Description: Background— There is increasing evidence that retinal microvascular diameters are associated with cardiovascular and cerebrovascular conditions. The shared genetic effects of these associations are currently unknown. The aim of this study was to increase our understanding of the genetic factors that mediate retinal vessel size. Methods and Results— This study extends previous genome-wide association study results using 24 000+ multiethnic participants from 7 discovery cohorts and 5000+ subjects of European ancestry from 2 replication cohorts. Using the Illumina HumanExome BeadChip, we investigate the association of single-nucleotide polymorphisms and variants collectively across genes with summary measures of retinal vessel diameters, referred to as the central retinal venule equivalent and the central retinal arteriole equivalent. We report 4 new loci associated with central retinal venule equivalent, one of which is also associated with central retinal arteriole equivalent. The 4 single-nucleotide polymorphisms are rs7926971 in TEAD1 ( P =3.1 x 10 – 11 ; minor allele frequency=0.43), rs201259422 in TSPAN10 ( P =4.4 x 10 –9 ; minor allele frequency=0.27), rs5442 in GNB3 ( P =7.0 x 10 –10 ; minor allele frequency=0.05), and rs1800407 in OCA2 ( P =3.4 x 10 –8 ; minor allele frequency=0.05). The latter single-nucleotide polymorphism, rs1800407, was also associated with central retinal arteriole equivalent ( P =6.5 x 10 –12 ). Results from the gene-based burden tests were null. In phenotype look-ups, single-nucleotide polymorphism rs201255422 was associated with both systolic ( P =0.001) and diastolic blood pressures ( P =8.3 x 10 –04 ). Conclusions— Our study expands the understanding of genetic factors influencing the size of the retinal microvasculature. These findings may also provide insight into the relationship between retinal and systemic microvascular disease.

Description: Objective— Tyrosine kinase receptor B (TrkB) is a high-affinity receptor for brain-derived neurotrophic factor. In addition to its nervous system functions, TrkB is also expressed in the cardiovascular system. However, the association of TrkB and coronary artery disease (CAD) remains unknown. We investigated the role of TrkB in the development of CAD and its mechanism. Approach and Results— We performed a case–control study in 2 independent cohort of Chinese subjects and found –69C〉G polymorphisms of TrkB gene significantly associated with CAD. TrkB –69C homozygotes, which corresponded to decreased TrkB expression by luciferase reporter assay, showed increased risk for CAD. Immunofluorescence analysis revealed that TrkB was expressed in the aortic endothelium in atherosclerotic lesions in humans and ApoE –/– mice. TrkB knockdown in the aortic endothelium resulted in vascular leakage in ApoE –/– mice. Mechanistic studies showed that TrkB regulated vascular endothelial cadherin (VE-cadherin) expression through induction and activation of Ets1 transcriptional factor. Importantly, TrkB activation attenuated proatherosclerotic factors induced-endothelial hyperpermeability in human vascular endothelial cells. Conclusions— Our data demonstrate that TrkB protects endothelial integrity during atherogenesis by promoting Ets1-mediated VE-cadherin expression and plays a previously unknown protective role in the development of CAD.

Description: Background and Purpose— Bryostatin, a potent protein kinase C (PKC) activator, has demonstrated therapeutic efficacy in preclinical models of associative memory, Alzheimer disease, global ischemia, and traumatic brain injury. In this study, we tested the hypothesis that administration of bryostatin provides a therapeutic benefit in reducing brain injury and improving stroke outcome using a clinically relevant model of cerebral ischemia with tissue plasminogen activator reperfusion in aged rats. Methods— Acute cerebral ischemia was produced by reversible occlusion of the right middle cerebral artery (MCAO) in 18- to 20-month-old female Sprague–Dawley rats using an autologous blood clot with tissue plasminogen activator–mediated reperfusion. Bryostatin was administered at 6 hours post-MCAO, then at 3, 6, 9, 12, 15, and 18 days after MCAO. Functional assessment was conducted at 2, 7, 14, and 21 days after MCAO. Lesion volume and hemispheric swelling/atrophy were performed at 2, 7, and 21 days post-MCAO. Histological assessment of PKC isozymes was performed at 24 hours post-MCAO. Results— Bryostatin-treated rats showed improved survival post-MCAO, especially during the first 4 days. Repeated administration of bryostatin post-MCAO resulted in reduced infarct volume, hemispheric swelling/atrophy, and improved neurological function at 21 days post-MCAO. Changes in αPKC expression and PKC expression in neurons were noted in bryostatin-treated rats at 24 hours post-MCAO. Conclusions— Repeated bryostatin administration post-MCAO protected the brain from severe neurological injury post-MCAO. Bryostatin treatment improved survival rate, reduced lesion volume, salvaged tissue in infarcted hemisphere by reducing necrosis and peri-infarct astrogliosis, and improved functional outcome after MCAO.

Description: Objective— Elevated serum phosphate has emerged as a major risk factor for vascular calcification. The sodium-dependent phosphate cotransporter, PiT-1, was previously shown to be required for phosphate-induced osteogenic differentiation and calcification of cultured human vascular smooth muscle cells (VSMCs), but its importance in vascular calcification in vivo and the potential role of its homologue, PiT-2, have not been determined. We investigated the in vivo requirement for PiT-1 in vascular calcification using a mouse model of chronic kidney disease and the potential compensatory role of PiT-2 using in vitro knockdown and overexpression strategies. Approach and Results— Mice with targeted deletion of PiT-1 in VSMCs were generated (PiT-1 sm ). PiT-1 mRNA levels were undetectable, whereas PiT-2 mRNA levels were increased 2-fold in the vascular aortic media of PiT-1 sm compared with PiT-1 flox/flox control. When arterial medial calcification was induced in PiT-1 sm and PiT-1 flox/flox by chronic kidney disease followed by dietary phosphate loading, the degree of aortic calcification was not different between genotypes, suggesting compensation by PiT-2. Consistent with this possibility, VSMCs isolated from PiT-1 sm mice had no PiT-1 mRNA expression, increased PiT-2 mRNA levels, and no difference in sodium-dependent phosphate uptake or phosphate-induced matrix calcification compared with PiT-1 flox/flox VSMCs. Knockdown of PiT-2 decreased phosphate uptake and phosphate-induced calcification of PiT-1 sm VSMCs. Furthermore, overexpression of PiT-2 restored these parameters in human PiT-1–deficient VSMCs. Conclusions— PiT-2 can mediate phosphate uptake and calcification of VSMCs in the absence of PiT-1. Mechanistically, PiT-1 and PiT-2 seem to serve redundant roles in phosphate-induced calcification of VSMCs.

Description: Background— The aim of this study was to determine the role of the chemokine receptor CXCR7 in atherosclerosis and vascular remodeling. CXCR7 is the alternative receptor of CXCL12, which regulates stem cell–mediated vascular repair and limits atherosclerosis via its receptor, CXCR4. Methods and Results— Wire-induced injury of the carotid artery was performed in mice with a ubiquitous, conditional deletion of CXCR7 and in mice treated with the synthetic CXCR7 ligand CCX771. The effect of CCX771 treatment on atherosclerosis was studied in apolipoprotein E–deficient ( Apoe –/– ) mice fed a high-fat diet for 12 weeks. Lipoprotein fractions were quantified in the plasma of Apoe –/– mice by fast protein liquid chromatography. Uptake of DiI-labeled very low-density lipoprotein to adipose tissue was determined by 2-photon microscopy. We show that genetic deficiency of Cxcr7 increased neointima formation and lesional macrophage accumulation in hyperlipidemic mice after vascular injury. This was related to increased serum cholesterol levels and subsequent hyperlipidemia-induced monocytosis. Conversely, administration of the CXCR7 ligand CCX771 to Apoe –/– mice inhibited lesion formation and ameliorated hyperlipidemia after vascular injury and during atherosclerosis. Treatment with CCX771 reduced circulating very low-density lipoprotein levels but not low-density lipoprotein or high-density lipoprotein levels and increased uptake of very low-density lipoprotein into Cxcr7 -expressing white adipose tissue. This effect of CCX771 was associated with an enhanced lipase activity and reduced expression of Angptl4 in adipose tissue. Conclusions— CXCR7 regulates blood cholesterol by promoting its uptake in adipose tissue. This unexpected cholesterol-lowering effect of CXCR7 is beneficial for atherosclerotic vascular diseases, presumably via amelioration of hyperlipidemia-induced monocytosis, and can be augmented with a synthetic CXCR7 ligand.

Description: Background— Cardiac development is a complex process resulting in an integrated, multilineage tissue with developmental corruption in early embryogenesis leading to congenital heart disease. Interrogation of individual genes has provided the backbone for cardiac developmental biology, yet a comprehensive transcriptome derived from natural cardiogenesis is required to gauge innate developmental milestones. Methods and Results— Stage-specific cardiac structures were dissected from 8 distinctive mouse embryonic time points to produce genome-wide expressome analysis across cardiogenesis. With reference to this native cardiogenic expression roadmap, divergent induced pluripotent stem cell–derived cardiac expression profiles were mapped from procardiogenic 3-factor (SOX2, OCT4, KLF4) and less-cardiogenic 4-factor (plus c-MYC) reprogrammed cells. Expression of cardiac-related genes from 3-factor–induced pluripotent stem cell differentiated in vitro at days 5 and 11 and recapitulated expression profiles of natural embryos at days E7.5-E8.5 and E14.5-E18.5, respectively. By contrast, 4-factor–induced pluripotent stem cells demonstrated incomplete cardiogenic gene expression profiles beginning at day 5 of differentiation. Differential gene expression within the pluripotent state revealed 23 distinguishing candidate genes among pluripotent cell lines with divergent cardiogenic potentials. A confirmed panel of 12 genes, differentially expressed between high and low cardiogenic lines, was transformed into a predictive score sufficient to discriminate individual induced pluripotent stem cell lines according to relative cardiogenic potential. Conclusions— Transcriptome analysis attuned to natural embryonic cardiogenesis provides a robust platform to probe coordinated cardiac specification and maturation from bioengineered stem cell–based model systems. A panel of developmental-related genes allowed differential prognosis of cardiogenic competency, thus prioritizing cell lines according to natural blueprint to streamline functional applications.

Description: Objective— Collateral arteriogenesis, the growth of existing arterial vessels to a larger diameter, is a fundamental adaptive response that is often critical for the perfusion and survival of tissues downstream of chronic arterial occlusion(s). Shear stress regulates arteriogenesis; however, the arteriogenic significance of reversed flow direction, occurring in numerous collateral artery segments after femoral artery ligation, is unknown. Our objective was to determine if reversed flow direction in collateral artery segments differentially regulates endothelial cell signaling and arteriogenesis. Approach and Results— Collateral segments experiencing reversed flow direction after femoral artery ligation in C57BL/6 mice exhibit increased pericollateral macrophage recruitment, amplified arteriogenesis (30% diameter and 2.8-fold conductance increases), and remarkably permanent (12 weeks post femoral artery ligation) remodeling. Genome-wide transcriptional analyses on human umbilical vein endothelial cells exposed to reversed flow conditions mimicking those occurring in vivo yielded 10-fold more significantly regulated transcripts, as well as enhanced activation of upstream regulators (nuclear factor B [NFB], vascular endothelial growth factor, fibroblast growth factor-2, and transforming growth factor-β) and arteriogenic canonical pathways (protein kinase A, phosphodiesterase, and mitogen-activated protein kinase). Augmented expression of key proarteriogenic molecules (Kruppel-like factor 2 [KLF2], intercellular adhesion molecule 1, and endothelial nitric oxide synthase) was also verified by quantitative real-time polymerase chain reaction, leading us to test whether intercellular adhesion molecule 1 or endothelial nitric oxide synthase regulate amplified arteriogenesis in flow-reversed collateral segments in vivo. Interestingly, enhanced pericollateral macrophage recruitment and amplified arteriogenesis was attenuated in flow-reversed collateral segments after femoral artery ligation in intercellular adhesion molecule 1 –/– mice; however, endothelial nitric oxide synthase –/– mice showed no such differences. Conclusions— Reversed flow leads to a broad amplification of proarteriogenic endothelial signaling and a sustained intercellular adhesion molecule 1–dependent augmentation of arteriogenesis. Further investigation of the endothelial mechanotransduction pathways activated by reversed flow may lead to more effective and durable therapeutic options for arterial occlusive diseases.

Description: The insufficiency of compensatory angiogenesis in the heart of patients with hypertension contributes to heart failure transition. The hypoxia-inducible factor 1α–vascular endothelial growth factor (HIF1α–VEGF) signaling cascade controls responsive angiogenesis. One of the challenges in reprograming the insufficient angiogenesis is to achieve a sustainable tissue exposure to the proangiogenic factors, such as HIF1α stabilization. In this study, we identified Rnd3, a small Rho GTPase, as a proangiogenic factor participating in the regulation of the HIF1α–VEGF signaling cascade. Rnd3 physically interacted with and stabilized HIF1α, and consequently promoted VEGFA expression and endothelial cell tube formation. To demonstrate this proangiogenic role of Rnd3 in vivo, we generated Rnd3 knockout mice. Rnd3 haploinsufficient (Rnd3 +/– ) mice were viable, yet developed dilated cardiomyopathy with heart failure after transverse aortic constriction stress. The poststress Rnd3 +/– hearts showed significantly impaired angiogenesis and decreased HIF1α and VEGFA expression. The angiogenesis defect and heart failure phenotype were partially rescued by cobalt chloride treatment, a HIF1α stabilizer, confirming a critical role of Rnd3 in stress-responsive angiogenesis. Furthermore, we generated Rnd3 transgenic mice and demonstrated that Rnd3 overexpression in heart had a cardioprotective effect through reserved cardiac function and preserved responsive angiogenesis after pressure overload. Finally, we assessed the expression levels of Rnd3 in the human heart and detected significant downregulation of Rnd3 in patients with end-stage heart failure. We concluded that Rnd3 acted as a novel proangiogenic factor involved in cardiac responsive angiogenesis through HIF1α–VEGFA signaling promotion. Rnd3 downregulation observed in patients with heart failure may explain the insufficient compensatory angiogenesis involved in the transition to heart failure.

Description: Objective— Cathepsin S (CatS) participates in atherogenesis through several putative mechanisms. The ability of cathepsins to modify histone tail is likely to contribute to stem cell development. Histone deacetylase 6 (HDAC6) is required in modulating the proliferation and migration of various types of cancer cells. Here, we investigated the cross talk between CatS and HADC6 in injury-related vascular repair in mice. Approach and Results— Ligation injury to the carotid artery in mice increased the CatS expression, and CatS-deficient mice showed reduced neointimal formation in injured arteries. CatS deficiency decreased the phosphorylation levels of p38 mitogen-activated protein kinase, Akt, and HDAC6 and toll-like receptor 2 expression in ligated arteries. The genetic or pharmacological inhibition of CatS also alleviated the increased phosphorylation of p38 mitogen-activated protein kinase, Akt, and HDAC6 induced by platelet-derived growth factor BB in cultured vascular smooth muscle cells (VSMCs), and p38 mitogen-activated protein kinase inhibition and Akt inhibition decreased the phospho-HDAC6 levels. Moreover, CatS inhibition caused decrease in the levels of the HDAC6 activity in VSMCs in response to platelet-derived growth factor BB. The HDAC6 inhibitor tubastatin A downregulated platelet-derived growth factor–induced VSMC proliferation and migration, whereas HDAC6 overexpression exerted the opposite effect. Tubastatin A also decreased the intimal VSMC proliferation and neointimal hyperplasia in response to injury. Toll-like receptor 2 silencing decreased the phosphorylation levels of p38 mitogen-activated protein kinase, Akt, and HDAC6 and VSMC migration and proliferation. Conclusions— This is the first report detailing cross-interaction between toll-like receptor 2–mediated CatS and HDAC6 during injury-related vascular repair. These data suggest that CatS/HDAC6 could be a potential therapeutic target for the control of vascular diseases that are involved in neointimal lesion formation.

Description: Background Trimethylamine- N -oxide (TMAO), a metabolite derived from gut microbes and dietary phosphatidylcholine, is linked to both coronary artery disease pathogenesis and increased cardiovascular risks. The ability of plasma TMAO to predict 5-year mortality risk in patients with stable coronary artery disease has not been reported. This study examined the clinical prognostic value of TMAO in patients with stable coronary artery disease who met eligibility criteria for a patient cohort similar to that of the Clinical Outcomes Utilizing Revascularization and Aggressive Drug Evaluation (COURAGE) trial. Methods and Results We examined the relationship between fasting plasma TMAO and all-cause mortality over 5-year follow-up in sequential patients with stable coronary artery disease (n=2235) who underwent elective coronary angiography. We identified the COURAGE-like patient cohort as patients who had evidence of significant coronary artery stenosis and who were managed with optimal medical treatment. Higher plasma TMAO levels were associated with a 4-fold increased mortality risk. Following adjustments for traditional risk factors, high-sensitivity C-reactive protein, and estimated glomerular filtration rate, elevated TMAO levels remained predictive of 5-year all-cause mortality risk (quartile 4 versus 1, adjusted hazard ratio 1.95, 95% CI 1.33–2.86; P =0.003). TMAO remained predictive of incident mortality risk following cardiorenal and inflammatory biomarker adjustments to the model (adjusted hazard ratio 1.71, 95% CI 1.11–2.61; P =0.0138) and provided significant incremental prognostic value for all-cause mortality (net reclassification index 42.37%, P 〈0.001; improvement in area under receiver operator characteristic curve 70.6–73.76%, P 〈0.001). Conclusions Elevated plasma TMAO levels portended higher long-term mortality risk among patients with stable coronary artery disease managed with optimal medical treatment.