Modar Kassan

Endoplasmic Reticulum Stress Is Involved in Cardiac Damage and Vascular Endothelial Dysfunction in Hypertensive Mice

Objective—Cardiac damage and vascular dysfunction are major causes of morbidity and mortality in hypertension. In the present study, we explored the beneficial therapeutic effect of endoplasmic reticulum (ER) stress inhibition on cardiac damage and vascular dysfunction in hypertension. Methods and Results—Mice were infused with angiotensin II (400 ng/kg per minute) with or without ER stress inhibitors (taurine-conjugated ursodeoxycholic acid and 4-phenylbutyric acid) for 2 weeks. Mice infused with angiotensin II displayed an increase in blood pressure, cardiac hypertrophy and fibrosis associated with enhanced collagen I content, transforming growth factor-&bgr;1 (TGF-&bgr;1) activity, and ER stress markers, which were blunted after ER stress inhibition. Hypertension induced ER stress in aorta and mesenteric resistance arteries (MRA), enhanced TGF-&bgr;1 activity in aorta but not in MRA, and reduced endothelial NO synthase phosphorylation and endothelium-dependent relaxation (EDR) in aorta and MRA. The inhibition of ER stress significantly reduced TGF-&bgr;1 activity, enhanced endothelial NO synthase phosphorylation, and improved EDR. The inhibition of TGF-&bgr;1 pathway improved EDR in aorta but not in MRA, whereas the reduction in reactive oxygen species levels ameliorated EDR in MRA only. Infusion of tunicamycin in control mice induced ER stress in aorta and MRA, and reduced EDR by a TGF-&bgr;1–dependent mechanism in aorta and reactive oxygen species–dependent mechanism in MRA. Conclusion—ER stress inhibition reduces cardiac damage and improves vascular function in hypertension. Therefore, ER stress could be a potential target for cardiovascular diseases.

Interleukin-10 Released by CD4+CD25+ Natural Regulatory T Cells Improves Microvascular Endothelial Function Through Inhibition of NADPH Oxidase Activity in Hypertensive Mice

Objective—We previously demonstrated that a reduced number of CD4+CD25+-regulatory T cells (Tregs) was associated with microvascular dysfunction in hypertension. However, the underlying mechanism by which Tregs regulate vascular endothelial function remains unknown. Methods and Results—Control and interleukin (IL)-10−/− knockout mice were infused with angiotensin II (400 ng/kg/min) for 2 weeks (hypertensive [HT] and HT-IL-10−/−). Endothelium-dependent relaxation (EDR) in response to acetylcholine was significantly reduced in mesenteric resistance artery (MRA) from HT and HT-IL-10−/− compared with control and IL-10−/− mice. Importantly, the incubation of MRA from HT mice with the conditioned media of cultured Tregs, isolated from control mice, reduced NADPH oxidase activity and improved EDR, whereas no effect was observed in MRA from control mice incubated with the same media. These effects were reversed when MRAs were preincubated with IL-10 antibody or IL-10 receptor antagonist, whereas incubation with transforming growth factor-&bgr; receptor antagonist had no effect. The transfer of cultured Tregs, isolated from control mice, into HT-IL-10−/− mice reduced systolic blood pressure (SBP) and NADPH oxidase activity and improved EDR in MRA compared with untreated HT-IL-10−/− mice. In vivo treatment of HT mice with IL-10 (1000 ng/mouse) significantly reduced SBP and NADPH oxidase activity and improved EDR in MRA compared with untreated HT mice. The transfer of cultured Tregs, isolated from IL-10−/− mice, into HT mice did not reduce SBP or NADPH oxidase activity or improve EDR. The incubation of MRA from HT mice with apocynin improved EDR, whereas NADPH oxidase substrate attenuated EDR in MRA from control mice, which was reversed with exogenous IL-10. Conclusion—These data demonstrate that IL-10 released from Tregs attenuates NADPH oxidase activity, which is a critical process in the improvement of microvascular endothelial function in hypertension, suggesting that Tregs/IL-10 could be a therapeutic target for treatment of vasculopathy in hypertension.

Natural Regulatory T Cells Control Coronary Arteriolar Endothelial Dysfunction in Hypertensive Mice

Coronary artery disease in patients with hypertension is increasing worldwide and leads to severe cardiovascular complications. The cellular and molecular mechanisms that underlie this pathologic condition are not well understood. Experimental and clinical research indicates that immune cells and inflammation play a central role in the pathogenesis of cardiovascular diseases. Recently, it has been reported that CD4(+)CD25(+) regulatory T cells (Tregs) regulate heart fibrosis in hypertension. In this study, we determined the role of Tregs in coronary arteriolar endothelial dysfunction in angiotensin II-dependent hypertensive mice. Mice infused with angiotensin II had significantly increased blood pressure, as determined using telemetry, and apoptotic Treg numbers, as measured using flow cytometry. The mice displayed inflammation, assessed by macrophage activation/infiltration into coronary arterioles and the heart, and increased local tumor necrosis factor-α release, which participates in reduced coronary arteriolar endothelial-dependent relaxation in response to acetylcholine using an arteriograph. Hypertensive mice injected with Tregs isolated from control mice had significantly reduced macrophage activation and infiltration, reduced tumor necrosis factor-α release, and improved coronary arteriolar endothelium-dependent relaxation. Our novel data indicate that Tregs are important in the development of coronary arteriolar endothelial dysfunction in hypertension. These results suggest a new direction in the investigation of vascular disease in hypertension and could lead to a therapeutic strategy that involves immune system modulation using Tregs.

Mechanism of endoplasmic reticulum stress-induced vascular endothelial dysfunction.

BACKGROUND We recently reported that ER stress plays a key role in vascular endothelial dysfunction during hypertension. In this study we aimed to elucidate the mechanisms by which ER stress induction and oxidative stress impair vascular endothelial function. METHODOLOGY/PRINCIPAL FINDINGS We conducted in vitro studies with primary endothelial cells from coronary arteries stimulated with tunicamycin, 1μg/mL, in the presence or absence of two ER stress inhibitors: tauroursodeoxycholic acid (Tudca), 500μg/mL, and 4-phenylbutyric acid (PBA), 5mM. ER stress induction was assessed by enhanced phosphorylation of PERK and eIF2α, and increased expression of CHOP, ATF6 and Grp78/Bip. The ER stress induction increased p38 MAPK phosphorylation, Nox2/4 mRNA levels and NADPH oxidase activity, and decreased eNOS promoter activity, eNOS expression and phosphorylation, and nitrite levels. Interestingly, the inhibition of p38 MAPK pathway reduced CHOP and Bip expressions enhanced by tunicamycin and restored eNOS promoter activation as well as phosphorylation. To study the effects of ER stress induction in vivo, we used C57BL/6J mice and p47phox(-/-) mice injected with tunicamycin or saline. The ER stress induction in mice significantly impaired vascular endothelium-dependent and independent relaxation in C57BL/6J mice compared with p47phox(-/-) mice indicating NADPH oxidase activity as an intermediate for ER stress in vascular endothelial dysfunction. CONCLUSION/SIGNIFICANCE We conclude that chemically induced ER stress leads to a downstream enhancement of p38 MAPK and oxidative stress causing vascular endothelial dysfunction. Our results indicate that inhibition of ER stress could be a novel therapeutic strategy to attenuate vascular dysfunction during cardiovascular diseases.

A Novel Role for Epidermal Growth Factor Receptor Tyrosine Kinase and Its Downstream Endoplasmic Reticulum Stress in Cardiac Damage and Microvascular Dysfunction in Type 1 Diabetes Mellitus

Epidermal growth factor receptor tyrosine kinase (EGFRtk) and endoplasmic reticulum (ER) stress are important factors in cardiovascular complications. Understanding whether enhanced EGFRtk activity and ER stress induction are involved in cardiac damage, and microvascular dysfunction in type 1 diabetes mellitus is an important question that has remained unanswered. Cardiac fibrosis and microvascular function were determined in C57BL/6J mice injected with streptozotocin only or in combination with EGFRtk inhibitor (AG1478), ER stress inhibitor (Tudca), or insulin for 2 weeks. In diabetic mice, we observed an increase in EGFRtk phosphorylation and ER stress marker expression (CHOP, ATF4, ATF6, and phosphorylated-eIF2&agr;) in heart and mesenteric resistance arteries, which were reduced with AG1478, Tudca, and insulin. Cardiac fibrosis, enhanced collagen type I, and plasminogen activator inhibitor 1 were decreased with AG1478, Tudca, and insulin treatments. The impaired endothelium-dependent relaxation and -independent relaxation responses were also restored after treatments. The inhibition of NO synthesis reduced endothelium-dependent relaxation in control and treated streptozotocin mice, whereas the inhibition of NADPH oxidase improved endothelium-dependent relaxation only in streptozotocin mice. Moreover, in mesenteric resistance arteries, the mRNA levels of Nox2 and Nox4 and the NADPH oxidase activity were augmented in streptozotocin mice and reduced with treatments. This study unveiled novel roles for enhanced EGFRtk phosphorylation and its downstream ER stress in cardiac fibrosis and microvascular endothelial dysfunction in type 1 diabetes mellitus.

Enhanced NF-κB Activity Impairs Vascular Function Through PARP-1–, SP-1–, and COX-2–Dependent Mechanisms in Type 2 Diabetes

Type 2 diabetes (T2D) is associated with vascular dysfunction. We hypothesized that increased nuclear factor-κB (NF-κB) signaling contributes to vascular dysfunction in T2D. We treated type 2 diabetic (db−/db−) and control (db−/db+) mice with two NF-κB inhibitors (6 mg/kg dehydroxymethylepoxyquinomicin twice a week and 500 μg/kg/day IKK-NBD peptide) for 4 weeks. Pressure-induced myogenic tone was significantly potentiated, while endothelium-dependent relaxation (EDR) was impaired in small coronary arterioles and mesenteric resistance artery from diabetic mice compared with controls. Interestingly, diabetic mice treated with NF-κB inhibitors had significantly reduced myogenic tone potentiation and improved EDR. Importantly, vascular function was also rescued in db−/db−p50NF-κB−/− and db−/db−PARP-1−/− double knockout mice compared with db−/db− mice. Additionally, the acute in vitro downregulation of NF-κB–p65 using p65NF-κB short hairpin RNA lentivirus in arteries from db−/db− mice also improved vascular function. The NF-κB inhibition did not affect blood glucose level or body weight. The RNA levels for Sp-1 and eNOS phosphorylation were decreased, while p65NF-κB phosphorylation, cleaved poly(ADP-ribose) polymerase (PARP)-1, and cyclooxygenase (COX)-2 expression were increased in arteries from diabetic mice, which were restored after NF-κB inhibition and in db−/db−p50NF-κB−/− and db−/db−PARP-1−/− mice. In the current study, we provided evidence that enhanced NF-κB activity impairs vascular function by PARP-1–, Sp-1–, and COX-2–dependent mechanisms in male type 2 diabetic mice. Therefore, NF-κB could be a potential target to overcome diabetes-induced vascular dysfunction.

Chronic inhibition of endoplasmic reticulum stress and inflammation prevents ischaemia-induced vascular pathology in type II diabetic mice

Endoplasmic reticulum (ER) stress and inflammation are important mechanisms that underlie many of the serious consequences of type II diabetes. However, the role of ER stress and inflammation in impaired ischaemia‐induced neovascularization in type II diabetes is unknown. We studied ischaemia‐induced neovascularization in the hind‐limb of 4‐week‐old db − /db− mice and their controls treated with or without the ER stress inhibitor (tauroursodeoxycholic acid, TUDCA, 150 mg/kg per day) and interleukin‐1 receptor antagonist (anakinra, 0.5 µg/mouse per day) for 4 weeks. Blood pressure was similar in all groups of mice. Blood glucose, insulin levels, and body weight were reduced in db − /db− mice treated with TUDCA. Increased cholesterol and reduced adiponectin in db − /db− mice were restored by TUDCA and anakinra treatment. ER stress and inflammation in the ischaemic hind‐limb in db − /db− mice were attenuated by TUDCA and anakinra treatment. Ischaemia‐induced neovascularization and blood flow recovery were significantly reduced in db − /db− mice compared to control. Interestingly, neovascularization and blood flow recovery were restored in db − /db− mice treated with TUDCA or anakinra compared to non‐treated db − /db− mice. TUDCA and anakinra enhanced eNOS‐cGMP, VEGFR2, and reduced ERK1/2 MAP‐kinase signalling, while endothelial progenitor cell number was similar in all groups of mice. Our findings demonstrate that the inhibition of ER stress and inflammation prevents impaired ischaemia‐induced neovascularization in type II diabetic mice. Thus, ER stress and inflammation could be potential targets for a novel therapeutic approach to prevent impaired ischaemia‐induced vascular pathology in type II diabetes. Copyright

Poly(ADP-Ribose) Polymerase 1 Inhibition Improves Coronary Arteriole Function in Type 2 Diabetes Mellitus

Type 2 diabetes mellitus (T2DM) is associated with microvascular dysfunction. We hypothesized that increased poly(ADP-ribose) polymerase 1 (PARP-1) activity contributes to microvascular dysfunction in T2DM. T2DM (db−/db−) and nondiabetic control (db−/db+) mice were treated with 2 different PARP-1 inhibitors (INO-1001, 5 mg/kg per day and ABT-888, 15 mg/kg per day) for 2 weeks. Isolated coronary arterioles were mounted in an arteriograph. Pressure-induced myogenic tone was significantly potentiated, whereas endothelium-dependent relaxation was significantly attenuated in diabetic mice compared with control mice. These results were associated with decreased endothelial NO synthase phosphorylation and cGMP level and increased PARP-1 activity in coronary arterioles from diabetic mice compared with control mice. Interestingly, PARP-1 inhibitors significantly reduced the potentiation of myogenic tone, improved endothelium-dependent relaxation, restored endothelial NO synthase phosphorylation and cGMP, and attenuated cleaved PARP-1. These results were supported by in vitro studies indicating that downregulation of PARP-1 in mesenteric resistance arteries using PARP-1 short hairpin RNA lentiviral particles significantly improved endothelium-dependent relaxation in mesenteric resistance arteries from diabetic mice compared with control mice. The inhibition of NO synthesis by NG-nitro-L-arginine methyl ester (L-NAME) significantly reduced the endothelium-dependent relaxation in coronary arterioles and mesenteric resistance arteries from control and diabetic mice treated with PARP-1 inhibitors and PARP-1 short hairpin RNA lentiviral particles. In addition, we demonstrated that enhanced cleaved PARP-1, its binding to DNA, and DNA damage were reduced after PARP-1 inhibition in cultured endothelial cells stimulated with high glucose. We provide evidence that T2DM impairs microvascular function by an enhanced PARP-1 activity-dependent mechanism. Therefore, PARP-1 could be a potential target for overcoming diabetic microvascular complications.

CD4+CD25+Foxp3 regulatory T cells and vascular dysfunction in hypertension.

Endothelial dysfunction plays a key role in the development and progression of cardiovascular disease. In patients with hypertension, endothelial dysfunction is characterized by a decrease of vasodilator factors release. Recent evidence highlights the involvement of regulatory T cell in the cardiovascular physiology and pathology. An increasing body of data suggest that an imbalance in the immune system triggers inflammation and compromises the cardiovascular homeostasis. In this mini-review, we will highlight the role of immune regulatory T cells in hypertension-induced vascular dysfunction.

Vascular microRNA-204 is remotely governed by the microbiome and impairs endothelium-dependent vasorelaxation by downregulating Sirtuin1

Gut microbiota promotes atherosclerosis, and vascular endothelial dysfunction, signalled by impaired endothelium-dependent vasorelaxation, is an early marker of atherosclerosis. Here we show that vascular microRNA-204 (miR-204) expression is remotely regulated by the microbiome, and impairs endothelial function by targeting the Sirtuin1 lysine deacetylase (Sirt1). MiR-204 is downregulated, while Sirt1 is upregulated, in aortas of germ-free mice. Suppression of gut microbiome with broad-spectrum antibiotics decreases miR-204, increases Sirt1 and bioavailable vascular nitric oxide, and improves endothelium-dependent vasorelaxation in mouse aortas. Antibiotics curtail aortic miR-204 upregulation, and rescue decline of aortic Sirt1 and endothelium-dependent vasorelaxation, triggered by high-fat diet feeding. Improvement of endothelium-dependent vasorelaxation by antibiotics is lost in mice lacking endothelial Sirt1. Systemic antagonism of miR-204 rescues impaired endothelium-dependent vasorelaxation and vascular Sirt1, and decreases vascular inflammation induced by high-fat diet. These findings reveal a gut microbe-vascular microRNA–Sirtuin1 nexus that leads to endothelial dysfunction.