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Introduction: Morbidity and mortality attributable to hypertension are higher in African American (AAEH) compared to Caucasian essential hypertensive (EH) patients, possibly related to a differential effect on vascular injury and repair. While circulating endothelial progenitor cells (EPC) preserve endothelial integrity IEC detach from sites of injury and represent markers of vascular damage. We hypothesized that plasma levels of IEC and inflammatory markers would be higher in AAEH compared to EH patients. METHODS: Inferior vena cava levels of CD34+/KDR+ (EPC) and VAP-1+ (IEC) cells were measured by FACS in EH and AAEH under fixed sodium intake (150 mEq/d) and blockade of the renin-angiotensin-system, and compared to systemic levels in normotensive control subjects (n=19 each). Systemic levels of inflammatory cytokines and EPC homing factors were measured by Luminex. Results: Blood pressure, serum creatinine, lipids, antihypertensive medications, and EPC levels did not differ between EH and AAEH patients. Circulating IEC were higher in AAEH, and inversely correlated with EPC levels (Figure). Systemic levels of inflammatory cytokines and EPC homing factors were higher in AAEH compared to EH patients (Table), and correlated directly with IEC. Conclusion: Despite preserved kidney function and controlled BP, circulating inflammatory markers were elevated in AAEH and correlated with increased IEC and decreased EPC levels. Increased release of cytokines and IEC in AAEH may impair EPC reparative capacity and predispose to hypertensive vascular injury. This process may aggravate vascular damage and accelerate hypertension-related morbidity/mortality rates in AAEH.

Objective: Excessive autophagy (degradation of intracellular structures) characterizes cardiomyocyte injury in hypertensive heart disease. The effect of Angiotensin II receptor blocker Valsartan on RVH-induced myocardial autophagy remains unexplored. Method: Twenty-one domestic pigs (50-60 Kg) were randomized into 3 groups: RVH, RVH+Valsartan, and Control (n=7 each), and observed for 10 wks. Valsartan (320mg/d PO) was administered for 4 wks starting at 6 wks of RVH. After 10 wks, myocardial fibrosis was examined by trichrome staining, and inflammation by M1 macrophage (M1 Ø, CD163+/iNOS+) staining and TNF-α expression (immunoblotting). Autophagy was examined for Atg 12-Atg 5, Beclin-1 and its inhibitor mTOR. Result: At 6 wks, MAP was higher in both RVH and RVH+Valsartan compared to Control (147.6 ±14.0 and 139.1±11.2 vs. 105.9±11.4 mmHg, respectively, P<0.05). At 10 wks, Valsartan did not decrease MAP compared to RVH (P=0.31), Both Beclin-1 and Atg 12-Atg 15 expressions that were elevated in RVH (P<0.05 vs. Control), indicating increased autophagy, were restored by Valsartan (Figure). Valsartan restored a compensatory increase in mTOR expression in RVH, and ameliorated myocardial fibrosis, M1 Ø, and TNF-α expression (Figure). Conclusion: Valsartan decreased myocardial autophagy, fibrosis and inflammation in pigs with RVH, independent of effective blood pressure control. Valsartan has novel pleiotropic effects on turnover of cellular structures in RVH-induced myocardial damage.

Introduction: Stem cells have poor survival after transplantation, what can limit the use of these therapies. Mitochondrial dysfunction has been recognized as a limiting event that can lead to cell death. Here, we hypothesized that the mitochondrial function of progenitor cells can be monitored in vivo using reporter gene-based molecular imaging. A better understanding of the biology of transplanted stem cells in the ischemic myocardium can lead to optimization of cell therapies. Methods: Using RT-PCR we identified the antioxidant NAD(P)H:Quinone oxidoreductase (NQO1) enzyme as a marker of mitochondrial dysfunction in mouse Mesenchymal Stem Cells (MSCs) that were treated with a mitochondrial stressor, diethyl maleate (DEM). We then constructed a luciferase reporter gene driven by the NQO1 promoter. After in vitro validation, MSCs were transplanted into a mouse model of ischemia/reperfusion (IR, n=4) and mitochondrial function was monitored by bioluminescence imaging. Results: MSCs treated with DEM had an increase in NQO1 reporter gene signal that inversely correlated with the amount of the mitochondrial by-product adenosine tri-phosphate (ATP, r = -0.9643, p = 0.0028; Fig 1A). Mitochondrial dysfunction was preserved by the use of a mitochondria-targeted antioxidant Mito-Tempo (MT), correcting ATP and NQO1 luciferase signal. In vivo studies showed that MSCs yielded higher Firefly luciferase activity in IR mice compared to sham and this signal was normalized when cells were pre-treated with MT (Fig 1B). Data was confirmed by ex vivo measurement of Firefly luciferase activity, which was 4.9-fold higher in the IR group than in shams, after correction for cell number (Fig 1C). Conclusions: We have developed a novel imaging strategy that allows noninvasive assessment of the mitochondrial status of transplanted stem cells in the ischemic myocardium. Knowing when transplanted cells “suffer’ from mitochondrial dysfunction may allow optimization of these therapies.

Background: Cardiovascular risk factors have an impact on coronary atherosclerosis evolution. However, there is only limited information about the relationship between risk and atherosclerotic plaque as measured invasively by Intravascular Ultrasound Radiofrequency (IVUS-RF) analysis. The Aim of this study was to assess the impact of cardiovascular risk factors not only on overall coronary plaque volume but on coronary plaque composition. Methods: 990 patients from 42 centers were enrolled in the prospective, multicenter, non randomized, global VH-IVUS registry. Coronary lesions were measured by conventional and IVUS-VH parameters using 20MHz Intravascular Ultrasound catheter. The four IVUS-VH plaque components (Dense Calcium-DC, Necrotic Core-NC, Fibrous tissue-F, Fibro Fatty-FF) were analyzed in every recorded frame. The results were expressed as mean cross-sectional areas, and percentage of total plaque volume. Risk factor assessment included correlation with family history of myocardial infarction, past or current smoking, diabetes mellitus, hypertension, and laboratory measurements. Results: Patients with diabetes have an increased relative amount of NC (6.47±0.28% vs. 5.86±0.14%, p=0.037) and DC (4.58±0.27% vs. 3.90±0.14%, p=0.017), Patients with hypertension have an increased relative amount of FF, DC (4.35±0.16% vs. 3.57±0.17%, p=0.02) and NC (6.24±0.17% vs. 5.60±0.19%, p=0.01). Compared with patients with LDL cholesterol 160 mg/dl had higher plaque volume (342.1±26.2mm 3 vs. 318.6±10.7mm 3 ). Linear Regression Analysis showed a correlation between the level of HDL and F (r=−0.149, p<0.01), FF (r=−0 –106, p< 0.01), and NC (r=−0.90, p<0.05). The level of LDL correlated with F (r = 0.110, p < 0.01). Patients with prior MI have an increased percentage of F (30.03±0.59 % vs. 28.20±0.37 %, p<0.009). Smoking had no relevant effect on plaque composition. Conclusion: IVUS-RF analysis detects marked differences in coronary plaque depending on the risk factor profile with particular focus on lipid levels. Patients with greater amounts of necrotic core were associated with hypertension, myocardial infarction, and low HDL. This may have implications for prognosis and systemic treatment of patients with coronary artery disease.

Background : Renovascular disease (RVD) and impaired renal function are important risk factors for cardiac disease, but it remained unclear whether selective recovery of renal function can reverse cardiac dysfunction. Aims: This study tested the hypothesis that improvement in renal function using selective intra-renal administration of endothelial progenitor cells (EPC) in swine renovascular hypertension would improve myocardial microvascular (MV) integrity. Methods: The functions of the myocardial MV (response to IV adenosine) and the stenotic kidney of 7 pigs were assessed in-vivo using multi-detector CT after 6 weeks of RVD and hypertension. Autologous EPC expanded in culture were then infused (10x10 6 cells) intra-renally, and in vivo studies repeated 4 wks later. Six normal pigs served as controls. Results : Glomerular filtration rate was lower in RVD compared to normal (47.9±10.1 vs. 70.8±4.3 mL/min, p<0.05) and improved after EPC (to 62.7±1.5 mL/min, p=NS vs. normal), as did renal blood flow. Mean arterial pressure remained elevated after EPC (120.7±11 and 129.4±6 vs. 99.9±4.7 mmHg in normal, p<0.05), and plasma renin activity was unchanged. Myocardial MV permeability-surface area increased in RVD in response to adenosine (0.009±0.001 to 0.011±0.002 AU, p=0.01), while MV volume fraction decreased (4.3±0.8 to 2.7±0.6%, p=0.01), indicating impaired MV barrier function and integrity. Contrarily, both remained unchanged in response to adenosine 4 wks after EPC (0.01±0.002 to 0.01±0.002 AU, p=0.24, and 3.5±0.8 to 3.4±0.5%, p=0.46, respectively), as did normal MV. Conclusion : A single intra-renal infusion of EPC that improved renal function in experimental RVD also preserved remote myocardial MV function, despite sustained systemic hypertension. These findings underscore the cross talk between renal and cardiac function, and the potential of selective renal EPC intervention to preserve both the kidney and heart in RVD. Renal and myocardial microvascular function change in normal and RVD before and after EPC treatment

Introduction: Cardiovascular disease (CVD) is the leading cause of morbidity/mortality in the US. Despite being a class 1A evidence-based recommendation, secondary prevention efforts in the period following cardiac rehabilitation (CR) lack a proven and successful intervention. A previously validated digital health intervention (DHI) delivered Mayo Clinic’s CR, including educational material, tracking CVD indices, and diet/exercise adherence, for 3 months after usual CR. Hypothesis: We hypothesized DHI use would improve CVD risk factors and rehospitalizations/ emergency department (ED) visits. Methods: Patients undergoing usual Mayo Clinic CR were recruited to use the application following usual CR (n=17) and compared to a similar cohort undergoing standard post-CR care, but not assigned to the application (n=11). DHI patients performed daily informational tasks, entered their own risk factor information (weight, blood pressure (BP), glucose, lipids, physical activity, and diet) at baseline and for three months post-CR. Changes in risk factors, re-hospitalizations/ED visits were assessed after three months. Results: There were no differences in baseline demographics between the two groups. There was no difference in weight change between the two groups during the three months of usual CR, yet in the three months following usual CR the DHI group had significant reductions in weight (-2.5±3.8 kg, p=0.04) and systolic BP (-12.6±12.4 mmHg, p=0.001) compared to the control group. The DHI group also showed significantly lower rate of rehospitalizations/ED visits (-28%, p=0.04, Figure). Conclusions: A DHI post-CR program can enhance secondary prevention strategies compared to standard post-CR care by augmenting weight and BP reduction and reducing the healthcare burden of repeat CVD events. This study thus supports development and implementation of DHI programs.

MicroRNAs are a class of small non-coding RNAs that regulate gene expression by binding to discrete sites of genes and inhibiting translation. The objective of this study was to identify microRNAs and downstream enriched targets associated with symptomatic carotid plaques. Carotid plaque samples from patients were defined as symptomatic if they were associated with a prior ischemic stroke or transient ischemic attack, or as asymptomatic if there were no prior ischemic events. Digital microRNA profiling was performed on the NanoString nCounter analysis system (NanoString Technologies, Seattle, WA) on carotid plaques from symptomatic (n=6) and asymptomatic (n=6) patients. We used the BioConductor/R package "edgeR" to perform moderated-ANOVA testing for differences in gene abundance between groups, assuming a negative binomial distribution of gene counts, with Benjamini-Hochberg multiple hypothesis correction to control false discovery rate. Analysis revealed five microRNAs that were significantly up-regulated in the symptomatic cohort (see Table 1) including miR-16, which was confirmed by RTqPCR (p=0.02). Predicted downstream targets of miR-16 were enriched for proteoglycan related genes including hyaluronan synthase 2, perlecan and betaglycan. Additionally, RTqPCR demonstrated up-regulation of CathepsinB transcript in symptomatic patients (n=46-50 per cohort, p=0.02) while CathepsinL and heparanase transcripts were not significantly different. CathepsinL enzyme activity was also not significantly different (n=46-50 per cohort). Taken together, these findings may reveal novel proteoglycan mediated signaling pathways, mediated through miR-16, regulating the progression of carotid plaque formation.

Introduction: Venous thromboembolism (VTE) is a cause of significant morbidity and mortality. The vascular endothelium may be involved in the pathogenesis of both VTE and arterial thrombosis, as endothelial injury and dysfunction may disrupt normal anticoagulation mechanisms. We aimed to test the hypothesis that coronary endothelial dysfunction (CED) is associated with development of VTE in prospective follow-up. Methods: Coronary vascular reactivity was evaluated in 502 patients with non-obstructive coronary artery disease by administration of intracoronary acetylcholine during diagnostic angiography. Microvascular CED was defined as ≤50% increase in coronary blood flow (CBF) from baseline in response to maximal dose of acetylcholine. After median follow-up of 6.3 years (IQR 3.5, 10.7), patients were assessed by standardized questionnaire, phone-call and review of medical records for development of VTE. Results: Median age of the population was 53 years (IQR 45, 62); 68% were females. Hypertension was prevalent in 40.8%, diabetes in 8.4%, and hyperlipidemia in 58.3% of patients. Of 502 patients, 279 had CED. There were no significant differences in baseline characteristics including cardiovascular and VTE risk factors between patients with and without CED (p>0.05). Eighty-nine percents of patients who developed VTE had CED. Patients who developed spontaneous or provoked VTE both had significantly lower % change in CBF than patients who did not develop VTE (p<0.05) (Figure 1). In univariate analysis, microvascular dysfunction was associated with increased risk of VTE (OR 6.55 (95% confidence interval (0.81, 52.79); p=0.04). Conclusion: We found a seven-fold increased risk of VTE with CED, suggesting a link between the two. Endothelial injury disrupting vascular homeostasis may predispose patients to VTE.

Polycystic kidney disease (PKD) is the most common monogenic kidney disorder and an important cause of end stage renal disease. Vascular abnormalities are the most important non‐cystic complication and contribute to renal disease progression. Homocysteine (Hcy)‐mediated endothelial dysfunction (ED) and oxidative stress are evident early on, but the underlying mechanisms remain unknown. We hypothesized that abnormalities in Hcy metabolism are responsible for the increased levels, leading to Hcy‐induced ED from early stages of PKD. Methods We investigated the Hcy metabolic pathway (transulfuration/remethylation fig. 1) and downstream effects ‐main sources of renal tubular (RT) and endothelial cell (EC) reactive oxygen species (ROS), NADPH oxidase 4, (NOX4) and mitochondria‐ in 4‐week‐old PCK and Sprague‐Dawley (SD) control rats (n=12 each). Kidneys were harvested, frozen in liquid nitrogen, or preserved in formalin for metabolomics and ex‐vivo studies. Twenty‐four‐hour urine and terminal blood samples were collected for metabolite analysis and chemistries. Plasma folic acid, vitamin B12, and Hcy were determined by ELISA. Hcy pathway metabolites (betaine, dimethylglycine) were determined by 1 HNMR. Endothelial NO (eNOS) and oxidative stress were assessed by double immunofluorescence staining for CD31/eNOS and CD31/NOX4, respectively. RT and EC mitochondrial structure was assessed by electron microscopy. Results Serum creatinine and BUN remained unchanged, yet kidney weight/body weight ratio were increased in PCK. eNOS immunoreactivity was lower in PCK vs SD. Plasma Hcy and urine excretion of methyl donor betaine were elevated in PCK, and Hcy positively correlated with urine betaine. Plasma betaine was similar between the groups, but its tissue concentration was lower in PCK, which may lead to betaine insufficiency, impaired betaine dependent remethylation, and hyperhomocysteinemia. Contrarily, tissue glutathione concentration was higher in PCK vs SD, and plasma folate and vitamin B12 were similar, arguing against a defect in the transsulfuration or folate‐dependent remethylation pathways. RT NOX4 expression and immunoreactivity were increased in PCK compared to SD, in association with renal oxidative stress. PCK had increased number of CD31/NOX4 positive cells in peritubular capillaries, indicating increased EC ROS production. Collecting duct and EC mitochondria were swollen and showed cristae remodeling. Conclusion In PKD, early elevation in Hcy is likely related to betaine insufficiency and impaired betaine‐dependent remethylation may be responsible for the early elevation of Hcy. NOX4 and mitochondria abnormalities may be important contributors to oxidative stress and disease severity. Our data suggests these may not only be early vascular disease markers, but also play an important pathogenic role in vascular complications associated with PKD, and contribute to renal disease progression. Support or Funding Information R25‐DK101405, NIDDK P30 DK090728, NIDDK P30 DK 106912 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Background 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, blunting their capacity to preserve the post‐stenotic kidney in pigs with metabolic syndrome (MetS) and renal artery stenosis (RAS). Methods We compared the protein cargo of EVs to their parent MSCs obtained from pigs with 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 LC‐MS/MS proteomics, clustering analysis was performed to identify primary functional categories of proteins enriched in Lean‐ and MetS‐EVs. In addition, MetS pigs were studied after 16 weeks of RAS untreated or treated 4 weeks earlier with a single intrarenal delivery of allogeneic labeled Lean‐ or MetS‐EVs (2.5x10^5/Kg). Lean and MetS Sham served as controls (n=6 each). Single‐kidney volume, renal blood flow (RBF), and glomerular filtration rate (GFR) were studied in‐vivo (multi‐detector CT), and tubular injury (Periodic Acid Schiff) and fibrosis (Trichrome) ex‐vivo. Results Proteomics analysis identified 6,690 and 6,790 distinct proteins in Lean‐ and MetS‐EVs, respectively. Differential expression analysis revealed that 91 proteins were upregulated in Lean‐EVs and 130 proteins in MetS‐EVs compared to their parent MSCs (FDR<0.05 and log2 fold‐change EVs/MSCs>2). 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 (Figure 1A). Contrarily, proteins enriched only in MetS‐EVs are linked to pro‐inflammatory pathways, including acute inflammatory response, leukocyte transendothelial migration, and cytokine production (Figure 1B). Blood pressure was elevated in all MetS groups and all RAS groups develop moderate, but significant stenosis of a similar degree (Table). EVs were detected in the stenotic‐kidney 4 weeks after injection (Figure 2A). Renal volume, RBF, and GFR, which increased in MetS compared to Lean (consistent with hyperfiltration), fell in MetS+RAS, but restored in pigs treated with Lean‐EVs. Lean‐EVs also attenuated tubular injury and renal fibrosis (Figure 2B). Yet, these renoprotective effects were blunted in pigs treated with MetS‐EVs. Conclusion MetS alters the protein cargo of porcine MSC‐derived EVs, selectively packaging specific pro‐inflammatory signatures. Intrarenal delivery of Lean‐EVs attenuated renal injury and fibrosis and improved function in pigs with MetS+RAS, whereas these beneficial effects were blunted in pigs treated with MetS‐EVs. Therefore, our observations suggest that approaches to improve the therapeutic efficacy of autologous MSC‐derived EVs might be useful for patients with MetS+RAS. Support or Funding Information This study was partly supported by the National Institutes of Health grant numbers: DK104273, HL123160, DK102325, and DK106427. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .