Lerman, Amir

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 .

Renovascular disease (RVD) and hypertension (HT) are cardiovascular risk factors, but if recovery of renal function can reverse cardiac dysfunction remained unclear. We hypothesized that improved stenotic kidney function would rescue myocardial microvascular (MMV) function in renovascular HT by reducing oxidative stress and inflammation. Single‐kidney hemodynamics and function and MMV permeability were assessed in pigs in vivo using CT after 6 weeks of RVD. Autologous endothelial progenitor cells (EPC) were then infused intra‐renally, and studies repeated 4 weeks later (RVD+EPC, n=7). Normal and untreated RVD (n=7 each) served as controls. Blood pressure was similarly increased in RVD and RVD+EPC pigs. Compared to normal, RVD showed elevated systemic TNF‐alpha (142.3± 3.7 vs 43.3 ± 3.5 pg/ml, p<0.001) and oxidized‐LDL, blunted glomerular filtration rate (47.9±10.1 vs 70.8 ± 4.3 ml/min, p<0.05), decreased MMV volume fraction, increased MMV permeability, and myocardial fibrosis. EPC were rarely observed in myocardium. However, RVD+EPC showed a decrease in TNF‐alpha (90.5± 2.9, p<0.01 vs RVD) and oxidized‐LDL, implying decreased inflammation and oxidative stress. Furthermore, not only renal, but also MMV function were significantly improved in RVD+EPC. In conclusion, selective improvement in renal function reduces oxidative stress and inflammation and preserves remote MMV function, underscoring functionally important cardiorenal crosstalk.

Background: The mechanisms responsible for cardiac injury in the early stage of metabolic syndrome (MetS) remain unknown. Mitochondria are intimately associated with myofibrils, where the cytoskeleton functions as a linkage coordinator. We hypothesized that early MetS is characterized by cytoskeletal-mitochondrial disorganization, and that mitoprotection with mitochondria-targeted peptides (MTP) would preserve cytoskeletal-mitochondrial structure and attenuate myocardial injury in early swine MetS. Methods: Pigs were studied after 16wks of diet-induced MetS, MetS treated for the last 4wks with the MTP Elamipretide (0.1mg/kg SC q.d), and Lean controls (n=6 each). Cardiac function (multidetector CT) was assessed in-vivo, and mitochondrial structure (electron microscopy), the mitochondrial inner membrane phospholipid cardiolipin (mass spectrometry), cytoskeletal proteins, oxidative stress, and apoptosis ex-vivo. Results: MetS pigs developed hypertension and insulin resistance, yet cardiac function was preserved. MetS induced loss of desmin and tubulin that was paralleled by mitochondrial disorganization, decreased 18:2 cardiolipin, and increased oxidative stress and apoptosis. MTP slightly improved cardiolipin species profile, restored mitochondrial organization, and upregulated cytoskeletal proteins, attenuating apoptosis and oxidative stress. Conclusion: Early MetS leads to disorganization of the cardiomyocyte cytoskeletal- mitochondrial architecture and cardiac injury. MTP may provide a novel therapeutic potential for improving cardiac injury in early MetS, and potentially preventing future deterioration in cardiac function.

Significance Statement Renal artery stenosis (RAS) engenders stenotic-kidney ischemia, dysfunction, and injury, but whether these are mediated by cellular senescence has not been elucidated. INK-ATTAC transgenic mice, high-resolution imaging, and unbiased single-cell RNA sequencing of murine kidneys demonstrated cellular senescence as an important mechanism of progressive injury to epithelial/stromal cells within poststenotic kidneys. Both p16-specific and broad quercetin/dasatinib interventions to blunt senescence improved renal function and structure, underscoring the central role of senescence in the pathogenesis. Furthermore, this mechanism was conserved in human subjects with RAS. These observations reveal new mechanisms that contribute to the pathogenesis of chronic ischemic renal injury, and support the development of senolytic therapy to reduce senescent cell burden and delay renal injury. Background Peripheral vascular diseases may induce chronic ischemia and cellular injury distal to the arterial obstruction. Cellular senescence involves proliferation arrest in response to stress, which can damage neighboring cells. Renal artery stenosis (RAS) induces stenotic-kidney dysfunction and injury, but whether these arise from cellular senescenceand their temporal pattern remain unknown. Methods Chronic renal ischemia was induced in transgenic INK-ATTAC and wild type C57BL/6 mice by unilateral RAS, and kidney function ( in vivo micro-MRI) and tissue damage were assessed. Mouse healthy and stenotic kidneys were analyzed using unbiased single-cell RNA-sequencing. To demonstrate translational relevance, cellular senescence was studied in human stenotic kidneys. Results Using intraperitoneal AP20187 injections starting 1, 2, or 4 weeks after RAS, selective clearance of cells highly expressing p16 Ink4a attenuated cellular senescence and improved stenotic-kidney function; however, starting treatment immediately after RAS induction was unsuccessful. Broader clearance of senescent cells, using the oral senolytic combination dasatinib and quercetin, in C57BL/6 RAS mice was more effective in clearing cells positive for p21 ( Cdkn1a ) and alleviating renal dysfunction and damage. Unbiased, single-cell RNA sequencing in freshly dissociated cells from healthy and stenotic mouse kidneys identified stenotic-kidney epithelial cells undergoing both mesenchymal transition and senescence. As in mice, injured human stenotic kidneys exhibited cellular senescence, suggesting this process is conserved. Conclusions Maladaptive tubular cell senescence, involving upregulated p16 ( Cdkn2a ), p19 ( Cdkn2d ), and p21 ( Cdkn1a ) expression, is associated with renal dysfunction and injury in chronic ischemia. These findings support development of senolytic strategies to delay chronic ischemic renal injury.

AbstractBackgroundWe assessed the long‐term clinical outcomes of an intermediate lesion (IL) according to the presence of a combined culprit lesion (CCL).HypothesisLong‐term clinical outcomes of IL may be affected by the presence of a CCL.MethodsAngiographic findings (n = 1096) and medical chart were reviewed. Patients with IL were divided into two groups: IL without CCL group (n = 383, 64.5%) and IL with CCL group (n = 211, 35.5%).ResultsThe major adverse cardiovascular events (MACE) in the IL with CCL group were significantly higher than those in the IL without CCL group (death: 12.3% vs. 7.0%, myocardial infarction: 3.3%vs. 0.5%, stroke: 6.6% vs. 2.6%, and revascularization [RVSC]: 25.1% vs. 7.6%) during a mean follow up period of 118.4 ± 5.5 months. IL related RVSC rate in the IL with CCL group was higher than that in the IL without CCL group (5.7% vs. 2.1%, p = 0.020). RVSC rate related to IL in total subjects was lower than that related to stented lesion (3.4% vs. 6.4%). The important predictors of total MACE in total subjects were the presence of CCL, IL percent diameter stenosis, hypertension, history of percutaneous coronary intervention, blood glucose and ejection fraction. The predictors of IL related RVSC were IL percent diameter stenosis and IL located in the right coronary artery.Conclusion10‐year clinical outcomes of an IL (especially IL without CCL) were better than those of stented lesions. This study suggests that the IL can be safely followed up in sites that do not have ability to assess functional study.

Percutaneous transluminal renal angioplasty and stenting (PTRS) for RVH may not revert the secondary cardiac dysfunction and remodeling. In experimental RVH, infusion of the tetrapeptide Bendavia (that inhibits mitochondrial permeability transition pore opening) during PTRS attenuated stenotic kidney inflammatory injury and improved its function. We hypothesized that adjunct Bendavia during PTRS would also attenuate cardiac remodeling after reversal of RVH. RVH was induced in 18 pigs, of which 6 were sham treated (RVH) and 12 underwent PTRS plus a 4h IV infusion of Bendavia (0.05 mg/kg) or vehicle (n=6, each); 6 normal pigs served as controls. Cardiac oxygenation and function were assessed 4 wks later by blood oxygen level‐dependent (BOLD)‐MRI and CT, and tissue studies performed. PTRS normalized blood pressure (Table). Systolic function was unchanged. Apoptotic activity (caspase‐3), hypoxia (BOLD‐R2*), inflammation (TNF‐α, IL‐6), and fibrosis (trichrome) remained elevated and diastolic function (E/A) impaired in PTRS+Vehicle, but all improved in PTRS+Bendavia (Figure). Thus, novel cardioprotective properties of Bendavia enhance cardiac recovery after reversal of experimental RVH, and may provide a novel therapeutic option.