Lerman, Lilach O.

Abstract Aims Cardiovascular disease and cancer share common pathogenesis and risk factors. Coronary microvascular dysfunction (CMD), reflecting impaired coronary microvascular dilation in response to stress, is related to a higher risk of major cardiovascular events; however, its association with cancer has not been explored. Methods and results A retrospective study on 1042 patients with non-obstructive coronary artery diseases (NOCADs) was performed. Data regarding demographic, clinical history, diagnostic coronary reactivity test, and cancer occurrence were collected. Coronary microvascular dysfunction was defined as coronary flow reserve (the ratio of hyperaemic blood flow to resting blood flow) ≤2.5. Thirty-four per cent had CMD (67.4% female and the average age was 52.4 ± 12.2 years). Of 917 patients with no history of cancer, 15.5% developed cancer during follow-up [median of 9 (4, 16) years]. Kaplan–Meier analysis showed that CMD patients had lower cancer-free survival compared with those without CMD (log-rank P = 0.005). Cox proportional hazard analyses showed that after adjusting for age, sex, hypertension, diabetes, smoking, and glomerular filtration rate, CMD is independently associated with cancer [hazard ratio, 1.4; 95% confidence interval (CI), 1.09–2.04; P = 0.04]. The rate of major adverse cardiovascular events (MACE) was significantly higher in CMD patients compared with that in non-CMD patients who had a previous history of cancer [odds ratio (OR), 2.5; 95% CI, 1–6.2; P = 0.04] and those with no history of cancer (OR, 1.4; 95% CI, 1.01–1.9; P = 0.044). Conclusion Coronary microvascular dysfunction is associated with cancer incidence in patients presenting with NOCADs. This study emphasizes follow-up in patients with CMD to evaluate the risk of MACE as well as potential malignant diseases.

Background: Uric acid (UA) concentration within carotid plaque and its association with cerebrovascular events have not been detected or quantified. Systemically, serum UA is a marker of inflammation and risk factor for atherosclerosis. However, its association with carotid plaque instability and stroke pathogenesis remains unclear. In patients undergoing carotid endarterectomy, we aimed to determine whether UA is present differentially in symptomatic versus asymptomatic carotid plaques and whether serum UA is associated with cerebrovascular symptoms (stroke, transient ischemic attack, or amaurosis fugax). Methods: Carotid atherosclerotic plaques were collected during carotid endarterectomy. The presence of UA was assessed using Gomori methenamine silver staining as well as anti-UA immunohistochemical staining and its quantity measured using an enzymatic colorimetric assay. Clinical information was obtained through a retrospective review of data. Results: UA was more commonly detected in symptomatic (n=23) compared with asymptomatic (n=9) carotid plaques by Gomori methenamine silver (20 [86.9%] versus 2 [22.2%]; P =0.001) and anti-UA immunohistochemistry (16 [69.5%] versus 1 [11.1%]; P =0.004). UA concentration was higher in symptomatic rather than asymptomatic plaques (25.1 [9.5] versus 17.9 [3.8] µg/g; P =0.021). Before carotid endarterectomy, serum UA levels were higher in symptomatic (n=341) compared with asymptomatic (n=146) patients (5.9 [interquartile range, 4.6–6.9] mg/dL versus 5.2 [interquartile range, 4.6–6.2] mg/dL; P =0.009). Conclusions: The current study supports a potential role of UA as a potential tissue participant and a systemic biomarker in the pathogenesis of carotid atherosclerosis. UA may provide a mechanistic explanation for plaque instability and subsequent ischemic cerebrovascular events.

Background: Scattered tubular-like cells (STCs) are dedifferentiated renal tubular cells endowed with progenitor-like characteristics to repair injured parenchymal cells. STCs may be damaged and rendered ineffective by renal artery stenosis (RAS), but the underlying processes remain unclear. We hypothesized that RAS alters the epigenetic landscape on DNA and the ensuing gene transcriptional profile of swine STCs. Methods: CD24+/CD133+ STCs were isolated from pig kidneys after 10 weeks of RAS or sham (n = 3 each) and their whole 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) profiles were examined by 5mC and 5hmC immunoprecipitation sequencing (MeDIP-/hMeDIP-seq, respectively). A subsequent integrated (MeDIP/hMeDIP-seq/mRNA-seq) analysis was performed by comparing all online available gene sets using Gene Set Enrichment Analysis. Apoptosis, proteolysis, and mitochondrial structure and function were subsequently evaluated in vitro. Results: Differential expression (DE) analysis revealed 239 genes with higher and 236 with lower 5mC levels and 275 genes with higher and 315 with lower 5hmC levels in RAS-STCs compared to Normal-STCs (fold change ≥1.4 or ≤0.7, p ≤ 0.05). Integrated MeDIP-/hMeDIP-seq/mRNA-seq analysis identified several overlapping (DE-5mC/mRNA and DE-5hmC/mRNA levels) genes primarily implicated in apoptosis, proteolysis, and mitochondrial functions. Furthermore, RAS-STCs exhibited decreased apoptosis, mitochondrial matrix density, and ATP production, and increased intracellular amino acid concentration and ubiquitin expression. Conclusions: Renal ischemia induces epigenetic changes in apoptosis-, proteolysis-, and mitochondria-related genes, which correlate with alterations in the transcriptomic profile and corresponding function of swine STCs. These observations may contribute to developing novel targeted interventions to preserve the reparative potency of STCs in renal disease.

ABSTRACT Background Renal artery stenosis (RAS) is an important cause of chronic kidney disease and secondary hypertension. In animal models, renal ischemia leads to downregulation of growth factor expression and loss of intrarenal microcirculation. However, little is known about the sequelae of large-vessel occlusive disease on the microcirculation within human kidneys. Method This study included five patients who underwent nephrectomy due to renovascular occlusion and seven nonstenotic discarded donor kidneys (four deceased donors). Micro-computed tomography was performed to assess microvascular spatial densities and tortuosity, an index of microvascular immaturity. Renal protein expression, gene expression and histology were studied in vitro using immunoblotting, polymerase chain reaction and staining. Results RAS demonstrated a loss of medium-sized vessels (0.2–0.3 mm) compared with donor kidneys (P = 0.037) and increased microvascular tortuosity. RAS kidneys had greater protein expression of angiopoietin-1, hypoxia-inducible factor-1α and thrombospondin-1 but lower protein expression of vascular endothelial growth factor (VEGF) than donor kidneys. Renal fibrosis, loss of peritubular capillaries (PTCs) and pericyte detachment were greater in RAS, yet they had more newly formed PTCs than donor kidneys. Therefore, our study quantified significant microvascular remodeling in the poststenotic human kidney. RAS induced renal microvascular loss, vascular remodeling and fibrosis. Despite downregulated VEGF, stenotic kidneys upregulated compensatory angiogenic pathways related to angiopoietin-1. Conclusions These observations underscore the nature of human RAS as a microvascular disease distal to main vessel stenosis and support therapeutic strategies directly targeting the poststenotic kidney microcirculation in patients with RAS.

Introduction: The metabolic syndrome (MetS) leads to cardiac vascular injury, which may reflect in increased retention of endothelial progenitor cells (EPC). Coronary endothelial cell (EC) mitochondria partly regulate vascular function and structure. We hypothesized that chronic mitoprotection would preserve EC mitochondria and attenuate coronary vascular injury and dysfunction in swine MetS. Methods: Pigs were studied after 16 weeks of diet-induced MetS, MetS treated for the last 4 weeks with the mitochondria-targeted peptide elamipretide (ELAM, 0.1mg/kg SC q.d), and lean controls (n=6 each). Coronary artery and sinus blood samples were collected. EC mitochondrial density (electron microscopy), endothelial nitric oxide (eNOS) immunoreactivity (staining), myocardial microvascular density (3D micro-CT), and coronary endothelial function (organ bath) were assessed ex-vivo. The number and arteriovenous gradient of CD34+/KDR+ EPC was calculated by FACS (a negative gradient indicating EPC retention). Results: MetS and MetS+ELAM pigs developed similar MetS (obesity, hyperlipidemia, insulin resistance, and hypertension). EC mitochondrial density decreased in MetS compared to lean, but normalized in MetS+ELAM. ELAM also improved eNOS immunoreactivity, subendocardial microvascular density, and coronary endothelial function. ELAM-induced vasculoprotection was reflected in decreased coronary retention of EPCs. Conclusions: These observations underscore the role of mitochondria in regulating the cardiac circulation in experimental MetS, and the benefits of mitoprotection to preserve EC myocardial microvascular integrity.

Determinants of Intra-renal oxygenation in human RVD are poorly understood. Blood oxygen level-dependent (BOLD) MRI measures deoxyhemoglobin (R2*) to determine cortical and medullary R2* which have focal variations and gradients. We hypothesized that tissue fractional hypoxia (% of axial kidney area with R2*>30s -1 ) would correlate better with tissue perfusion, renal blood flow (RBF), and O2 consumption than using regions of interest (ROI). METHODS: Inpatient studies were performed in EH (n=68 kidneys), and high-grade RVD (n=18 kidneys, 71±5.5% occlusion) before and 3 months after stents. All were treated with ACE/ARB Rx and thiazides during 150 mEq/d Na+ intake. BOLD MR images were obtained at 3T before and after furosemide (20mg IV), and axial images analyzed to estimate R2* levels above 30s -1 (fractional hypoxia) over the entire kidney section. Maximal R2* levels using regions of interest (ROIs) also were determined. Single-kidney cortical and medullary RBF (ml/min) perfusion (ml/min/cc) were determined by MDCT. Results: RBF was lower in kidneys with untreated RVD than EH (285.6±141 vs. 399±159 ml/min p<0.05), as was cortical perfusion (2.58±.63 vs. 3.50±1.05, p<.05 ). Cortical and medullary R2* did not differ, but fractional hypoxia was greater in RVD as compared to EH (18.3±12 vs. 11.5±8.5%, p<.05). Fractional hypoxia was inversely correlated with blood flow (R=-.29, p<.01) and perfusion (R=-.26, p<.01). R2* fell after furosemide (p<0.01) indicating a role for O2 consumption in determining tissue hypoxia. RBF and cortical perfusion increased after stenting (285±141 vs. 318±178 ml/min and 2.58±0.6 vs. 2.9±0.7ml/min/cc, p<0.05) respectively. Fractional hypoxia, but not cortical or medullary R2* based on ROI-methods, decreased in patients 3 months after renal revascularization (18.3 ±12 vs. 10.52%, p<0.05) Conclusions: These data demonstrate for the first time that fractional hypoxia in the kidney is directly related to chronically reduced blood flow and cortical perfusion in human RVD, whereas selective ROI-based measures identified only minor changes in peak R2* gradients between cortex and medulla. The reversibility of fractional hypoxia after stenting supports the use of this new method to identify critical renovascular lesions.

Background: Atherosclerotic renal artery stenosis (ARAS) reduces renal blood flow (RBF) and amplifies regional kidney hypoxia, but their relationship to tissue inflammation and injury markers is poorly understood. We tested the hypothesis that renal injury, reflected by renal vein levels of neutrophil gelatinase associated lipocalin (NGAL) and inflammatory cytokines monocyte chemoattractant protein-1 (MCP-1) would be magnified with severe occlusive vascular disease in humans with ARAS. Methods: Inpatient studies performed in patients with ARAS (n=40, 71.3±11% occlusion by CT angiography) or essential hypertension (EH) (n=30), during fixed Na+ intake and ACE/ARB Rx. Single-kidney cortical and medullary perfusion and RBF were measured using multidetector CT, and glomerular filtration rate (GFR) by iothalamate clearance. Tissue deoxyhemoglobin levels (R2*) and fractional kidney hypoxia (% of axial area with R2*>30/s) were measured by BOLD-MRI at 3T. Results: Single kidney RBF, cortical and medullary perfusion were reduced in the post-stenotic kidney, as was GFR. Renal vein NGAL, MCP-1 and fractional hypoxia were higher in patients with ARAS than EH. NGAL and MCP-1 levels correlated directly with cortical R2* and fractional hypoxia (r = 0.52, P <0 .0001 and r = 0.4, P = 0.0006) and with the degree of artey stenosis (r = 0.37, P = 0.04 and r = 0.35, P = 0.05). GFR correlated inversely with NGAL (r= - 0.5, P <0.0001). Conclusions: Our results demonstrate that elevated renal venous markers of kidney inflammation (MCP-1) and injury (NGAL) in ARAS are correlated with the severity of tissue hypoxia as measured by BOLD MR, suggesting a possible link between kidney inflammation and hypoxia.

Background: Renal hypoxia is a complex function of post-glomerular medullary blood flow, active solute transport and is considered a major risk element for acute kidney injury. Previous studies indicate that sodium balance, diuretic administration and some drugs affect tissue oxygenation. Water loading in younger subjects lowers medullary deoxyhemoglobin. We sought to evaluate the effects of water loading on the day of BOLD imaging in older subjects undergoing protocol evaluation of atherosclerotic renovascular disease (ARVD). METHODS: Inpatient studies were performed in patients with hemodynamically severe ARVD (n=15, Doppler US velocity >270 cm/sec), during 150 mEq Na+ intake and ACE/ARB Rx. Two groups were studied: Group A (n=10) received oral water loading (20 ml/kg), whereas Group B (n=5) had none. Tissue R2* (index of deoxyhemoglobin levels) and fractional kidney hypoxia (medullary estimate) (% of renal coronal area with R2*>20/s) were measured in stenotic kidneys by BOLD-MRI at 3T, before and after IV injection of Furosemide (20gm). RESULTS: Cortical R2* and fractional tissue hypoxia were similar for the 2 groups (P =0.6 and 0.7). Both groups reduced cortical R2* and fractional hypoxia after furosemide injection to the same degree. Table CONCLUSION: These data demonstrate that water loading on the day of BOLD evaluation in older subjects with ARVD does not materially alter cortical or medullary estimates of oxygenation. Our results extend previous studies indicating that water loading in older individuals fails to alter medullary oxygenation unlike alterations in sodium intake, diuretic administration or blockade of the renin-angiotensin system.

Introduction: Coronary vasomotor response is different between males and females. Overall, females have lower coronary flow reserve (CFR) than males due to higher baseline coronary flow than males. Hypothesis: Sex has an essential role in the interaction between age and CFR. Methods: We included 668 patients with nonobstructive coronary artery disease who underwent invasive coronary vasoreactivity testing using intracoronary incremental doses of adenosine (18-72 μg). Changes in CFR (hyperemic flow velocity/baseline flow velocity) with aging were separately analyzed between males (N=207, mean age 53±13 years) and females (N=461, mean age 54±12 years). We further divided patients by age of 53 which is average age of menopause. Results: In males, CFR decreased with aging in younger patients <53 years and tended to decrease in older male patients (all ages [N=207]: r=-0.33, P <0.0001; <53 years [N=99]: r=-0.27, P =0.01; ≥53 years [N=108]: r=-0.17, P =0.07) ( Figure A ). In contrast, CFR did not decrease with aging in younger females <53 years, whereas there was a significant negative correlation between CFR and age in older females ≥53 years (all ages [N=461]: r=-0.15, P =0.001; <53 years [N=211]: r=-0.006, P =0.39; ≥53 years [N=250]: r=-0.25, P <0.0001) ( Figure B ). Baseline microvascular resistance (BMR) was similar between younger and older males ( P =0.30), whereas older females had higher baseline BMR than younger females ( P <0.0001) ( Figure C ). Conclusions: CFR decreased with aging in males and older females, but not in younger females, potentially highlighting the role of protective hormonal differences in maintaining low baseline coronary tone in younger females.