Lerman, Amir

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.

Objectives: To investigate the role of endothelial function in patients with previous spontaneous coronary artery dissection. Background: Mechanisms underlying spontaneous coronary artery dissection, including a possible contribution from endothelial dysfunction, remain poorly understood. Methods: This was a single center, retrospective study of patients with a prior spontaneous coronary artery dissection episode who underwent invasive endothelial function testing in the cardiac catheterization laboratory for evaluation of recurrent chest pain. Coronary epicardial and microvascular responses to acetylcholine, adenosine, and nitroglycerine were assessed. Findings were compared to a reference group of normal controls ( n=232). Results: A total of 10 patients with prior angiographically confirmed spontaneous coronary artery dissection were referred for coronary endothelial function testing. The median coronary flow reserve was 2.8 (interquartile range (IQR) 2.3, 3.6). The median change in coronary diameter with acetylcholine was −0.9% (IQR −23.9, 4.2). The median increase in peak coronary blood flow following acetylcholine administration was 91.4% (IQR 9.1, 105.7), which was similar to the response observed in a reference group of patients (median age 51 years, 96% women) from our laboratory with normal microvascular responses to acetylcholine: 107.4% (IQR 75.5, 165.7; P=0.20). Four patients (40%) had an abnormal microvascular response to acetylcholine, with less than a 50% increase in coronary blood flow, and all but one patient had left anterior descending artery or multivessel spontaneous coronary artery dissection. Conclusion: Coronary epicardial and microvascular vasomotor dysfunction is not a predominant feature of spontaneous coronary artery dissection. Endothelial dysfunction is not implicated as the principal underlying mechanism.

Abstract BACKGROUND The collateral circulation is important in maintenance of blood supply to the ischemic kidney distal to renal artery stenosis (RAS). Obesity metabolic syndrome (MetS) preserves renal blood flow (RBF) in the stenotic kidney, but whether this is related to an increase of collateral vessel growth is unknown. We hypothesized that MetS increased collateral circulation around the renal artery. METHODS Twenty-one domestic pigs were randomly divided into unilateral RAS fed an atherogenic (high-fat/high-fructose, MetS-RAS) or standard diet, or controls (n = 7 each). RBF, glomerular filtration rate (GFR), and the peristenotic collateral circulation were assessed after 10 weeks using multidetector computed tomography (CT) and the intrarenal microcirculation by micro-CT. Vascular endothelial growth factor (VEGF) expression was studied in the renal artery wall, kidney, and perirenal fat. Renal fibrosis and stiffness were examined by trichrome and magnetic resonance elastography. RESULTS Compared with controls, RBF and GFR were decreased in RAS, but not in MetS-RAS. MetS-RAS formed peristenotic collaterals to the same extent as RAS pigs but induced greater intrarenal microvascular loss, fibrosis, stiffness, and inflammation. MetS-RAS also attenuated VEGF expression in the renal tissue compared with RAS, despite increased expression in the perirenal fat. CONCLUSIONS MetS does not interfere with collateral vessel formation in the stenotic kidney, possibly because decreased renal arterial VEGF expression offsets its upregulation in perirenal fat, arguing against a major contribution of the collateral circulation to preserve renal function in MetS-RAS. Furthermore, preserved renal function does not protect the poststenotic kidney from parenchymal injury.

Abstract Aims Early studies from the 1990s have shown that statins improve survival and attenuate cardiac allograft vasculopathy (CAV). However, little contemporary data are available on the incremental benefit of statins with the current use of new-generation immunosuppressive agents and the use of coronary intravascular ultrasound for assessment of CAV. We sought to investigate the effect of early statin (ES) as compared with late statin (LS) initiation after heart transplantation (HT) on long-term CAV progression and clinical outcomes in a large contemporary HT cohort. Methods and results We analysed a cohort of 409 adult HT recipients. CAV progression was assessed by serial coronary intravascular ultrasound volumetric measurements of the differences between baseline and last follow-up plaque volume (PV) and plaque index (PV/vessel volume ratio). CAV progression and clinical outcomes were compared between the ES (<2 years after HT) and the LS (>2 years after HT) groups. During a median follow-up of 8.2 years, ES resulted in significantly lower change (Δ) of plaque index (+3.8% ± 1.7% vs. +8.2% ± 3.6%; P = 0.0008) and PV (+0.8 ± 0.3 vs. +1.9 ± 1.2; P = 0.045) compared with LS group. In a Cox proportional hazards regression model and after adjustment for baseline characteristics, ES was associated with a 52% decreased risk of CAV-associated events (hazard ratio 0.48, 95% confidence interval: 0.27–0.91; P = 0.025) and a 42% decreased risk of the composite endpoint of all-cause mortality and CAV-associated events (hazard ratio 0.58, 95% confidence interval: 0.38–0.91; P = 0.019). Conclusions Early initiation of statin therapy after HT results in attenuated CAV progression as well as in decreased CAV-related events and mortality.

Cardiac fibrosis is a biological process that increases with age and contributes to myocardial dysfunction. Humanin (HN) is an endogenous mitochondria-derived peptide that has cytoprotective effects and reduces oxidative stress. The present study aimed to test the hypothesis that chronic supplementation of exogenous HN in middle-aged mice could prevent and reverse cardiac fibrosis and apoptosis in the aging heart. Female C57BL/6N mice at 18 mo of age received 14-mo intraperitoneal injections of vehicle (old group; n = 6) or HN analog (HNG; 4 mg/kg 2 times/wk, old + HNG group, n = 8) and were euthanized at 32 mo of age. C57BL/6N female mice (young group, n = 5) at 5 mo of age were used as young controls. HNG treatment significantly increased the ratio of cardiomyocytes to fibroblasts in aging hearts, as shown by the percentage of each cell type in randomly chosen fields after immunofluorescence staining. Furthermore, the increased collagen deposition in aged hearts was significantly reduced after HNG treatment, as indicated by picrosirius red staining. HNG treatment also reduced in aging mice cardiac fibroblast proliferation (5′-bromo-2-deoxyuridine staining) and attenuated transforming growth factor-β1, fibroblast growth factor-2, and matrix metalloproteinase-2 expression (immunohistochemistry or real-time PCR). Myocardial apoptosis was inhibited in HNG-treated aged mice (TUNEL staining). To decipher the pathway involved in the attenuation of the myocardial fibrosis by HNG, Western blot analysis was done and showed that HNG upregulated the Akt/glycogen synthase kinase -3β pathway in aged mice. Exogenous HNG treatment attenuated myocardial fibrosis and apoptosis in aged mice. The results of the present study suggest a role for the mitochondria-derived peptide HN in the cardioprotection associated with aging. NEW & NOTEWORTHY Cardiac fibrosis is a biological process that increases with age and contributes to myocardial dysfunction. Humanin is an endogenous mitochondria-derived peptide that has cytoprotective effects and reduces oxidative stress. Here, we demonstrate, for the first time, that exogenous humanin treatment attenuated myocardial fibrosis and apoptosis in aging mice. We also detected upregulated Akt/glycogen synthase kinase-3β pathway in humanin analog-treated mice, which might be the mechanism involved in the cardioprotective effect of humanin analog in aging mice.

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.