sequencing (miRNA-seq) was performed to identify miRNAs up-or down-regulated in MetS-MSCs compared with Lean-MSCs. Functional pathways of SA genes targeted by miRNAs were analyzed using gene ontology. MSC senescence was evaluated by p16 and p21 immunoreactivity, H2AX protein expression, and SA-β-Galactosidase activity. In addition, gene expression of p16, p21, MAPK3 (ERK1) and MAPK14, and MSC migration were studied after inhibition of SA-miR-27b. Senescence biomarkers were significantly elevated in MetS-MSCs. We found seven upregulated miRNAs, including miR-27b, and three downregulated miRNAs in MetS-MSCs, which regulate 35 SA genes, particularly MAPK signaling. Inhibition of miR-27b in cultured MSCs downregulated p16 and MARP3 genes, and increased MSC migration. MetS modulates MSC expression of SA-miRNAs that may regulate their senescence, and the p16 pathway seems to play an important role in MetS-induced MSC senescence.

Background: Extracellular vesicles (EVs) released from mesenchymal stem/stromal cells (MSCs) mediate their paracrine effect, but their efficacy to protect the microcirculation of the kidney is unknown. Using a novel swine model of unilateral renovascular disease (RVD) complicated by metabolic syndrome (MetS), we tested the hypothesis that EVs would attenuate renal microvascular loss. Methods: Four groups of pigs ( n = 7 each) were studied after 16 weeks of diet-induced MetS and RVD (MetS+RVD), MetS+RVD treated 4 weeks earlier with a single intra-renal delivery of EVs harvested from autologous adipose tissue-derived MSCs, and Lean and MetS Sham controls. Stenotic-kidney renal blood flow (RBF) and glomerular filtration rate (GFR) were measured in-vivo (fast CT), whereas EV characteristics, renal microvascular architecture (micro-CT), and injury pathways were studied ex-vivo. Results: mRNA sequencing and proteomic analysis revealed that EVs are packed with several pro-angiogenic genes and proteins, such as vascular endothelial growth factor. Labeled EVs were detected in the stenotic kidney 4 weeks after injection internalized by tubular and endothelial cells. EVs restored renal expression of angiogenic factors and improved cortical microvascular and peritubular capillary density. Renal apoptosis, oxidative stress, tubular injury, and fibrosis were also attenuated in EV-treated pigs. RBF and GFR decreased in MetS+RVD compared with MetS, but normalized in MetS+RVD+EVs. Conclusions: Intra-renal delivery of MSC-derived EVs bearing pro-angiogenic properties restored the renal microcirculation and in turn hemodynamics and function in chronic experimental MetS+RVD. Our study suggests a novel therapeutic potential for MSC-derived EVs in restoring renal hemodynamics in experimental MetS+RVD.

New Findings What is the central question of this study ? We hypothesized that chronic mitoprotection would decrease renal vascular remodelling and dysfunction in swine metabolic syndrome. What is the main finding and its importance ? This study shows that experimental metabolic syndrome exerts renal microvascular and endothelial cell mitochondrial injury, which were attenuated by mitoprotection, underscoring the contribution of mitochondrial injury to the pathogenesis of metabolic syndrome‐induced vascular damage. Abstract The metabolic syndrome (MetS) induces intrarenal microvascular disease, which may involve mitochondrial injury. The mitochondrial cardiolipin‐targeting peptide elamipretide (ELAM) improves the microcirculation in post‐stenotic kidneys, but its ability to attenuate MetS‐induced renal vascular damage is unknown. We hypothesized that chronic treatment with ELAM would decrease renal vascular remodelling and function in swine MetS. Pigs were studied after 16 weeks of diet‐induced MetS, MetS treated for the last 4 weeks with daily injections of ELAM (0.1 mg kg −1 ), and lean control (Lean) animals ( n = 6 each). Single‐kidney regional perfusion, blood flow and glomerular filtration rate were measured with multi‐detector computed tomography (CT). Peritubular capillary (PTC) endothelial cell (EC) mitochondrial density and cardiolipin content were assessed in situ , as were PTC‐EC apoptosis and oxidative stress. The spatial density of PTCs (Haematoxylin and Eosin staining) and renal microvessels (micro‐CT), and renal artery endothelial function (organ bath) were characterized. Regional perfusion and serum creatinine were preserved in MetS pigs, but renal blood flow and glomerular filtration rate were higher compared with Lean. Mitochondrial density and cardiolipin content were diminished in MetS PTC‐ECs, but improved in ELAM‐treated pigs, as did PTC density. Elamipretide also attenuated PTC‐EC oxidative stress and apoptosis. Furthermore, ELAM improved renal microvascular density, decreased microvascular remodelling and restored endothelial nitric oxide expression and endothelium‐dependent relaxation of renal artery segments. In conclusion, MetS‐induced mitochondrial alterations might contribute to renal PTC and microvascular loss and might impair renal artery endothelial function in pigs. Mitoprotection with ELAM preserved a hierarchy of renal vessels, underscoring its potential to ameliorate renal vascular injury in MetS.

Transplantation of autologous mesenchymal stem cells (MSCs) may be a viable option for treatment of several diseases. MSCs efficacy depends on adequate function of their mitochondria, which might be impaired in a noxious milieu. We hypothesized that obesity compromises MSCs mitochondrial structure and function, possibly via micro‐RNA (miRNA)‐based mechanisms. MSCs were collected from swine abdominal adipose tissue after 16 weeks of Lean or Obese diet (n = 7 each). Mitochondrial structure was assessed by electron microscopy and function by membrane potential and cytochrome‐c oxidase (COX)‐IV activity. Oxidative stress was assessed by Mito‐SOX and dihydroethidium staining. Next‐generation sequencing (RNA‐seq) was performed to identify miRNAs expression in MSCs, and predicted mitochondrial target genes were then identified (MitoCarta). Compared to Lean‐MSCs, mitochondria from Obese‐MSCs were smaller and showed cristae remodeling and loss. Mitochondrial membrane potential and COX‐IV activity decreased in Obese‐MSCs, associated with increased mitochondrial oxidative stress. RNA‐seq generated reads for 413 miRNAs, of which 5 miRNAs were upregulated in Obese‐MSCs (fold change >2, p < 0.05) and found to target 43 specific mitochondrial genes. Obesity impairs MSC mitochondrial structure and function, possibly mediated partly through miRNA‐induced mitochondrial gene regulation, leading to increased oxidative stress. Importantly, these alterations may limit the therapeutic use of autologous MSCs in subjects with obesity.

Background— Atherosclerotic renal artery stenosis reduces renal blood flow (RBF) and amplifies stenotic kidney hypoxia. Revascularization with percutaneous transluminal renal angioplasty (PTRA) and stenting often fails to recover renal function, possibly because of ischemia/reperfusion injury developing after PTRA. Elamipretide is a mitochondrial-targeted peptide that binds to cardiolipin and stabilizes mitochondrial function. We tested the hypothesis that elamipretide plus PTRA would improve renal function, oxygenation, and RBF in patients with atherosclerotic renal artery stenosis undergoing PTRA. Methods and Results— Inpatient studies were performed in patients with severe atherosclerotic renal artery stenosis scheduled for PTRA. Patients were treated before and during PTRA with elamipretide (0.05 mg/kg per hour intravenous infusion, n=6) or placebo (n=8). Stenotic kidney cortical/medullary perfusion and RBF were measured using contrast-enhanced multidetector CT, and renal oxygenation by 3-T blood oxygen level−dependent magnetic resonance imaging before and 3 months after PTRA. Age and basal glomerular filtration rate did not differ between groups. Blood oxygen level−dependent imaging demonstrated increased fractional hypoxia 24 hours after angiography and stenting in placebo (+47%) versus elamipretide (−6%). These were reverted to baseline 3 months later. Stenotic kidney RBF rose (202±29–262±115 mL/min; P =0.04) 3 months after PTRA in the elamipretide-treated group only. Over 3 months, systolic blood pressure decreased, and estimated glomerular filtration rate increased ( P =0.003) more in the elamipretide group than in the placebo group ( P =0.11). Conclusions— Adjunctive elamipretide during PTRA was associated with attenuated postprocedural hypoxia, increased RBF, and improved kidney function in this pilot trial. These data support a role for targeted mitochondrial protection to minimize procedure-associated ischemic injury and to improve outcomes of revascularization for human atherosclerotic renal artery stenosis. Clinical Trial Registration— URL: https://www.clinicaltrials.gov . Unique identifier: NCT01755858.