Cultured Rat Aortic Endothelial Cells Research Articles

Piezo1 channel activation stimulates ATP production through enhancing mitochondrial respiration and glycolysis in vascular endothelial cells.

Piezo1 channels are mechanosensitive cationic channels that are activated by mechanical stretch or shear stress. Endothelial Piezo1 activation by shear stress caused by blood flow induces ATP release from endothelial cells; however, the link between shear stress and endothelial ATP production is unclear. The mitochondrial respiratory function of cells was measured by using high-resolution respirometry system Oxygraph-2k. The intracellular Ca2+ concentration was evaluated by using Fluo-4/AM and mitochondrial Ca2+ concentration by Rhod-2/AM. The specific Piezo1 channel activator Yoda1 or its analogue Dooku1 increased [Ca2+ ]i in human umbilical vein endothelial cells (HUVECs), and both Yoda1 and Dooku1 increased mitochondrial oxygen consumption rates (OCRs) and mitochondrial ATP production in HUVECs and primary cultured rat aortic endothelial cells (RAECs). Knockdown of Piezo1 inhibited Yoda1- and Dooku1-induced increases of mitochondrial OCRs and mitochondrial ATP production in HUVECs. The shear stress mimetics, Yoda1 and Dooku1, and the Piezo1 knock-down technique also demonstrated that Piezo1 activation increased glycolysis in HUVECs. Chelating extracellular Ca2+ with EGTA or chelating cytosolic Ca2+ with BAPTA-AM did not affect Yoda1- and Dooku1-induced increases of mitochondrial OCRs and ATP production, but chelating cytosolic Ca2+ inhibited Yoda1- and Dooku1-induced increase of glycolysis. Confocal microscopy showed that Piezo1 channels are present in mitochondria of endothelial cells, and Yoda1 and Dooku1 increased mitochondrial Ca2+ in endothelial cells. Piezo1 channel activation stimulates ATP production through enhancing mitochondrial respiration and glycolysis in vascular endothelial cells, suggesting a novel role of Piezo1 channel in endothelial ATP production.

PS-BPB01-1: SPARC IS INDUCED AT THE EARLY STAGE OF VASCULAR ENDOTHELIAL DYSFUNCTION TO COMPENSATE INFLAMMATION IN DOCA-SALT HYPERTENSIVE RATS

Objective: Extracellular matrix (ECM) regulates various cellular functions as well as provides structural support. SPARC (secreted protein acidic and rich in cysteine) is one of the ECM components, which participates in various cellular functions, especially during morphogenesis and tissue remodeling. We have reported that 1) SPARC, which increases in accordance with renal injury, causes collagen deposition through ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin type 1 motif) production, and 2) renin-angiotensin system is placed in the upstream of SPARC overexpression in the hypertensive rat kidney. The present study investigated the roles for SPARC in vascular endothelial cell dysfunction and inflammation in the setting of hypertension. Design and method: Rats were treated with deoxycorticosterone acetate (DOCA) and 1% salt water for 0, 1, 2 or 3 weeks. Blood pressure was measured by tail-cuff methods, and thoracic aorta was isolated to examine acetylcholine-induced vasodilatory response, macrophage infiltration by immunohistochemistry, and protein expression of SPARC and ADAMTS1 by Western blotting. In the separate experiment, angiotensin II (10–7mol/L, 6hr) or SPARC recombinant protein (100nmol/L, 24hr) was treated to cultured rat aortic endothelial cells (RAECs) with/without siRNA targeting SPARC and ADAMTS1. MCP-1 and LOX-1 mRNA levels were measured by real-time RT-PCR. Result: DOCA-salt caused time-dependent increases in systolic blood pressure, impairment of endothelium-dependent vasodilation, and macrophage infiltration into the adventitial area of the aorta. The expression of SPARC in the DOCA-salt rat aorta was enhanced at week1 and week2 compared to week0. ADAMTS1 expression tended to increase from week1 and showed significant increases at week3. In RAECs, SPARC and ADAMTS1 gene knockdown aggravated angiotensin II-induced overexpression of LOX-1 and MCP-1 mRNA, indicating anti-inflammatory effects of SPARC. On the other hand, SPARC recombinant protein treatment induced the upregulation of LOX-1 and MCP-1. Conclusions: SPARC may be induced at the early stage of vascular injury to compensate inflammation under the existence of angiotensin II. We need further investigation into the diverse effects of SPARC and the relationship between SPARC and ADAMTS1.

Endothelial cell specific knockout of caveolin‐1 attenuates AngII/BAPN‐induced vascular remodeling in mice

Caveolin‐1 null mice (Cav1‐/‐) are protected from developing vascular diseases while mice expressing Cav1 solely in the endothelium are capable of disease progression. To explore potential mechanisms through which endothelial caveolin‐1 contribute to vascular disease, we subjected Cav1 fl/fl Cre ‐ and endothelial cell specific, caveolin‐1 knockout mice (Cav1 fl/fl Cre+) to Angiotensin II (AngII: infusion at 1ug/kg/min for 4 weeks) and β‐aminopropionitrile (BAPN: administered in drinking water at 0.625mg/ml for 2 weeks) to induce vascular remodeling associated with aortic aneurysm formation. We found that the inner aortic diameter significantly increased (>50%) in Cav1 fl/fl Cre‐ mice in response to AngII/BAPN. Both outer diameter and vessel wall thickness, while trending towards increased values, were not statistically different in these mice compared to aortas from saline infused mice. These remodeling events were not observed in EC‐Cav1 null mice. Immuno‐histochemical analysis of vessel cross‐sections revealed significant enhancement of macrophage accumulation (F4/80 staining) and expression of MMP2 only within the tunica media of Cav1 fl/fl Cre‐ aortas exposed to AngII/BAPN. In addition, we found that AngII/BAPN enhanced adhesion molecule expression (ICAM‐1/VCAM‐1) on the endothelial cell surface of these but not EC‐Cav1‐/‐ mice. This observation correlated with activation of the NFkB pathway in cultured rat aortic endothelial cells in response to AngII/BAPN, an event that was abated in cells depleted of Cav1 via pretreatment with a Cav‐1 miRNA. These findings indicate that endothelial cell caveolin‐1 plays a role in vascular disease through regulation of inflammatory processes that contribute to vascular remodeling.

Arginase inhibition by (−)-Epicatechin reverses endothelial cell aging

Endothelial dysfunction (EnD) occurs with aging and endothelial nitric oxide (NO) production by NO synthase (NOS) can be impaired. Low NO levels have been linked to increased arginase (Ar) activity as Ar competes with NOS for L-arginine. The inhibition of Ar activity can reverse EnD and (−)-epicatechin (Epi) inhibits myocardial Ar activity. In this study, through in silico modeling we demonstrate that Epi interacts with Ar similarly to its inhibitor Norvaline (Norv). Using in vitro and in vivo models of aging, we examined Epi and Norv-inhibition of Ar activity and its endothelium-protective effects. Bovine coronary artery endothelial cells (BCAEC) were treated with Norv (10 μM), Epi (1 μM) or the combination (Epi + Norv) for 48 h. Ar activity increased in aged BCAEC, with decreased NO generation. Treatment decreased Ar activity to levels seen in young cells. Epi and Epi + Norv decreased nitrosylated Ar levels by ~25% in aged cells with lower oxidative stress (~25%) (dihydroethidium) levels. In aged cells, Epi and Epi + Norv restored the eNOS monomer/dimer ratio, protein expression levels and NO production to those of young cells. Furthermore, using 18 month old rats 15 days of treatment with either Epi (1 mg/kg), Norv (10 mg/kg) or combo, decreased hypertension and improved aorta vasorelaxation to acetylcholine, blood NO levels and tetra/dihydribiopterin ratios in cultured rat aortic endothelial cells. In conclusion, results provide evidence that inhibiting Ar with Epi reverses aged-related loss of eNOS function and improves vascular function through the modulation of Ar and eNOS protein levels and activity.

Involvement of Senescence and Mitochondrial Fission in Endothelial Cell Pro-Inflammatory Phenotype Induced by Angiotensin II.

Angiotensin II (AngII) has a crucial role in cardiovascular pathologies, including endothelial inflammation and premature vascular aging. However, the precise molecular mechanism underlying aging-related endothelial inflammation induced by AngII remains elusive. Here, we have tested a hypothesis in cultured rat aortic endothelial cells (ECs) that the removal of AngII-induced senescent cells, preservation of proteostasis, or inhibition of mitochondrial fission attenuates the pro-inflammatory EC phenotype. AngII stimulation in ECs resulted in cellular senescence assessed by senescence-associated β galactosidase activity. The number of β galactosidase-positive ECs induced by AngII was attenuated by treatment with a senolytic drug ABT737 or the chemical chaperone 4-phenylbutyrate. Monocyte adhesion assay revealed that the pro-inflammatory phenotype in ECs induced by AngII was alleviated by these treatments. AngII stimulation also increased mitochondrial fission in ECs, which was mitigated by mitochondrial division inhibitor-1. Pretreatment with mitochondrial division inhibitor-1 attenuated AngII-induced senescence and monocyte adhesion in ECs. These findings suggest that mitochondrial fission and endoplasmic reticulum stress have causative roles in endothelial senescence-associated inflammatory phenotype induced by AngII exposure, thus providing potential therapeutic targets in age-related cardiovascular diseases.

Anti-streptococcal antibody and T-cell interactions with vascular endothelial cells initiate the development of rheumatic carditis.

The role of group A streptococcal and Streptococcus dysgalactiae subspecies equisimilis M-protein specific Abs and T-cells in endothelial cell activation was investigated using cultured rat aortic endothelial cells, and in a rat model of autoimmune valvulitis. Heat inactivated serum and mononuclear cells from streptococcal M-protein immunized rats independently induced upregulation of the endothelial cell adhesion molecules, vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 in cultured cells. We also observed T-cell migration across endothelial cell monolayers incubated with serum from M-protein-immunized rats. Furthermore, we observed VCAM-1 and ICAM-1 expression in the myocardium of rats injected with M-protein compared to control animals. These observations support the contention that initial interactions between streptococcal M-protein specific Abs and/or T-cells with the heart endothelium lead to endothelial cell activation followed by transmigration of M-protein specific T-cells into heart tissue leading to an inflammatory process that leads to carditis in rheumatic fever and rheumatic heart disease.

Exercise restores impaired endothelium-derived hyperpolarizing factor-mediated vasodilation in aged rat aortic arteries via the TRPV4-KCa2.3 signaling complex.

BackgroundAging leads to structural and functional changes in the vasculature characterized by arterial endothelial dysfunction and stiffening of large elastic arteries and is a predominant risk factor for cardiovascular disease, the leading cause of morbidity and mortality in modern societies. Although exercise reduces the risk of many age-related diseases, including cardiovascular disease, the mechanisms underlying the beneficial effects of exercise on age-related endothelial function fully elucidated.PurposeThe present study explored the effects of exercise on the impaired endothelium-derived hyperpolarizing factor (EDHF)–mediated vasodilation in aged arteries and on the involvement of the transient receptor potential vanilloid 4 (TRPV4) channel and the small-conductance calcium-activated potassium (KCa2.3) channel signaling in this process.MethodsMale Sprague-Dawley rats aged 19–21 months were randomly assigned to a sedentary group or to an exercise group. Two-month-old rats were used as young controls.ResultsWe found that TRPV4 and KCa2.3 isolated from primary cultured rat aortic endothelial cells pulled each other down in co-immunoprecipitation assays, indicating that the two channels could physically interact. Using ex vivo functional arterial tension assays, we found that EDHF-mediated relaxation induced by acetylcholine or by the TRPV4 activator GSK1016790A was markedly decreased in aged rats compared with that in young rats and was significantly inhibited by TRPV4 or KCa2.3 blockers in both young and aged rats. However, exercise restored both the age-related and the TRPV4-mediated and KCa2.3-mediated EDHF responses.ConclusionThese results suggest an important role for the TRPV4-KCa2.3 signaling undergirding the beneficial effect of exercise to ameliorate age-related arterial dysfunction.

Abstract P279: Regulation Of H 2 S Production By Cystathionine Gamma-lyase In Endothelial Cells.

Hydrogen sulfide (H 2 S) is the most recently described endothelium-derived vasodilator. In the endothelium, H 2 S is predominantly produced by cystathionine γ-lyase (CSE). We and others have previously shown CSE inhibition diminishes acetylcholine (ACh)-induced dilation in aortic ring segments. However, the mechanism of CSE regulation is not well-defined. The goal of this study was to identify endogenous regulators of CSE in endothelial cells. Previous reports suggest that [Ca 2+ ] and phosphorylation regulate CSE activity. We previously reported that there was an increase in H 2 S production in rat aortic endothelial cells loaded with an H 2 S fluorescence indicator, sulfide fluor-7 acetoxymethylester (SF7-AM, 10 μM) after exposure to either ACh (10 μM) or an H 2 S donor (NaHS, 10 μM). Pretreatment with the CSE inhibitor, β-cyanoalanine (BCA, 100 μM) prevented the ACh-induced but not the NaHS-induced increases in SF 7 flourescence. Our current study tested the hypothesis that increasing intracellular calcium stimulates CSE-dependent H 2 S production. Cultured rat aortic endothelial cells were loaded with SF 7 and a nuclear dye (Nuclear-ID Red DNA stain, to correct for cell number) and fluorescence was measured at baseline and in response to a calcium ionophore (ionomycin, 100 nM). Ionomycin increased H 2 S production (Control: 0.8293, +ionomycin: 1.0365, p = 0.0001, n=3 of 7-15 wells per replicate). Pretreatment with BCA significantly decreased ionomycin-induced H 2 S production (+ionomycin: 1.0365, +BCA: 0.6600, p < 0.0001, n=3 of 7-15 wells per replicate). Preliminary ongoing studies in pressurized mesenteric arteries suggest a tendency of CSE inhibition to reduce ACh-induced dilation (Control: 77.06%, +BCA: 42.04%, p=0.08, n=3-4 arteries per group). However, a larger sample size is needed. Taken together, these studies suggest that ACh elicits vasodilation through a Ca 2+ -dependent activation of CSE.

Neuregulin-1 attenuates stress-induced vascular senescence.

Cardiovascular ageing is a key determinant of life expectancy. Cellular senescence, a state of irreversible cell cycle arrest, is an important contributor to ageing due to the accumulation of damaged cells. Targeting cellular senescence could prevent age-related cardiovascular diseases. In this study, we investigated the effects of neuregulin-1 (NRG-1), an epidermal growth factor with cardioprotective and anti-atherosclerotic effects, on cellular senescence. Senescence was induced in cultured rat aortic endothelial cells (ECs) and aortic smooth muscle cells (SMCs) by 2 h exposure to 30 µM hydrogen peroxide (H2O2). Cellular senescence was confirmed after 72 h using senescence-associated-β-galactosidase staining (SA-β-gal), cell surface area, and western blot analyses of SA pathways (acetyl-p53, p21). Recombinant human NRG-1 (rhNRG-1, 20 ng/mL) significantly reduced H2O2-induced senescence, as shown by a lower number of SA-β-gal positive cells, smaller surface area and lower expression of acetyl-p53. In C57BL/6 male mice rendered diabetic with streptozotocin (STZ), rhNRG-1 attenuated cellular senescence in aortic ECs and SMCs. Next, we created mice with SMC-specific knockdown of the NRG-1 receptor ErbB4. Aortic SMCs isolated from SMC-specific ErbB4 deficient mice (ErbB4f/+ SM22α-Cre+) showed earlier cellular senescence in vitro compared with wild-type (ErbB4+/+ SM22α-Cre+) SMCs. Furthermore, when rendered diabetic with STZ, ErbB4f/+ SM22α-Cre+ male mice showed significantly more vascular senescence than their diabetic wild-type littermates and had increased mortality. This study is the first to explore the role of NRG-1 in vascular senescence. Our data demonstrate that NRG-1 markedly inhibits stress-induced premature senescence in vascular cells in vitro and in the aorta of diabetic mice in vivo. Consistently, deficiency in the NRG-1 receptor ErbB4 provokes cellular senescence in vitro as well as in vivo.

Matrine‐Type Alkaloids Inhibit Advanced Glycation End Products Induced Reactive Oxygen Species‐Mediated Apoptosis of Aortic Endothelial Cells In Vivo and In Vitro by Targeting MKK3 and p38MAPK Signaling

BackgroundThe matrine‐type alkaloids are bioactive components extracted from Sophora flavescens, which is used in treatment of diabetes mellitus in traditional Chinese medicine. Advanced glycation end products mediate diabetic vascular complications. This study was aimed to investigate the protective effects and molecular mechanisms of matrine‐type alkaloids on advanced glycation end products–induced reactive oxygen species–mediated endothelial apoptosis.Methods and ResultsRats aorta and cultured rat aortic endothelial cells were exposed to advanced glycation end products. Matrine‐type alkaloids, p38 mitogen‐activated protein kinase (MAPK) inhibitor, and small interference RNAs against p38 MAPK kinases MAPK kinase kinase (MKK)3 and MKK6 were administrated. Intracellular reactive oxygen species production, cell apoptosis, phosphorylation of MKKs/p38 MAPK, and expression levels of heme oxygenase/NADPH quinone oxidoreductase were assessed. The nuclear factor erythroid 2‐related factor 2 nuclear translocation and the binding activity of nuclear factor erythroid 2‐related factor 2 with antioxidant response element were also evaluated. Matrine‐type alkaloids suppressed intracellular reactive oxygen species production and inhibited endothelial cell apoptosis in vivo and in vitro by recovering phosphorylation of MKK3/6 and p38 MAPK, nuclear factor erythroid 2‐related factor 2 nuclear translocation, and antioxidant response element binding activity, as well as the expression levels of heme oxygenase/NADPH quinone oxidoreductase. p38 MAPK inhibitor treatment impaired the effects of matrine‐type alkaloids in vivo and in vitro. MKK3/6 silencing impaired the effects of matrine‐type alkaloids in vitro.ConclusionsMatrine‐type alkaloids exert endothelial protective effects against advanced glycation end products induced reactive oxygen species–mediated apoptosis by targeting MKK3/6 and enhancing their phosphorylation.

NFAT regulation of cystathionine γ-lyase expression in endothelial cells is impaired in rats exposed to intermittent hypoxia.

Sleep apnea is a risk factor for cardiovascular disease, and intermittent hypoxia (IH, 20 episodes/h of 5% O2-5% CO2 for 7 h/day) to mimic sleep apnea increases blood pressure and impairs hydrogen sulfide (H2S)-induced vasodilation in rats. The enzyme that produces H2S, cystathionine γ-lyase (CSE), is decreased in rat mesenteric artery endothelial cells (EC) following in vivo IH exposure. In silico analysis identified putative nuclear factor of activated T cell (NFAT) binding sites in the CSE promoter. Therefore, we hypothesized that IH exposure reduces Ca2+ concentration ([Ca2+]) activation of calcineurin/NFAT to lower CSE expression and impair vasodilation. In cultured rat aortic EC, inhibiting calcineurin with cyclosporine A reduced CSE mRNA, CSE protein, and luciferase activity driven by a full-length but not a truncated CSE promoter. In male rats exposed to sham or IH conditions for 2 wk, [Ca2+] in EC in small mesenteric arteries from IH rats was lower than in EC from sham rat arteries (Δfura 2 ratio of fluorescence at 340 to 380 nm from Ca2+ free: IH = 0.05 ± 0.02, sham = 0.17 ± 0.03, P < 0.05), and fewer EC were NFATc3 nuclear positive in IH rat arteries than in sham rat arteries (IH = 13 ± 3, sham = 59 ± 11%, P < 0.05). H2S production was also lower in mesenteric tissue from IH rats vs. sham rats. Endothelium-dependent vasodilation to acetylcholine (ACh) was lower in mesenteric arteries from IH rats than in arteries from sham rats, and inhibiting CSE with β-cyanoalanine diminished ACh-induced vasodilation in arteries from sham but not IH rats but did not affect dilation to the H2S donor NaHS. Thus, IH lowers EC [Ca2+], NFAT activity, CSE expression and activity, and H2S production while inhibiting NFAT activation lowers CSE expression. The observations that IH exposure decreases NFATc3 activation and CSE-dependent vasodilation support a role for NFAT in regulating endothelial H2S production.NEW & NOTEWORTHY This study identifies the calcium-regulated transcription factor nuclear factor of activated T cells as a novel regulator of cystathionine γ-lyase (CSE). This pathway is basally active in mesenteric artery endothelial cells, but, after exposure to intermittent hypoxia to mimic sleep apnea, nuclear factor of activated T cells c3 nuclear translocation and CSE expression are decreased, concomitant with decreased CSE-dependent vasodilation.

Gene Expression Analysis of Cultured Rat-Endothelial Cells after Nd:YAG Laser Irradiation by Affymetrix GeneChip Array.

Endothelial cells and dental pulp cells enhance osteo-/odontogenic and angiogenic differentiation. In our previous study, rat pulp cells migrated to Nd:YAG laser-irradiated endothelial cells in an insert cell culture system. The purpose of this study was to examine the possible changes in the gene expression of cultured rat aortic endothelial cells after Nd:YAG laser irradiation using affymetrix GeneChip Array. Total RNA was extracted from the cells at 5 h after laser irradiation. Gene expressions were evaluated by DNA array chip. Up-regulated genes were related to cell migration and cell structure (membrane stretch, actin regulation and junctional complexes), neurotransmission and inflammation. Heat-shock 70 kDa protein (Hsp70) was related to the development of tooth germ. This study offers candidate genes for understanding the relationship between the laser-stimulated endothelial cells and dental pulp cells.

低浓度胰蛋白酶原代培养大鼠血管内皮细胞实验方法研究

目的：探索改进一种简单、经济和高纯度的大鼠血管内皮细胞分离培养方法。方法：采用2%戊巴比妥钠麻醉Wistar大鼠，在无菌条件下截取主动脉约3~4 cm，再将主动脉血管外翻使内膜暴露，用丝线结扎动脉两端，以0.125%胰蛋白酶消化18~20 min，在倒置相差显微镜下直接观察细胞生长和细胞形态特征，分别应用免疫组化和免疫荧光方法检测第VIII因子相关抗原抗体结合情况，对培养传代的血管内皮细胞进行鉴定。结果：采用0.125%胰蛋白酶直接消化可获得高纯度的大鼠主动脉血管内皮细胞。经过3~4天培养，细胞呈集落样散在分布于六孔培养板底，到10~12天时，细胞贴壁增长至覆盖大部分培养板底，约85%的细胞汇合成单层，呈现典型的“鹅卵石”样内皮细胞特征；此外，免疫组化和免疫荧光方法检测第VIII因子相关抗原抗体结合情况，发现大多数细胞胞浆呈现棕黄色和绿色荧光的阳性反应，显示分离培养的细胞主要是血管内皮细胞。结论：本改良的细胞培养方法操作简便，只需浓度低至0.125%胰蛋白酶即可获取较高纯度的血管内皮细胞，不需要较昂贵的胶原酶及内皮细胞生长因子，与现有的其他培养方法相比更为经济实用。 Objective: To explore a simple, economic and highly purified modified method to isolate and cul-ture the endothelial cells of rat thoracic aorta. Methods: Under an aseptic condition, the thoracic aorta was harvested in a male adult Wistar rat anesthetized with 2% pentobarbital sodium. After having removed the peripheral connective tissue and fat, the thoracic aorta was turned inside out to expose its intima. Then, the thoracic aorta ends were ligated with the sterile silk thread, and thoracic aorta intima was digested with 0.125% trypsin for 18 min - 20 min in centrifugal tube. Cell growth and cell morphology was directly observed under inverted phase contrast microscope; immunohistochemistry and immunofluorescence method is applied to detect and identify if the primary culture rat aortic endothelia cells (RAECs) are the vascular endothelial cells by their combining reactivity with VIII factor related antigen antibody. Results: After 3 - 4 days, the cultured rat aortic endothelial cells were distributed on the basement of six-well plates in a cluster. 10 - 12 days later, the adherent cells spread to cover most bottom of culture plate with about 85% of the adherent cells in a confluent monolayer, and show a typical cobblestone appearance which is the characteristic of the vascular endothelial cells. In addition, immunohistochemical staining and mmunofluorescence staining with monoclonal anti-Factor-VIII antigen antibody in culturing RAECs were shown by the tan and green fluorescence positive reactive particulate matter found in most of the cell cytoplasm, suggesting that the primary culture RAECs are the vascular endothelial cells. Conclusions: Just using the concentrations as low as 0.125% trypsin, our modified cultural method of vascular endothelial cells is a convenient, economical and applicable one with a higher cell activity and purity, no needing the expensive collagen enzyme and endothelial cell growth factor.

Abstract 087: Mitochondrial Fission Inducer, Dynamin-Related Protein 1 (DRP1), is Required for Endothelial Inflammatory Responses

Among various cardiovascular diseases, hypertension (HTN) is considered to be a disease plagued by chronic low-grade inflammation associated with endothelial dysfunction. Interestingly, recent studies have identified mitochondrial adaptation and/or dysfunction as components to hypertensive vascular dysfunction. While mitochondria are indispensable to maintain cellular metabolism, they also participate in adaptive and maladaptive cell/tissue responses via several retro grade signaling pathways. DRP1 plays a major role in mitochondrial quality control. However, whether DRP1 is involved in mitochondrial dysfunction and endothelial inflammation during development of HTN remains unknown. In the present study, we tested the hypothesis that inflammatory stimuli, through DRP1-dependent mitochondrial alteration, enhance endothelial inflammation. In cultured rat aortic endothelial cells (RAECs), TNFα (10 μg/mL) transiently induced mitochondrial fission maximally at 3h which was inhibited using a mitochondrial fission inhibitor, Mdivi1 (10 μM) (0.16±0.04 vs 0.10±0.02 mitochondria fragmentation count with MitoTracker, p&lt;.01 ). TNFα and FCCP (a fission agonist, 10 μM) increased THP-1 monocyte adhesion to RAECs, which was also inhibited with Mdivi1 (256±17 vs 139±16 for TNFα, 238±30 vs 156±14 for FCCP, attached cells per field scanned, p&lt;.01 ). Likewise, mdivi1 and adenoviruses encoding siRNA for DRP1 or dominant-negative K38A DRP1 (50 moi) attenuated TNFα-induced VCAM-1 induction in RAECs. TNFα increased aerobic respiration, which was prevented by mdivi1 or ER stress inhibitor PBA (10 mM). Inhibition of ER stress, glycolysis or mitochondrial respiration using PBA, 2-DG (1 mg/mL) or oligomycin (1 μM) prevented VCAM-1 induction. However, suppression of TNFα-induced mitochondrial ROS production by mito-Tempo (25 nM) was unable to prevent VCAM-1 induction. In C57BL6 mice receiving AngII (1000 ng/kg/min, 2 weeks) infusion, treatment with Mdivi-1 (25 mg/kg ip every other day) or PBA (1g/kg/day) prevented vascular VCAM-1 induction. In conclusion, our data suggests a critical role for ER stress and subsequent functional and structural remodeling of mitochondria induced by DRP1 in mediating endothelial inflammatory activation in HTN.

Establishment and evaluation of oxidative stress injury model of in vitro rat aortic endothelial cells

To establish a model of oxidative stress injury in cultured rat aortic endothelial cells, and to provide a basis for the research of cell injury and apoptosis. The rats were decapitated to get the aorta in thoracic operation under aseptic conditions. By subculture after tissue block culture method to get sufficient aortic endothelial cells, cultured in 96-well plates or grow on cover glass for the following test. Without H2O2 group as a control group, with different doses of H2O2 (100,200,300,400,500 μmol/L) treated endothelialcells in 12 h to screen the optimal dose. Based on the results, with the same dose of H2O2 (100 or 200 μmol/L) acted on endothelial cells respectively in different time (3, 6, 9, 12 and 24 h) to screen the optimal duration. Each group was made in sextuplicate. The establishment of the model was evaluated by immunofluorescence,cell viability testing, biochemical indicators detection (lactate dehydrogenase(LDH), nitric oxide(NO), malondialdehyde(MDA), superoxidedismutase(SOD))and apoptosis index testing. Endothelial cells were cultured successfully and verified by immunofluorescence staining of intracellular antigen Ⅷ collagen. With the increase of H2O2 doses at the same action time 12 h, the cell viability was significantly decreased (77.63%±5.20% to 40.90%±2.10%). The same dose(100 μmol/L group and 200 μmol/L group)with the action time increasing, the cellviability was significantly decreased (100 μmol/L group was 86.83%±12.11% to 44.26%±5.70%, 200 μmol/L group was 78.28%±11.98% to 34.45%±5.87%). At dose of H2O2 was 100 μmol/L and treated in 3,6,9,12 and 24 h, LDH-L and MDA were significantly increased after 9 h while NO and SOD were significantly decreased. In H2O2 dose of 100 μmol/L and action time 12 h, flow cytometry showed endothelial cellapoptosis rate was 16.92%±2.37%, significantly higher than the control group of 2.68%±0.47%(P<0.01); TUNEL detected endothelial cell apoptosis index was17.65%±2.36%, which was significantly higher than that in the control group of 3.23%±0.57%(P<0.01). The method was successfullyestablished a model of oxidative stress injury in cultured rat aortic endothelial cells, explore the moderate conditions that induced cells injury and apoptosis which could be a basis for the research.

Black tea protects against hypertension-associated endothelial dysfunction through alleviation of endoplasmic reticulum stress.

Hypertensive patients have been found to be associated with elevated levels of homocysteine, known as hyperhomocysteinemia. Homocysteine (Hcy) can induce endoplasmic reticulum (ER) stress in endothelial cells. This study aims to investigate whether black tea (BT) protects against hypertension-associated endothelial dysfunction through alleviation of ER stress. Rat aortae and cultured rat aortic endothelial cells were treated with Hcy, BT extract, and theaflavin-3,3’-digallate (TF3). Male Sprague Dawley rats were infused with angiotensin II (Ang II) to induce hypertension and orally administrated with BT extract at 15 mg/kg/day for 2 weeks. Hcy impaired endothelium-dependent relaxations of rat aortae and led to ER stress in endothelial cells, which were ameliorated by co-incubation of BT extract and TF3. The blood pressure of Ang II-infused rats and plasma Hcy level were normalized by BT consumption. Impaired endothelium-dependent relaxations in renal arteries, carotid arteries and aortae, and flow-mediated dilatations in third-order mesenteric resistance arteries were improved. Elevations of ER stress markers and ROS level, plus down-regulation of Hcy metabolic enzymes in aortae from Ang II-infused rats were prevented by BT treatment. Our data reveal the novel cardiovascular benefits of BT in ameliorating vascular dysfunctions, providing insight into developing BT into beneficial dietary supplements in hypertensive patients.

Beneficial paracrine effects of adipocytes from obese rats on cultured endothelial cells

The purpose was to test the hypothesis that adipocytes from obese rats would exert pro-atherogenic paracrine effects on cultured endothelial cells compared to adipocytes from lean rats, and that the adverse obesity-associated paracrine effects of adipocytes would be more pronounced in visceral than subcutaneous adipose tissue. Epididymal and subcutaneous adipose tissues were harvested from 32-wk old obese Otsuka Long Evans Tokushima Fatty (OLETF) and lean Long Evans Tokushima Otsuka (LETO) rats. Cultured rat aortic endothelial cells were treated for 24 h with media conditioned with LETO subcutaneous adipocytes (LSA), OLETF subcutaneous adipocytes (OSA), LETO epididymal adipocytes (LEA), and OLETF epididymal adipocytes (OEA). The amount of key adipokines secreted by ATs was measured in the supernatant fluid with ELISA and mRNA levels of a number of pro- and anti-atherogenic genes were assessed in treated endothelial cells via quantitative real-time PCR. Compared to adipocytes from other depots, secretion of leptin and TNFα was highest and vaspin secretion was lowest from OEA. However, endothelial cells treated with OEA conditioned media exhibited lower expression of several pro-atherogenic genes. These data suggest that adipocytes isolated from obese visceral adipose tissue secrete some as-yet unidentified factor(s) that confers a beneficial effect on transcription of pro- and anti-atherogenic genes of endothelial cells.

Down-regulation of DDAH2 and eNOS induces endothelial dysfunction in sinoaortic-denervated rats

The aim of present study was to investigate whether downregulation of dimethylarginine dimethylaminohydrolase (DDAH2) and endothelial nitric oxide synthase (eNOS) induced endothelial dysfunction in sinoaortic-denervated (SAD) rats. SAD rats exhibited significantly higher blood pressure (BP) variability and markedly lower baroreflex sensitivity. However, there was no significant difference in BP between SAD rats and sham-operated rats. In SAD rats, ultrastructural analysis revealed that endothelial cells were degenerated and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) study showed that apoptotic aortic endothelial cells increased. Circulating angiotensinII (AngII), asymmetric dimethylarginine (ADMA) and malondialdehyde (MDA) levels in SAD rats were similar to sham-operated rats, but aortic AngII and MDA levels locally increased. Endothelium-mediated relaxation of thoracic aorta isolated from SAD rats was impaired compared to sham-operated rats, whereas the sodium nitroprusside-induced relaxation was quite similar. Western blotting results showed that DDAH2 and eNOS expressions decreased significantly in the aortae of SAD rats. Treatment of primary cultured rat aortic endothelial cells with AngII (1 μM) resulted in a marked reduction of DDAH2 and eNOS expressions, and coadministration of losartan (1 μM), an AT 1 receptor antagonist, abolished the effect. In conclusion, downregulation of DDAH2 and eNOS induced endothelial dysfunction in SAD rats. DDAH2 and eNOS may be the potential targets for treatment of endothelial dysfunction.

Inflammatory Markers in Isolated Endothelial Cells of the Spontaneously Hypertensive Rat

Besides an elevated blood pressure, the spontaneously hypertensive rat (SHR) has inflammation, extensive cell injury, microvascular and parenchymal cell dysfunction and vascular lesions. Current evidence in‐vivo shows that both excessive oxidative stress and proteolytic activity are involved, but their source is uncertain, We hypothesize that isolated endothelial cells of the SHR without the involvement of other sources (e.g. adrenal hormones) have elevated levels of oxygen free radical and degrading enzyme. To separate endothelial mechanisms from others, we isolated and cultured rat aortic endothelial cells (RAOEC) of adult SHR and control Wistar rats and propagated them with minimal loss of morphologic and phenotypic characteristics. The expression of oxidative enzymes (xanthine oxidase, XO; nicotinamide adenine dinucleotide phosphate, NADPH), apoptotic enzymes (BAX; BCl‐2) and matrix metalloproteinase (MMP‐9) was determined in cultured second passage RAOEC by immunoperoxidase detection of RAOEC using a standardized avidin/biotin immunolabeling technique and digitally measured by optical intensity. Compared to Wistar endothelium measurements, protein levels of XO, NADPH, MMP‐9 as well as the apoptotic marker BAX, were significantly enhanced in the SHR endothelium with attenuated BCL‐2 protein levels. These results suggest that even isolated aortic endothelium of the SHR has upregulation of proteins as seen previously in‐vivo that may promote inflammation and lesion formation and even in the absence of an elevated blood pressure.(Supported by: 1Grant of “Knowledge Innovation Project”, Academy of Science, China No. KJCX1‐SW‐07, 1Grant of IM0608, CAMS and 2NIH HL‐10081)

Thyroid hormone receptor‐beta promotes angiogenesis stimulating ERK phosphorylation

Deficient angiogenesis is one of the causes leading to tissue ischemia and dysfunction. Thyroid hormones have been known to possess angiogenic properties; however, the detailed mechanisms involved in this effect are unknown. The activation of the extracellular signal‐regulated kinase (ERK) by phosphorylation in the mitogen‐activated protein kinase (MAPK) pathway is known to directly link growth factor signaling and cell migration and proliferation. We hypothesized that the angiogenic effect of thyroid hormones is mediated by a specific thyroid hormone receptor (TR) linked to the activation of ERK in endothelial cells. We analyzed ERK phosphorylation in cultured rat aortic endothelial cells by Western blot after exposure to thyroid hormone (T3) or adenoviral expression of either TRα or TRβ. In addition, we investigated the effect of these treatments on aortic sprouting. Our results showed that T3 increased ERK phosphorylation in a dose response manner. Only TRβ induced ERK phosphorylation. Aortic sprouting was increased by T3 and TRβ expression. In conclusion, the angiogenic effect of thyroid hormone is mediated by specific activation of TRβ through activation of the MAPK pathway.