Mouse Coronary Endothelial Cells Research Articles

Abstract Or110: Inflammation-induced endothelial cell activation and angiogenic sprouting are downmodulated by ubiquitin-specific peptidase 20

The deubiquitinase ubiquitin-specific peptidase 20 (USP20) reverses lysine-63 ubiquitination of the signaling adaptor TRAF6 and suppresses nuclear-factor-κB (NFκB) activation induced by proatherogenic stimuli in vascular smooth muscle cells (SMCs). Dephosphorylation of USP20 at Ser 334 augments USP20/TRAF6 binding and USP20-mediated TRAF6 deubiquitination, consequently decreasing NFκB signaling. SMC USP20 activity also attenuates atherosclerosis in Ldlr -/- mice. NFκB is a key mediator of vascular endothelial cell (EC) activation and inflammation-driven angiogenesis, which promotes growth and rupture of atherosclerotic plaques. We tested the hypothesis that USP20 attenuates EC NFκB signaling and decreases angiogenesis, with both in vitro and ex vivo models to study the role of USP20 in EC activation and ensuing angiogenesis. We analyzed experimental data by one-way ANOVA followed by Holm-Šídák's multiple comparisons test. Cytokine-induced NFκB activity was elevated in primary ECs isolated from Usp20 -/- mice as compared with ECs from congenic wild type (WT) mice (N=3, p<0.001). Assessed by scratch-wound healing and spheroid assays, respectively, migration and angiogenesis of mouse coronary endothelial cells (MCECs) transduced with recombinant adenoviruses was (a) increased by inactive USP20 or phospho-mimetic USP20(S334D) (migration, N=4, P<0.05; angiogenesis, N=6, P<0.01), and (b) decreased by transduction with WT USP20 or phospho-resistant USP20(S334A) (migration, N=4, P< 0.05; angiogenesis, N=6, P<0.01). Additionally, chemical inhibition of NFκB activation with TPCA-1 (which inhibits the upstream kinase IKK-2) attenuated MCEC migration (N=4, P<0.0001) and angiogenesis (N=6, P<0.005), as compared with untreated cells. Angiogenesis assessed by aortic ring sprouting was increased in Usp20 -/- compared with WT specimens (males, N=3, P<0.0001; females, N=2, P<0.0001), and it was also suppressed by TPCA-1 (males, N=2, WT; N=3 Usp20 -/- , P<0.0001; females, N=2 WT; N=3, Usp20 -/- P<0.0005). Angiogenesis was enhanced in aortic rings from USP20(S334D) CRISPR/Cas9 gene-edited mice (males, N=3, P<0.01; females, N=3 WT & N=4, USP20(S334D) P<0.0001), but reduced in aortic rings from USP20(S334A) mice (males, N=3, P<0.05; females, N=3 WT & N=5 USP20(S334A) P<0.0001). We conclude that preserving EC USP20 activity by preventing phosphorylation of USP20 on Ser 334 diminishes endothelial activation and angiogenic sprouting, which can decrease atherosclerosis.

Exposure to the Dioxin-like Pollutant PCB 126 Afflicts Coronary Endothelial Cells via Increasing 4-Hydroxy-2 Nonenal: A Role for Aldehyde Dehydrogenase 2.

Exposure to environmental pollutants, including dioxin-like polychlorinated biphenyls (PCBs), play an important role in vascular inflammation and cardiometabolic diseases (CMDs) by inducing oxidative stress. Earlier, we demonstrated that oxidative stress-mediated lipid peroxidation derived 4-hydroxy-2-nonenal (4HNE) contributes to CMDs by decreasing the angiogenesis of coronary endothelial cells (CECs). By detoxifying 4HNE, aldehyde dehydrogenase 2 (ALDH2), a mitochondrial enzyme, enhances CEC angiogenesis. Therefore, we hypothesize that ALDH2 activation attenuates a PCB 126-mediated 4HNE-induced decrease in CEC angiogenesis. To test our hypothesis, we treated cultured mouse CECs with 4.4 µM PCB 126 and performed spheroid and aortic ring sprouting assays, the ALDH2 activity assay, and Western blotting for the 4HNE adduct levels and real-time qPCR to determine the expression levels of Cyp1b1 and oxidative stress-related genes. PCB 126 increased the gene expression and 4HNE adduct levels, whereas it decreased the ALDH2 activity and angiogenesis significantly in MCECs. However, pretreatment with 2.5 µM disulfiram (DSF), an ALDH2 inhibitor, or 10 µM Alda 1, an ALDH2 activator, before the PCB 126 challenge exacerbated and rescued the PCB 126-mediated decrease in coronary angiogenesis by modulating the 4HNE adduct levels respectively. Finally, we conclude that ALDH2 can be a therapeutic target to alleviate environmental pollutant-induced CMDs.

Abstract 14365: Identification of MicroRNAs and Their Target Genes in Microvascular Disease in Diabetes

Cardiovascular disease is the leading cause of death in patients with diabetes. Among coronary vascular defects, obstructive coronary artery disease (CAD) is the primary cause of cardiac mortality. However, there is growing evidence that patients with coronary microvascular disease (CMD, also known as non-obstructive coronary disease) also experience chest pain and occasional heart attack. Furthermore, recent studies show that diabetes is the risk factor of CMD. However, the molecular mechanisms by which diabetes develops CMD is not fully understood. MicroRNAs (miRNAs) are a non-coding small RNA that regulates gene expression at the post-transcriptional level, and the change of miRNA profile is implicated in many diseases. In this study, we identified the core miRNAs which are involved in the development of coronary endothelial dysfunction in diabetes. We used an inducible type 2 diabetic (T2D) mouse model generated by high-fat diet and a single injection of low-dose streptozotocin. Our T2D mice did not show detectable atherosclerotic plaque, but developed CMD evidenced by reduced coronary flow velocity reserve. We conducted a miRNA array using mouse coronary endothelial cells (MCECs) isolated from diabetic and control mice, and selected seven miRNAs based on the result. Next, real-time PCR assays were carried out with seven miRNAs and found that miR210 and miR342-5p were downregulated, and miR378 was upregulated in MCECs of diabetic mice compared to the control. To identify the target genes of miR342-5p and miR378, we inhibited miR342-5p or overexpressed miR378 in MCECs. After testing 93 genes that are involved in endothelial functions, we found that inhibition of miR342-5p decreased the levels of Atp2a3 , Opa1 , Sod3 , and Vegfb , whereas miR378 overexpression significantly decreased Aggf1, HK1, Mapk3, Pak1, Panx1, Stim1, Stim2, Vcam1, and Vegfb and increased Casp2 and Gja1 . Western blotting data revealed that the expression levels of Affg1, Opa1, and Pak1 were significantly decreased in the MCECs from diabetic mice compared to the control. In summary, these data suggest that miR342-5p and miR378 could be a potential therapeutic target for CMD in diabetic patients, and Affg1 Opa1 and Pak1 are the downstream mRNAs of miR342-5p or miR378.

Abstract 14425: Coronary Microvascular Disease in Type 2 Diabetes is Linked to the Upregulation of P53

Diabetes mellitus is a prominent risk factor of coronary microvascular disease (CMD) and the comorbidity of CMD in diabetic patients leads to an increasing occurrence of cardiac mortality. Increased protein O -linked β-N-acetylglucosaminylation ( O -GlcNAcylation), a post-translational modification of proteins, is implicated in the development of vascular complications in diabetes. Therefore, we investigated the molecular mechanisms in which excess protein O -GlcNAcylation advances the progression of CMD in diabetes through coronary endothelial cell dysfunction. We hypothesize that excess protein O -GlcNAcylation will up-regulate p53 which will induce the development of CMD. We had conducted in vivo and in vitro experiments in control mice, TALLYHO/Jng (TH) mice, a polygenic type 2 diabetic mouse model, and endothelial cell-specific O -GlcNAcase (OGA, a regulatory enzyme that catalyzes the removal of O- GlcNAc from proteins)-overexpressing TH mice. We found that compared to control mice, TH mice exhibited a decrease in coronary flow velocity reserve (CFVR, an indicator of coronary microvascular function), reduced capillary density, and increased endothelial cell apoptosis in the left ventricle. In addition, TH mice showed a significant increase of p53 protein expression in mouse coronary endothelial cells (MCECs) compared to control mice. Moreover, overexpression of OGA lowered protein O -GlcNAcylation and decreased p53 levels in MCECs. p53 inhibition with pifithrin-α restoratively increased CFVR and cardiac contractility in TH mice. From this data, we conclude OGA overexpression lowers p53 expression levels and thus attenuates endothelial cell apoptosis and restores CFVR and cardiac function in diabetes. The downregulation of p53 through OGA overexpression could be a potential therapeutic target for CMD in diabetic patients.

Orai-IGFBP3 signaling complex regulates high-glucose exposure-induced increased proliferation, permeability, and migration of human coronary artery endothelial cells

IntroductionDiabetes-associated endothelium dysfunction might be linked to disturbances in Ca2+ homeostasis. Our main objective is to reveal the potential mechanisms by which high-glucose (HG) exposure promotes increased proliferation of human...

Gap Junction Intercellular Communication and Coronary Microvascular Disease in Type 2 Diabetes

Recent reports demonstrate that diabetic patients with coronary microvascular disease (CMD, also known as non‐obstructive coronary artery disease) exhibit higher cardiac mortality than diabetic patients without CMD. However, the molecular mechanisms in which diabetes leads to CMD are not well understood. In this study, we examine the role of gap junction intercellular communication in the development of CMD in diabetes. We used type 2 diabetic (T2D) mice induced by a single injection of low‐dose streptozotocin (75 mg/kg) with a high‐fat diet (60 % kcal). Coronary flow velocity reserve (CFVR) was measured in mice to assess coronary microvascular function and T2D mice showed significant decrease in CFVR compared to control mice, suggesting that T2D mice are suffering from CMD. Mouse coronary endothelial cells (MCECs) isolated from T2D mice and human coronary endothelial cells (HCECs) treated with high‐glucose exhibited the reduction of gap junction activity compared to their controls. Gap junction is composed of connexins (Cxs) and coronary endothelial cells express Cx37, Cx40, Cx43, and Cx45. Among those Cxs, Cx40 mRNA level was significantly decreased in MCECs isolated from T2D mice. Furthermore, Cx40 protein levels were significantly decreased in MCECs from inducible T2D mice, MCECs from spontaneous T2D mice (Tallyho mice), and HCECs from T2D patients compared to those controls. Cx40 knockout mice exhibited lower CFVR which was accompanied by decreased capillary density in the left ventricle (LV), when compared to the wild‐type mice. The potential causes of decreased capillary density include attenuated endothelial migration/proliferation and increased endothelial apoptosis. In ex vivo, high‐glucose treatment impaired capillary network formation in HCECs and Cx40 overexpression restored the level of capillary network in high‐glucose treated HCECs. We also found that Cx40 overexpression in T2D mice increased capillary density in the LV and CFVR. These data suggest that the downregulation of Cx40 decreases capillary density in the heart and leads to CMD in diabetes. Therefore, Cx40 overexpression in ECs could be the potential therapy for CMD in diabetic patients.Support or Funding InformationThis work was supported by grants from the National Heart, Lung, and Blood Institute of the National Institutes of Health (HL142214 and HL146764 to A. Makino).

Chloroquine differentially modulates coronary vasodilation in control and diabetic mice.

Chloroquine is a traditional medicine to treat malaria. There is increasing evidence that chloroquine not only induces phagocytosis but regulates vascular tone. Few reports investigating the effect of chloroquine on vascular responsiveness of coronary arteries have been made. In this study, we examined how chloroquine affected endothelium-dependent relaxation in coronary arteries under normal and diabetic conditions. We isolated coronary arteries from mice and examined endothelium-dependent relaxation (EDR). Human coronary endothelial cells and mouse coronary endothelial cells isolated from control and diabetic mouse (TALLYHO/Jng [TH] mice, a spontaneous type 2 diabetic mouse model) were used for the molecular biological or cytosolic NO and Ca2+ measurements. Chloroquine inhibited endothelium-derived NO-dependent relaxation but had negligible effect on endothelium-derived hyperpolarization (EDH)-dependent relaxation in coronary arteries of control mice. Chloroquine significantly decreased NO production in control human coronary endothelial cells partly by phosphorylating eNOSThr495 (an inhibitory phosphorylation site of eNOS) and attenuating the rise of cytosolic Ca2+ concentration after stimulation. EDR was significantly inhibited in diabetic mice in comparison to control mice. Interestingly, chloroquine enhanced EDR in diabetic coronary arteries by, specifically, increasing EDH-dependent relaxation due partly to its augmenting effect on gap junction activity in diabetic mouse coronary endothelial cells. These data indicate that chloroquine affects vascular relaxation differently under normal and diabetic conditions. Therefore, the patients' health condition such as coronary macrovascular or microvascular disease, with or without diabetes, must be taken account into the consideration when selecting chloroquine for the treatment of malaria.

MicroRNA Profiling in Coronary Endothelial Cells in Type 2 Diabetic Mice

Coronary endothelial dysfunction is a major cause of cardiac ischemia and it is commonly seen in patients with diabetes. MicroRNAs (miRNAs) are small noncoding RNAs that modulate the expression of multiple mRNAs. There is increasing evidence showing that the change in miRNA expression is implicated in the development and/or progression of cardiovascular diseases. However, the pathological role of miRNAs in coronary endothelial dysfunction in type 2 diabetes (T2D) is still unknown. MicroRNA array was used to profile the expression level of miRNAs in coronary endothelial cells (ECs) isolated from control and T2D mice. Real time PCR analysis was performed to confirm the miRNA array results. Among 491 murine miRNAs tested, 64 up‐regulated and 20 down‐regulated miRNAs were identified and seven miRNAs (miR106b, 210, 221, 222, 342‐5p, 378, 705, fold change>1.25) were selected to confirm the data using real‐time PCR. Real time PCR data revealed that miR378 expression was significantly higher and miR210 and miR342‐5p expression levels were significantly lower in diabetic ECs than in control ECs. To identify the target genes of miR378 and 342‐5p, we transfected miR378 mimic or miRNA342‐5p inhibitor in mouse coronary ECs. We chose 91 genes that are highly expressed in ECs and play crucial roles in endothelial functions, including a) endothelium‐derived relaxing factors and their regulators; b) modifiers of cytosolic, mitochondrial, and endoplasmic Ca2+ concentration; and c) regulators of EC proliferation/migration/apoptosis. The real‐time PCR data showed that miR378 mimic overexpression altered mRNA levels of 11 genes and miR342‐5p inhibitor in 5 genes. Among those genes, Vegfb, Atp3a3, Stim2 were decreased in coronary ECs transfected with miRNA mimic or inhibitor and in coronary ECs in T2D mice compared to their controls. These data suggest that miR378 and/or miR342‐5p are a potential target to restore coronary endothelial dysfunction in diabetes.Support or Funding InformationThis work was supported by a grant from the National Heart, Lung, and Blood Institute of the National Institutes of Health (HL142214 to A. Makino).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Methylglyoxal accumulation de-regulates HoxA5 expression, thereby impairing angiogenesis in glyoxalase 1 knock-down mouse aortic endothelial cells

Impaired angiogenesis leads to long-term complications and is a major contributor of the high morbidity in patients with Diabetes Mellitus (DM). Methylglyoxal (MGO) is a glycolysis byproduct that accumulates in DM and is detoxified by the Glyoxalase 1 (Glo1). Several studies suggest that MGO contributes to vascular complications through mechanisms that remain to be elucidated. In this study we have clarified for the first time the molecular mechanism involved in the impairment of angiogenesis induced by MGO accumulation.Angiogenesis was evaluated in mouse aortic endothelial cells isolated from Glo1-knockdown mice (Glo1KD MAECs) and their wild-type littermates (WT MAECs). Reduction in Glo1 expression led to an accumulation of MGO and MGO-modified proteins and impaired angiogenesis of Glo1KD MAECs. Both mRNA and protein levels of the anti-angiogenic HoxA5 gene were increased in Glo1KD MAECs and its silencing improved both their migration and invasion. Nuclear NF-ĸB-p65 was increased 2.5-fold in the Glo1KD as compared to WT MAECs. Interestingly, NF-ĸB-p65 binding to HoxA5 promoter was also 2-fold higher in Glo1KD MAECs and positively regulated HoxA5 expression in MAECs. Consistent with these data, both the exposure to a chemical inhibitor of Glo1 “SpBrBzGSHCp2” (GI) and to exogenous MGO led to the impairment of migration and the increase of HoxA5 mRNA and NF-ĸB-p65 protein levels in microvascular mouse coronary endothelial cells (MCECs).This study demonstrates, for the first time, that MGO accumulation increases the antiangiogenic factor HoxA5 via NF-ĸB-p65, thereby impairing the angiogenic ability of endothelial cells.

Connexin 40 Plays a Crucial Role in the Myocardial Ischemia Reperfusion Injury of Type 2 Diabetic Mice

Type 2 diabetes serves as an independent risk factor for the development of ischemic heart disease and ischemic heart diseases is a leading cause of high mortality in diabetes. In addition to atherosclerosis, microvascular disease due to endothelial dysfunction is implicated in the development and progression of ischemic heart disease. Connexin 40 (Cx40) predominantly expresses in vascular endothelial cells (ECs) and regulates endothelium‐dependent vascular relaxation and angiogenesis. We previously proved that downregulation of Cx40 is associated with coronary endothelial cell dysfunction in type 1 diabetic mice, however the involvement of Cx40 in cardiac ischemia reperfusion (I/R) injury in diabetes has never been revealed. In this study, we explore the role of Cx40 in cardiac I/R injury using type 2 diabetic mice (T2D mice). T2D mice were generated by a single injection of low‐dose streptozotocin (75 mg/kg) with a high‐fat diet (60 % kcal). T2D mice exhibited a significant decline of cardiac contractile function and larger infarction size after I/R compared to the control mice. T2D mice also showed attenuated endothelium‐dependent vascular relaxation in coronary arteries, decreased capillary density and enhanced endothelial apoptosis in the myocardium. Mouse coronary endothelial cells (MCECs) isolated from T2D mice exhibited significantly lower expression of Cx40 protein than in control MCECs. Therefore, we next examined the myocardial endothelial function using Cx40 knockout (Cx40−/−) mice. Our results demonstrated that a) Cx40 deletion impaired endothelium‐dependent relaxation via diminishing endothelium‐dependent hyperpolarization‐induced relaxation in coronary arteries, b) capillary density was decreased in the myocardium of Cx40−/− mice, c) cardiac contractility was attenuated in Cx40−/− mice, and d) infarction size after I/R was significantly increased in Cx40−/− mice compared to wild type mice. In conclusion, our data suggest that CX40 is an important protein to maintain myocardial endothelial function and decreased CX40 level might be one of crucial mechanisms which alter cardiac functions and lead to more severe damage after cardiac I/R in T2D mice.Support or Funding InformationNIH/HL115578

Direct Implications for Caveolin‐1 In TRPV1 Mediated NO production: Elucidating A Mechanism Of Vasoreactivity

Caveolin‐1 (Cav‐1), a scaffolding protein responsible for the formation of caveolae, is ubiquitously expressed and prominently in endothelial cells. Moreover, Cav‐1 is known to associate with several ion channels including various Kv and TRP subtypes. It is well established that when Cav‐1 and endothelial nitric oxide synthase (eNOS) are bound, Cav‐1 exerts an inhibitory effect on eNOS activity and thus decreases NO production until a Ca2+ dependent signal untethers the proteins. Our lab and others have shown that TRPV1 regulates eNOS activity and NO production; specifically, via a calcium‐mediated AKT‐CaMKII‐AMPK signaling cascade. Therefore, we hypothesize that TRPV1, Cav‐1, and eNOS form a regulatory protein complex in endothelial cells responsible for the modulation of NO production. Examination of the TRPV1 amino acid sequence revealed 4 potential Cav‐1 binding motifs (where Ø represents any aromatic amino acid and X represents any amino acid): ØXXØXXXXØØ, ØXXØXXXØ, ØXXXØXXØ, and ØXXXØXØ. Computational modeling predicted an interaction between TRPV1 and Cav‐1. Confirmation of a direct TRPV1/Cav‐1 association was illustrated using Proximity Ligation Assay (PLA) in mouse coronary endothelial cells, which was increased following treatment with the TRPV1 agonist capsaicin. Furthermore, capsaicin treatment decreased the interaction between Cav‐1 and eNOS visualized with PLA, further defining the nature of this NO mediated cellular response. Similarly, NO production increased following capsaicin treatment. In conclusion, this is the first evidence of a direct interaction between TRPV1 and Cav‐1 and its implications in the regulation of NO production and vascular function.

Mitochondrial connexin40 regulates mitochondrial calcium uptake in coronary endothelial cells.

Connexins (Cxs) are a group of integral membrane proteins that can form gap junctions between adjacent cells. Recently, it was reported that Cx43 is expressed not only in the plasma membrane but also in the inner mitochondrial membrane and that it regulates mitochondrial functions. Cx40 is predominantly expressed in vascular endothelial cells (ECs) and plays an important role in the electrical propagation between ECs and endothelial/smooth muscle cells. However, it is unknown whether Cx40 is expressed in the mitochondria and what the role of mitochondrial Cx40 is in endothelial functions. We observed in coronary ECs that Cx40 protein was expressed in the mitochondria, as determined by Western blot and immunofluorescence studies. We found that mouse coronary ECs (MCECs) isolated from Cx40 knockout (Cx40 KO) mice exhibited significantly lower resting mitochondrial calcium concentration ([Ca2+]mito) than MCECs from wild-type (WT) mice. After increase in cytosolic Ca2+ concentration ([Ca2+]cyto) with cyclopiazonic acid, calcium uptake into the mitochondria was significantly attenuated in MCECs from Cx40 KO mice compared with WT MCECs. There was no difference in resting [Ca2+]cyto and store-operated calcium entry in MCECs from WT and Cx40 KO mice. We also detected a significant decrease in the concentration of mitochondrial reactive oxygen species (ROS) in Cx40 KO MCECs. Cx40 overexpression in ECs significantly increased resting [Ca2+]mito level and calcium uptake by mitochondria in response to increased [Ca2+]cyto and augmented mitochondrial ROS production. These data suggest that mitochondrial Cx40 contributes to the regulation of mitochondrial calcium homeostasis.

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washed and incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 104 cells per one heart with over 90% purity.

Isolation of Mouse Coronary Endothelial Cells

Endothelial cells line the inner wall of blood vessels and play an important role in the regulation of vascular tone, vascular permeability, and new vascular formation. Endothelial cell dysfunction is implicated in the development and progression of many cardiovascular diseases including ischemic heart disease. To examine the function and characterization of coronary endothelial cells, cell isolation is the first step and it requires high purity and quantity to conduct subsequent experiments. This protocol describes an efficient method to isolate adult mouse coronary endothelial cells. The mouse heart is dissected and minced into small pieces. After the digestion of the heart using dispase and collagenase II, cells are washedand incubated with magnetic beads which are conjugated with anti-CD31 antibody. The beads with endothelial cells are washed several times and are ready to use in various applications, including imaging and molecular biological experiments. Efficient isolation yields approximately 10(4) cells per one heart with over 90% purity.

4‐HNE modified TRPV1 dysfunction leads to loss of capsaicin‐mediated eNOS activity and microvascular dysfunction

Our lab has previously demonstrated enhanced 4‐hydroxynonenal (4‐HNE) post‐translational modification (PTM) of TRPV1 decreases TRPV1 functional expression and contributes to microvascular dysfunction in diabetes. Furthermore, recent studies have shown TRPV1 activation elicits eNOS activation and NO production. Accordingly, we hypothesize that the enhanced 4‐HNE‐mediated PTM of TRPV1 contributes to diabetic microvascular dysfunction in db/db mice via decreased eNOS activation and resultant signaling. Contrast echocardiography demonstrated that 4‐HNE decreased capsaicin mediated increases in myocardial blood flow and decreased capsaicin‐mediated relaxation in isolated coronary microvessels. TRPV1 functional analysis using calcium imaging revealed blunted capsaicin‐mediated calcium influx in the presence of 4‐HNE in isolated mouse coronary endothelial cells (MCECs). Finally, 4‐HNE treatment of MCECs decreased eNOS phosphorylation at Ser1197 leading to decreased measure in capsaicin‐mediated nitrate production. Single point mutation, of a pore region cysteine residue 621, rescued channel activity and preserved capsaicin‐mediated eNOS phosphorylation and NO production. These data suggest that TRPV1 is targeted by redox‐active substances that directly modulate channel activity at numerous sites in diabetes to decrease TRPV1 functional expression and contribute to microvascular dysfunction. The results obtained demonstrate a signaling pattern aberrant in TRPV1 regulation of vascular control in diabetes.

Differential regulation of TRPV1 channels by H2O2: implications for diabetic microvascular dysfunction.

We demonstrated previously that TRPV1-dependent coupling of coronary blood flow (CBF) to metabolism is disrupted in diabetes. A critical amount of H2O2 contributes to CBF regulation; however, excessive H2O2 impairs responses. We sought to determine the extent to which differential regulation of TRPV1 by H2O2 modulates CBF and vascular reactivity in diabetes. We used contrast echocardiography to study TRPV1 knockout (V1KO), db/db diabetic, and wild type C57BKS/J (WT) mice. H2O2 dose-dependently increased CBF in WT mice, a response blocked by the TRPV1 antagonist SB366791. H2O2-induced vasodilation was significantly inhibited in db/db and V1KO mice. H2O2 caused robust SB366791-sensitive dilation in WT coronary microvessels; however, this response was attenuated in vessels from db/db and V1KO mice, suggesting H2O2-induced vasodilation occurs, in part, via TRPV1. Acute H2O2 exposure potentiated capsaicin-induced CBF responses and capsaicin-mediated vasodilation in WT mice, whereas prolonged luminal H2O2 exposure blunted capsaicin-induced vasodilation. Electrophysiology studies re-confirms acute H2O2 exposure activated TRPV1 in HEK293A and bovine aortic endothelial cells while establishing that H2O2 potentiate capsaicin-activated TRPV1 currents, whereas prolonged H2O2 exposure attenuated TRPV1 currents. Verification of H2O2-mediated activation of intrinsic TRPV1 specific currents were found in isolated mouse coronary endothelial cells from WT mice and decreased in endothelial cells from V1KO mice. These data suggest prolonged H2O2 exposure impairs TRPV1-dependent coronary vascular signaling. This may contribute to microvascular dysfunction and tissue perfusion deficits characteristic of diabetes.

O-GlcNAcase overexpression reverses coronary endothelial cell dysfunction in type 1 diabetic mice.

Cardiovascular disease is the primary cause of morbidity and mortality in diabetes, and endothelial dysfunction is commonly seen in these patients. Increased O-linked N-acetylglucosamine (O-GlcNAc) protein modification is one of the central pathogenic features of diabetes. Modification of proteins by O-GlcNAc (O-GlcNAcylation) is regulated by two key enzymes: β-N-acetylglucosaminidase [O-GlcNAcase (OGA)], which catalyzes the reduction of protein O-GlcNAcylation, and O-GlcNAc transferase (OGT), which induces O-GlcNAcylation. However, it is not known whether reducing O-GlcNAcylation can improve endothelial dysfunction in diabetes. To examine the effect of endothelium-specific OGA overexpression on protein O-GlcNAcylation and coronary endothelial function in diabetic mice, we generated tetracycline-inducible, endothelium-specific OGA transgenic mice, and induced OGA by doxycycline administration in streptozotocin-induced type 1 diabetic mice. OGA protein expression was significantly decreased in mouse coronary endothelial cells (MCECs) isolated from diabetic mice compared with control MCECs, whereas OGT protein level was markedly increased. The level of protein O-GlcNAcylation was increased in diabetic compared with control mice, and OGA overexpression significantly decreased the level of protein O-GlcNAcylation in MCECs from diabetic mice. Capillary density in the left ventricle and endothelium-dependent relaxation in coronary arteries were significantly decreased in diabetes, while OGA overexpression increased capillary density to the control level and restored endothelium-dependent relaxation without changing endothelium-independent relaxation. We found that connexin 40 could be the potential target of O-GlcNAcylation that regulates the endothelial functions in diabetes. These data suggest that OGA overexpression in endothelial cells improves endothelial function and may have a beneficial effect on coronary vascular complications in diabetes.

Loss of functional endothelial connexin40 results in exercise-induced hypertension in mice.

During activity, coordinated vasodilation of microcirculatory networks with upstream supply vessels increases blood flow to skeletal and cardiac muscles and reduces peripheral resistance. Endothelial dysfunction in humans attenuates activity-dependent vasodilation, resulting in exercise-induced hypertension in otherwise normotensive individuals. Underpinning activity-dependent hyperemia is an ascending vasodilation in which the endothelial gap junction protein, connexin (Cx)40, plays an essential role. Because exercise-induced hypertension is proposed as a forerunner to clinical hypertension, we hypothesized that endothelial disruption of Cx40 function in mice may create an animal model of this condition. To this end, we created mice in which a mutant Cx40T152A was expressed alongside wildtype Cx40 selectively in the endothelium. Expression of the Cx40T152A transgene in Xenopus oocytes and mouse coronary endothelial cells in vitro impaired both electric and chemical conductance and acted as a dominant-negative against wildtype Cx40, Cx43, and Cx45, but not Cx37. Endothelial expression of Cx40T152A in Cx40T152ATg mice attenuated ascending vasodilation, without effect on radial coupling through myoendothelial gap junctions. Using radiotelemetry, Cx40T152ATg mice showed an activity-dependent increase in blood pressure, which was significantly greater than in wildtype mice, but significantly less than in chronically hypertensive, Cx40knockout mice. The increase in heart rate with activity was also greater than in wildtype or Cx40knockout mice. We conclude that the endothelial Cx40T152A mutation attenuates activity-dependent vasodilation, producing a model of exercise-induced hypertension. These data highlight the importance of endothelial coupling through Cx40 in regulating blood pressure during activity.

O‐GlcNacase overexpression restores coronary endothelial dysfunction in type 1 diabetic mice (1076.1)

Cardiac ischemia is a leading cause of morbidity and mortality in diabetes, and endothelial cell (EC) dysfunction is implicated in coronary arterial disease. O‐linked N‐acetyglucosamine (O‐GlcNAcylation) is a post‐translational modification that is tightly controlled by two enzymes: O‐GlcNAc transferase (OGT, catalyzes the addition of an O‐GlcNAc to proteins) and O‐GlcNAcase (GCA, removes GlcNAc from proteins). In this study, we examine the role of GCA in coronary endothelial dysfunction in type 1 diabetic mice.We generated EC‐specific, inducible GCA transgenic mice and induced diabetes in these mice with streptozotocin. In diabetic mouse coronary endothelial cells (MCECs), GCA expression was significantly decreased and protein O‐GlcNAcylation was increased. GCA overexpression in diabetic mice decreased O‐GlcNAcylation in MCECs. Capillary density in the left ventricle was decreased in diabetes, while GCA overexpression significantly increased the density to the control level. Endothelium‐dependent relaxation was attenuated in diabetic coronary arteries (CAs) and restored by GCA overexpression. These data suggest that elevated protein O‐GlcNAcylation due to decreased GCA protein expression in MCECs contributes to coronary endothelial dysfunction in diabetes, whereas overexpression of GCA in ECs improves endothelial function and may have a beneficial effect on coronary vascular complications in diabetes.Grant Funding Source: supported by NIH/NHLBI(HL115578)

Expression of dysfunctional connexin40 in vascular endothelium enhances myogenic constriction and reduces artery distensibility

Mutant forms of connexin40 (Cx40) are known to exist in the human population and increase the risk of cardiovascular diseases such as atrial fibrillation. We hypothesised that the existence of dysfunctional Cx40 proteins amongst the native Cx40 population in the vascular endothelium may also impair arterial function. To this end, we created a new transgenic mouse strain expressing a mutant Cx40 (Cx40T202S) specifically in the endothelium; leaving native Cx40 expression intact. The ability of this mutation to impair gap junction function was tested in Xenopus Oocytes and mouse coronary endothelial cells while its effect on arterial function was tested in isolated small mesenteric arteries using pressure myography. Cell coupling studies found that Cx40T202S could form electrically but not chemically patent gap junctions. Analysis of the myogenic response of arteries from Cx40T202S mice revealed increased sensitivity to intraluminal pressure compared to wild type (P<0.05). This effect coincided with a loss of basally activated endothelium dependent hyperpolarisation (EDH) while nitric oxide remained intact. Examination of passive arterial properties showed Cx40T202S arteries were significantly stiffer than wild type (P<0.05). We conclude that the presence of a mutant Cx40 in the vascular endothelium is sufficient to prevent basal EDH, and increase both myogenic sensitivity and arterial stiffness.