Lerman, Lilach O.

Mesenchymal stromal/stem cells (MSCs) belong to the endogenous cellular reparative system, and can be used exogenously in cell-based therapy. MSCs release extracellular vesicles (EVs), including exosomes and microvesicles, which mediate some of their therapeutic activity through intercellular communication. We have previously demonstrated that metabolic syndrome (MetS) modifies the cargo packed within swine EV, but whether it influences their phenotypical characteristics remains unclear. This study tested the hypothesis that MetS shifts the size distribution of MSC-derived EVs. Adipose tissue-derived MSC-EV subpopulations from Lean ( n = 6) and MetS ( n = 6) pigs were characterized for number and size using nanoparticle-tracking analysis, flow cytometry, and transmission electron microscopy. Expression of exosomal genes was determined using next-generation RNA-sequencing (RNA-seq). The number of EV released from Lean and MetS pig MSCs was similar, yet MetS-MSCs yielded a higher proportion of small-size EVs (202.4 ± 17.7 nm vs. 280.3 ± 15.1 nm), consistent with exosomes. RNA-seq showed that their EVs were enriched with exosomal markers. Lysosomal activity remained unaltered in MetS-MSCs. Therefore, MetS alters the size distribution of MSC-derived EVs in favor of exosome release. These observations may reflect MSC injury and membrane recycling in MetS or increased expulsion of waste products, and may have important implications for development of adequate cell-based treatments.

Background Hypertension may be associated with renal cellular injury. Cells in distress release extracellular vesicles ( EV s), and their numbers in urine may reflect renal injury. Cellular senescence, an irreversible growth arrest in response to a noxious milieu, is characterized by release of proinflammatory cytokines. We hypothesized that EV s released by senescent nephron cells can be identified in urine of patients with hypertension. Methods and Results We recruited patients with essential hypertension ( EH ) or renovascular hypertension and healthy volunteers (n=14 each). Renal oxygenation was assessed using magnetic resonance imaging and blood samples collected from both renal veins for cytokine‐level measurements. EV s isolated from urine samples were characterized by imaging flow cytometry based on specific markers, including p16 (senescence marker), calyxin (podocytes), urate transporter 1 (proximal tubules), uromodulin (ascending limb of Henle's loop), and prominin‐2 (distal tubules). Overall percentage of urinary p16+ EV s was elevated in EH and renovascular hypertension patients compared with healthy volunteers and correlated inversely with renal function and directly with renal vein cytokine levels. Urinary levels of p16 + /urate transporter 1 + were elevated in all hypertensive subjects compared with healthy volunteers, whereas p16 + /prominin‐2 + levels were elevated only in EH versus healthy volunteers and p16 + /uromodulin + in renovascular hypertension versus EH . Conclusions Levels of p16 + EV s are elevated in urine of hypertensive patients and may reflect increased proximal tubular cellular senescence. In EH , EV s originate also from distal tubules and in renovascular hypertension from Henle's loop. Hence, urinary EV s levels may be useful to identify intrarenal sites of cellular senescence.

Metabolic syndrome (MetS) is associated with nutrient surplus and kidney hyperfiltration, accelerating chronic renal failure. The potential involvement of podocyte damage in early MetS remains unclear. Mitochondrial dysfunction is an important determinant of renal damage, but whether it contributes to MetS-related podocyte injury remains unknown. Domestic pigs were studied after 16 wk of diet-induced MetS, MetS treated with the mitochondria-targeted peptide elamipretide (ELAM; 0.1 mg·kg−1·day−1 sc) for the last month of diet, and lean controls ( n = 6 pigs/group). Glomerular filtration rate (GFR) and renal blood flow (RBF) were measured using multidetector computed tomography, and podocyte and mitochondrial injury were measured by light and electron microscopy. Urinary levels of podocyte-derived extracellular vesicles (pEVs; nephrin positive/podocalyxin positive) were characterized by flow cytometry. Body weight, blood pressure, RBF, and GFR were elevated in MetS. Glomerular size and glomerular injury score were also elevated in MetS and decreased after ELAM treatment. Evidence of podocyte injury, impaired podocyte mitochondria, and foot process width were all increased in MetS but restored with ELAM. The urinary concentration of pEVs was elevated in MetS pigs and directly correlated with renal dysfunction, glomerular injury, and fibrosis and inversely correlated with glomerular nephrin expression. Additionally, pEV numbers were elevated in the urine of obese compared with lean human patients. Early MetS induces podocyte injury and mitochondrial damage, which can be blunted by mitoprotection. Urinary pEVs reflecting podocyte injury might represent early markers of MetS-related kidney disease and a novel therapeutic target.

Background Women with non‐obstructive coronary artery disease have increased cardiovascular morbidity. The role of risk factors in this population has yet to be established. We aimed to study the predictive effect of triglycerides and the triglyceride/high‐density lipoprotein ratio on major adverse cardiovascular events (MACE) in patients with non‐obstructive coronary artery disease, and to explore the role of lipid lowering therapy in modifying this risk. Methods and Results This is a prospective cohort study enrolling patients with anginal symptoms referred to the cardiac catheterization laboratory for suspected ischemia, who were subsequently diagnosed with non‐obstructive coronary artery disease, defined as no stenosis >20% on angiography. All patients had baseline laboratory testing and were followed for 7.8±4.3 years for the development of major adverse cardiovascular events. We performed Cox proportional hazard testing to determine the effect of triglycerides on risk of major adverse cardiovascular events among men and women by baseline statin use. A total of 462 patients were included. Median age was 53 (Q1, Q3: 45, 62) years. In a Cox proportional hazard model stratified by statin use adjusting for confounders, among those not on baseline statins, triglycerides were independently predictive of major adverse cardiovascular events in women (per 50 mg/dL risk ratio: hazard ratio 1.25 [95% CI : 1.06, 1.47]; P =0.01). This was not true among men. The interaction between triglycerides and sex, and triglycerides and statin was statistically significant. Conclusions Triglyceride levels may play a key role in predicting cardiovascular‐specific risk in women, and statin use may be protective. Further investigation is necessary to better delineate the role of statin use in preventing cardiovascular risk.

Background: Serum uric acid may serve as a marker of cardiovascular events and outcomes in men, but further studies are required to determine its role in women. We aimed to determine differences in procedural characteristics and outcomes in women undergoing percutaneous coronary intervention(PCI). Methods: We retrospectively analyzed the Mayo Clinic Cardiovascular Catheterization Laboratory database, and included patients undergoing PCI from 1/1/2000 to 12/31/2007. There were 495 women who underwent PCI in this time period. Patients were divided into normal uric acid (n=240) and high uric acid (n=255) groups, and high uric acid was defined as >6.0 mg/dL. Patients were followed for adverse outcomes for a median of 47.1 (IQR 23.8, 62.8) months. Results: Mean age was 66.6 + 12.7 years in the normal group vs. 70.5 + 11.2 years in the elevated uric acid group(p=0.01). Hypertension and diabetes were more prevalent in the elevated uric acid group (p<0.01). BMI was significantly higher in the elevated uric acid group (p=0.001). There was a higher prevalence of calcified lesions among women with elevated uric acid when compared to those with normal uric acid levels (47% vs. 30% p<0.001). There was significantly lower TIMI=3 flow postprocedurally in those with elevated uric acid levels when compared to those with normal uric acid levels (91 % vs. 97%, p=0.003). After adjusting for potential confounders including age, hyperlipidemia, hypertension, BMI, diabetes, and chronic kidney disease, elevated uric acid was associated with increased likelihood of death (HR 1.8, p=0.01) and the composite endpoint of death or subsequent myocardial infarction (HR 1.5, p=0.03). Discussion: Uric acid may be an independent determinant of procedural characteristics and a predictor of outcomes after percutaneous intervention. Further studies are required to better understand the effect of uric acid on cardiovascular events in women.

Background: Serum uric acid is a marker of oxidative stress, and may serve as a marker of adverse cardiovascular events and outcomes including cardiovascular death, hospitalization and myocardial infarction. We aimed to determine whether uric acid predicts cardiovascular events and death in patients undergoing carotid endarterectomy (CEA). Methods: We enrolled 327 patients undergoing CEA between February 2002 and June 2017 who had complete laboratory work up at baseline. Patients were followed through the electronic chart as well as via regular nursing follow-up for a median of 10.9 years (IQR 7.3, 13.4). There were a total of 67 adverse cardiovascular events during follow-up. Results: Baseline serum uric acid levels were significantly higher in patients who developed cardiovascular events during the follow-up period vs. those that did not [6.87 + 2.29 vs. 6.39 + 1.61); p=0.047). Baseline serum uric was independently associated with cardiovascular events when adjusting for age, creatinine, diabetes mellitus, and hyperlipidemia (L-R 4.0, p=0.04)(Figure 1). Sex stratified analysis showed that serum uric acid was also an independent predictor of both cardiovascular events and death in men, not women undergoing after adjustment for confounders including age, cholesterol, systolic blood pressure and glucose (LR 4.97; p=0.03). Discussion: Higher uric acid levels are associated with cardiovascular events and death after CEA. Further investigation is necessary to determine the role of uric acid as a modifiable risk factor in these patients.

Background: Uric acid is a known risk factor for atherosclerosis. We aimed to determine whether baseline uric acid levels predict plaque stability on presentation and future cerebrovascular events (CVA) in patients undergoing carotid endarterectomy (CEA). Methods: We analyzed 317 patients who underwent CEA between 2/2002 and 6/2017. Patients were stratified according to plaque stability as unstable or stable --those who presented with a history of > 1 neurologic event were “unstable” and those with no history of CVA were classified as stable. All patients underwent comprehensive baseline laboratory testing at baseline prior to CEA and then were followed by a nurse coordinator and through the medical record for the development of cerebrovascular events including stroke and transient ischemic attack for a median of 10.9 years (IQR 7.3, 13.4). Results: Baseline uric acid was significantly higher in patients presenting with unstable plaque vs. stable plaque(6.5 (IQR 5.6, 7.9) vs. 5.8(IQR 4.8,6.6); p<0.001), and was an independent predictor of plaque stability when adjusting for potential confounders including glucose, BMI, hsCRP, cholesterol, and systolic blood pressure (L-R 6.9, p=0.0096). There were a total of 58 CVA during follow-up, and baseline uric acid was higher among patients who developed CVA vs those without CVA [(6.9 + 2.1) mg/dL vs. (6.4 + 1.7) mg/dL; p=0.037]. After adjusting for diabetes, cholesterol, systolic blood pressure, smoking , age, aspirin, and creatinine, uric acid and cholesterol were both independent predictors of stroke in this population [uric acid: (LR 4.84, p=0.03); cholesterol: (L-R 11.8, p=0.0006)]. Discussion: While cholesterol has been associated with poor outcomes in patients with vascular disease, our data suggest that uric acid may be a novel risk factor for stroke in patients undergoing CEA. Further investigation is necessary to understand the therapeutic potential of reducing uric acid levels in these patients and the potential benefit of reducing overall cerebrovascular risk.

Background: High sensitivity C-reactive protein is a risk factor atherosclerosis and plaque rupture. We aimed to determine the role of high-sensitivity C reactive protein in predicting future cerebrovascular events (CVA), including stroke and transient ischemic attack, in patients undergoing routine carotid endarterectomy (CEA). Methods: A total of 251 patients underwent routine CEA without any history of stroke. At baseline prior to CEA, patients underwent routine laboratory testing, including testing for novel biomarkers including hs-CRP. Patients were followed for a median of 10.9 years (IQR 7.3, 13.4) for the development of CVA with regular nursing follow-up and/or chart review. Results: Of 251 patients enrolled in the study, 19 patients without prior CVA subsequently developed CVA during follow-up. In univariate analysis, baseline hsCRP was significantly higher in patients who developed CVA during follow-up vs. those that did not (8.1 (IQR 2.2, 46.3) mg/L vs. 3.85(0.9, 11.75) mg/L; p=0.0054)(Figure 1). After adjusting for confounders including age. diabetes mellitus, cholesterol, systolic blood pressure, history of smoking, aspirin use and duration of follow-up, log hsCRP was an independent predictor of CVA during follow-up after CEA (L-R 9.9; p=0.0016). Discussion: Hs-CRP appears to be a risk factor for CVA during follow-up after CEA. Further investigation is necessary to determine the role of risk modification in these patients and the therapeutic potential of reducing inflammation in this population.