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4-Hydroxynonenal dependent alteration of TRPV1-mediated coronary microvascular signaling.

ABSTRACT: We demonstrated previously that TRPV1-dependent regulation of coronary blood flow (CBF) is disrupted in diabetes. Further, we have shown that endothelial TRPV1 is differentially regulated, ultimately leading to the inactivation of TRPV1, when exposed to a prolonged pathophysiological oxidative environment. This environment has been shown to increase lipid peroxidation byproducts including 4-Hydroxynonenal (4-HNE). 4-HNE is notorious for producing protein post-translation modification (PTM) via reactions with the amino acids: cysteine, histidine and lysine. Thus, we sought to determine if 4-HNE mediated post-translational modification of TRPV1 could account for dysfunctional TRPV1-mediated signaling observed in diabetes. Our initial studies demonstrate 4-HNE infusion decreases TRPV1-dependent coronary blood flow in C57BKS/J (WT) mice. Further, we found that TRPV1-dependent vasorelaxation was suppressed after 4-HNE treatment in isolated mouse coronary arterioles. Moreover, we demonstrate 4-HNE significantly inhibited TRPV1 currents and Ca2+ entry utilizing patch-clamp electrophysiology and calcium imaging respectively. Using molecular modeling, we identified potential pore cysteines residues that, when mutated, could restore TRPV1 function in the presence of 4-HNE. Specifically, complete rescue of capsaicin-mediated activation of TRPV1 was obtained following mutation of pore Cysteine 621. Finally, His tag pull-down of TRPV1 in HEK cells treated with 4-HNE demonstrated a significant increase in 4-HNE binding to TRPV1, which was reduced in the TRPV1 C621G mutant. Taken together these data suggest that 4-HNE decreases TRPV1-mediated responses, at both the in vivo and in vitro levels and this dysfunction can be rescued via mutation of the pore Cysteine 621. Our results show the first evidence of an amino acid specific modification of TRPV1 by 4-HNE suggesting this 4-HNE-dependent modification of TRPV1 may contribute to microvascular dysfunction and tissue perfusion deficits characteristic of diabetes.

SUBMITTER: DelloStritto DJ

PROVIDER: S-EPMC5490661 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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4-Hydroxynonenal dependent alteration of TRPV1-mediated coronary microvascular signaling.

DelloStritto Daniel J DJ Sinharoy Pritam P Connell Patrick J PJ Fahmy Joseph N JN Cappelli Holly C HC Thodeti Charles K CK Geldenhuys Werner J WJ Damron Derek S DS Bratz Ian N IN

Free radical biology & medicine 20160925

We demonstrated previously that TRPV1-dependent regulation of coronary blood flow (CBF) is disrupted in diabetes. Further, we have shown that endothelial TRPV1 is differentially regulated, ultimately leading to the inactivation of TRPV1, when exposed to a prolonged pathophysiological oxidative environment. This environment has been shown to increase lipid peroxidation byproducts including 4-Hydroxynonenal (4-HNE). 4-HNE is notorious for producing protein post-translation modification (PTM) via r ...[more]

PMID: 27682362

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Project description:BackgroundAccumulating evidence indicates that coronary microvascular dysfunction (CMD) caused by hypercholesterolemia can lead to myocardial ischemia, with or without obstructive atherosclerotic coronary artery disease. However, the molecular pathways associated with compromised coronary microvascular function before the development of myocardial ischemic injury remain poorly defined. In this study, we investigated the effects of hypercholesterolemia on the function and integrity of the coronary microcirculation in mice and the underlying mechanisms.Methods and resultsMice were fed a hypercholesterolemic Paigen's diet for 8 weeks. Echocardiography data showed that Paigen's diet caused CMD, characterized by significant reductions in coronary blood flow and coronary flow reserve, but did not affect cardiac remodeling or dysfunction. Immunofluorescence studies revealed that Paigen's diet-induced CMD was associated with activation of coronary arterioles inflammation and increased myocardial inflammatory cell infiltration. These pathological changes occurred in parallel with the upregulation of lysosomal signaling pathways in endothelial cells (ECs). Treating hypercholesterolemic mice with the cholesterol-lowering drug ezetimibe significantly ameliorated Paigen's diet-induced adverse effects, including hypercholesterolemia, steatohepatitis, reduced coronary flow reserve, coronary endothelial cell inflammation, and myocardial inflammatory cell infiltration. In cultured mouse cardiac ECs, 7-ketocholesterol increased mitochondrial reactive oxygen species and inflammatory responses. Meanwhile, 7-ketocholesterol induced the activation of transcriptional factor EB and lysosomal signaling in mouse cardiac ECs, whereas the lysosome inhibitor bafilomycin A1 blocked 7-ketocholesterol-induced transcriptional factor EB activation and exacerbated 7-ketocholesterol-induced inflammation and cell death. Interestingly, ezetimibe synergistically enhanced 7-ketocholesterol-induced transcriptional factor EB activation and attenuated 7-ketocholesterol-induced mitochondrial reactive oxygen species and inflammatory responses in mouse cardiac ECs.ConclusionsThese results suggest that CMD can develop and precede detectable cardiac functional or structural changes in the setting of hypercholesterolemia and that upregulation of transcriptional factor EB-mediated lysosomal signaling in endothelial cells plays a protective role against CMD.

Project description:BackgroundMicrovascular complications are associated with an overtly increased risk of adverse outcomes in patients with diabetes including coronary microvascular injury which manifested as disruption of adherens junctions between cardiac microvascular endothelial cells (CMECs). However, particular mechanism leading to diabetic coronary microvascular hyperpermeability remains elusive.MethodsExperimental diabetes was induced in mice with adipose tissue-specific Adipsin overexpression (AdipsinLSL/LSL-Cre) and their respective control (AdipsinLSL/LSL). In addition, cultured CMECs were subjected to high glucose/palmitic acid (HG + PA) treatment to simulate diabetes for a mechanistic approach.ResultsThe results showed that Adipsin overexpression significantly reduced cardiac microvascular permeability, preserved coronary microvascular integrity, and increased coronary microvascular density. Adipsin overexpression also attenuated cardiac dysfunction in diabetic mice. E/A ratio, an indicator of cardiac diastolic function, was improved by Adipsin. Adipsin overexpression retarded left ventricular adverse remodeling, enhanced LVEF, and improved cardiac systolic function. Adipsin-enriched exosomes were taken up by CMECs, inhibited CMECs apoptosis, and increased CMECs proliferation under HG + PA treatment. Adipsin-enriched exosomes also accelerated wound healing, rescued cell migration defects, and promoted tube formation in response to HG + PA challenge. Furthermore, Adipsin-enriched exosomes maintained adherens junctions at endothelial cell borders and reversed endothelial hyperpermeability disrupted by HG + PA insult. Mechanistically, Adipsin blocked HG + PA-induced Src phosphorylation (Tyr416), VE-cadherin phosphorylation (Tyr685 and Tyr731), and VE-cadherin internalization, thus maintaining CMECs adherens junctions integrity. LC-MS/MS analysis and co-immunoprecipitation analysis (Co-IP) unveiled Csk as a direct downstream regulator of Adipsin. Csk knockdown increased Src phosphorylation (Tyr416) and VE-cadherin phosphorylation (Tyr685 and Tyr731), while abolishing Adipsin-induced inhibition of VE-cadherin internalization. Furthermore, Csk knockdown counteracted Adipsin-induced protective effects on endothelial hyperpermeability in vitro and endothelial barrier integrity of coronary microvessels in vivo.ConclusionsTogether, these findings favor the vital role of Adipsin in the regulation of CMECs adherens junctions integrity, revealing its promises as a treatment target against diabetic coronary microvascular dysfunction. Graphical abstract depicting the mechanisms of action behind Adipsin-induced regulation of diabetic coronary microvascular dysfunction.

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4-Hydroxynonenal dependent alteration of TRPV1-mediated coronary microvascular signaling.

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4-Hydroxynonenal dependent alteration of TRPV1-mediated coronary microvascular signaling.

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