Mesenteric Artery Endothelial Cells Research Articles

Total flavone of flowers of Abelmoschus manihot (L.) Medic inhibits the expression of adhesion molecules in primary mesenteric arterial endothelial cells and ameliorates dextran sodium sulphate-induced ulcerative colitis in mice

BackgroundFlowers of Abelmoschus manihot (L.) medic (AM) is a traditional Chinese medicine used to treat chronic nephritis, nephrotic syndrome, diabetic nephropathy, and colonic inflammation. PurposeThis study aimed to explore the influence of the total flavone of AM flowers (TFA) on acute ulcerative colitis (UC) and the potential underlying mechanism. MethodsEfficacy of TFA (30, 60, 120 mg/kg) on UC was evaluated in a dextran sodium sulphate (DSS)-induced colonic inflammatory mouse model by analyzing disease activity index (DAI), histopathological score, colon length, and cytokine expression. Expression levels of critical adhesion molecules and nuclear factor kappa B (NF-κB) were examined by qRT-PCR, Western blotting, or immunofluorescence labeling. Myeloperoxidase activity was examined using ELISA. In vitro THP-1 adhesion assay was used to evaluate monocyte adhesion. ResultsTFA significantly reduced DAI score, prevented colon shortening, and ameliorated histological injuries of colons in DSS-treated mice. TFA inhibited the expression of cytokines (IL-1β and TNF-α) and adhesion molecules (ICAM-1, VCAM-1, and MAdCAM-1) in colon tissues of DSS mice. In vitro studies on mesenteric arterial endothelial cells (MAECs) showed that TFA attenuated TNF-α-induced upregulation of ICAM-1, VCAM-1, and MAdCAM-1, as well as THP-1 cell adhesion to MAECs. TFA also suppressed the phosphorylation and nuclear translocation of NF-κB in MAECs. ConclusionTFA efficaciously ameliorates UC possibly by inhibiting monocyte adhesion through blocking TNF-α-induced NF-κB activation, which in turn suppresses the upregulation of adhesive molecules in colon endothelial cells. Inhibiting the expression of adhesion molecule in MAECs may represent a useful strategy for therapeutic development to treat UC, with TFA being a safe and efficacious therapeutic agent.

Silybin induces endothelium-dependent vasodilation via TRPV4 channels in mouse mesenteric arteries.

Silybin is a flavonolignan extracted from the seeds of Silybum marianum that has been used as a dietary supplement for treating hepatic diseases and components of metabolic syndrome such as diabetes, obesity and hypertension. Transient receptor potential vanilloid 4 (TRPV4) channels are Ca2+-permeable, nonselective cation channels that regulate vascular endothelial function and blood flow. However, the relationship between silybin and TRPV4 channels in small mesenteric arteries remains unknown. In our study, we carried out a molecular docking experiment by using Discovery Studio v3.5 to predict the binding of silybin to TRPV4. Activation of TRPV4 with silybin was detected via intracellular Ca2+ concentration ([Ca2+]i) measurement and patch clamp experiments. The molecular docking results showed that silybin was likely to bind to the ankyrin repeat domain of TPRV4. [Ca2+]i measurements in mesenteric arterial endothelial cells (MAECs) and TRPV4-overexpressing HEK293 (TRPV4-HEK293) cells demonstrated that silybin induced Ca2+ influx by activating TRPV4 channels. The patch clamp experiments indicated that in TRPV4-HEK293 cells, silybin induced TRPV4-mediated cation currents. In addition, in high-salt-induced hypertensive mice, oral administration of silybin decreased systolic blood pressure (SBP) and significantly improved the arterial dilatory response to acetylcholine. Our findings provide the first evidence that silybin could induce mesenteric endothelium-dependent vasodilation and reduce blood pressure in high-salt-induced hypertensive mice via TRPV4 channels, thereby revealing the potential effect of silybin on preventing endothelial dysfunction-related cardiovascular diseases.

Abstract 045: Soluble Protein Oligomers Induce Endoplasmic Reticulum Stress In Endothelial Cells And Lead To Decreased Calcium Levels In The Endoplasmic Reticulum.

Soluble protein oligomers (SPOs) have powerful vascular effects suggesting a link between Alzheimer’s disease (AD) and cardiovascular diseases (CVD). SPOs accumulation induces endoplasmic reticulum (ER) stress hence cell death in several CVDs. Our previous data showed that in mesenteric resistance arteries (MRA), SPOs induce ER stress leading to pathologically exacerbated endothelium-dependent vasodilation. We therefore hypothesized that the SPOs will cause ER calcium dysregulation in endothelial cells isolated from MRA. To confirm the presence of ER stress and SPO subcellular localization, endothelial cells were treated with SPOs (0.1 υM) in the presence and absence of the ER stress inhibitor 4-PBA (2 mM) and vehicle (F-12 media). Immunostaining with KDEL antibody specific for ER resident chaperones Grp78/94 was performed for 19 hr prior to visualization. Endothelial cells treated with SPOs showed increased anti-KDEL fluorescence (AU: Vehicle: 4.74 ± 0.3 vs. SPO: 15.74 ± 1.7; n= 3; p= 0.018, one-way ANOVA). This is consistent with the increased Grp78/94 protein concentration during ER stress. Interestingly, 4-PBA treated cells reduced anti-KDEL fluorescence to the same level as the control (AU: Vehicle: 4.74 ± 0.3 vs. 4-PBA: 8.74 ± 2.9; n= 3; p= 0.39; one-way ANOVA) suggesting that SPOs bind to the ER-anchored proteins during ER stress. Additionally, changes in Ca 2+ concentrations have been implicated as a mediator between ER stress and SPOs. Calcium levels in endothelial cells was investigated by transfecting a plasmid expressing a Ca 2+ indicator pCMV G-CEPIA1 er for 72 hr . Cells were treated with vehicle or SPOs in the presence or absence of 4-PBA (45 min) prior to fluorescence imaging. Cells treated with SPOs+ACh had a decreased fluorescence in the ER from the basal Ca 2+ levels (%Δ Ff-F0/F0; SPO: 53% reduction vs. vehicle), but treatment with 4-PBA+ACh did not improve this response (%Δ F f -F 0 /F 0 ; SPO+4-PBA: 48.18% reduction vs. vehicle n=2). This reveals that SPOs induce ER stress and depletion of ER calcium. However, ER stress inhibitor did not improve Ca 2+ levels. These data suggest that ER stress, is the mechanism by which SPO’s exacerbate vasodilation in the mesenteric resistance artery endothelial cells regardless of changes in Ca 2+ levels.

Endothelial inwardly rectifying potassium channels regulates receptor‐mediated vasodilation via PI3K/Akt/eNOS signal pathway

We recently demonstrated that endothelial inwardly rectifying potassium (Kir) channels play a key role in flow‐induced vasodilation (FIV) of resistance arteries via regulating nitric oxide (NO) production with Akt/eNOS pathway. However, the role of endothelial Kir channels in receptor‐mediated vasodilation remains unknown. Therefore, we tested the role of Kir channels in receptor‐mediated vasodilation of mesenteric arteries. A significant reduction in the vasodilation to bradykinin was observed in mesenteric arteries from Kir2.1+/‐ mice, which we have shown earlier to lack functional expression of Kir2.1 in endothelial cells, as compared to arteries from WT mice. Moreover, blocking Kir channels with barium, Ba2+, significantly reduced bradykinin‐induced vasodilation in WT but not in Kir2.1+/‐ mice. Inhibition of eNOS withN(G)‐Nitro‐L‐arginine methyl ester (L‐NAME), which is known to reduce bradykinin‐induced vasodilation in WT mice, also had no effect in Kir2.1+/‐ mice. However, the vasodilation was completely blocked in Kir2.1+/‐ mice with apamin, a well‐known inhibitor of small‐conductance calcium activated potassium (SK) channels. These findings indicate that endothelial Kir2.1 channels play a key role in receptor‐mediated NO production, an effect that is independent of SK channel‐dependent dilation. In mechanistic terms, we tested the activation of PI3K/Akt1/eNOS pathway known to be a major contributor of receptor‐mediated NO production and vasodilation. In the mesenteric arterial endothelial cells (ECs) derived from Kir deficient mouse, there was no significant activation of PI3K, Akt, and eNOS in response to bradykinin (10‐6 M), whereas the ECs from WT mice showed significant activation in 3 minutes after the application of bradykinin. The application of adenoviral construct overexpressing WT Kir2.1 resulted in the recovery of the activation of PI3K/Akt/eNOS in Kir deficient ECs, whereas no further increase of activation in WT ECs was observed. In addition, bradykinin‐induced NO production was significantly reduced in Kir deficient ECs compared to WT ECs, and it was recovered by overexpressing WT Kir2.1. Taken together, we conclude that Kir2.1 channels regulate bradykinin‐induced NO production in ECs through PI3K/Akt/eNOS pathway, similar to our observations regarding flow‐induced vasodilation.

Isolation and Profiling of Human Primary Mesenteric Arterial Endothelial Cells at the Transcriptome Level.

Endothelial cells (ECs) are crucial for vascular and whole-body function through their dynamic response to environmental cues. Elucidating the transcriptome and epigenome of ECs is paramount to understanding their roles in development, health, and disease, but is limited in the availability of isolated primary cells. Recent technologies have enabled the high-throughput profiling of EC transcriptome and epigenome, leading to the identification of previously unknown EC cell subpopulations and developmental trajectories. While EC cultures are a useful tool in the exploration of EC function and dysfunction, the culture conditions and multiple passages can introduce external variables that alter the properties of native EC, including morphology, epigenetic state, and gene expression program. To overcome this limitation, the present paper demonstrates a method of isolating human primary ECs from donor mesenteric arteries aiming to capture their native state. ECs in the intimal layer are dissociated mechanically and biochemically with the use of particular enzymes. The resultant cells can be directly used for bulk RNA or single-cell RNA-sequencing or plated for culture. In addition, a workflow is described for the preparation of human arterial tissue for spatial transcriptomics, specifically for a commercially available platform, although this method is also suitable for other spatial transcriptome profiling techniques. This methodology can be applied to different vessels collected from a variety of donors in health or disease states to gain insights into EC transcriptional and epigenetic regulation, a pivotal aspect of endothelial cell biology.

Isolation and Profiling of Human Primary Mesenteric Arterial Endothelial Cells at the Transcriptome Level

Isolation and Profiling of Human Primary Mesenteric Arterial Endothelial Cells at the Transcriptome Level

Activation of Nav channels by veratridine in mesenteric arteries: Contractile or relaxation responses?

Veratridine (VTD), a selective activator of voltage-gated Na + (Nav) channels, triggers contractile responses in mesenteric arteries (MA), by increasing intracellular Ca2+ mediated by Na + /Ca 2+ exchanger (NCX) in smooth muscle cells (SMC). However, VTD induces endothelium-dependent relaxation of retinal and cremaster arteries. These opposite VTD-responses gives rise to the question as to whether Nav channels play a role in vascular tone. We aim to characterize the contribution of Nav channels to vascular tone in MA. Since Nav channels are expressed in SMC, endothelial cells (EC) and nerve terminal endings of arteries, our aim is also to identify those which are responsible of VTD-evoked vasomotor responses. The VTD-induced contractile responses of first order mesenteric arteries (FOMA) and cecolic arteries (CA) of mice were recorded by wire myography. EC were used to characterize Ca 2+ responses using FURA-2 probe. Western blot and absolute RT-qPCR were used to characterize Nav channels and NCX expression. Nav1.2 channels are expressed in both SMC and EC of MA. VTD elicits contraction of FOMA, which is abolished by prazosin. Surprisingly, VTD induces relaxation of CA. This vasorelaxation is resistant to prazosin and supressed by atropine. VTD-induced relaxation is inhibited by eNO-synthase inhibitor (L-NNA) and NCX antagonists. Morever, NCX1 is the main NCX isoform expressed in EC. Finally, acetylcholine-induced Ca 2+ reponses are inhibited by NCX antagonists in endothelial cells. VTD induces opposite contractile responses in isolated mesenteric arteries. In fact, VTD activates Nav channels of sympathetic neurons in FOMA, and parasympathetic neurons in CA, leading to noradrenaline and acetylcholine release, respectively. Thus, the vascular tone of both arteries are differently regulated by the autonomic nervous system. Finally, acetylcholine triggers Ca 2+ -activation of eNO-synthase mediated by NCX1 in CA.

Insulin Resistance and Pellino-1 Mediated Decrease in the Activities of Vasodilator Signaling Contributes to Sunitinib-Induced Hypertension.

Antiangiogenic tyrosine kinases inhibitors induce hypertension, which may increase the incidents of cardiovascular complications and limit their use. However, the mechanisms by which usage of TKIs results in hypertension have not been fully understood. Here, we report the potential mechanisms of how sunitinib, a widely used TKI, induces hypertension. Male SD rats were randomly divided into control group and sunitinib-administrated group. We show that sunitinib administration for seven days caused a significant increase in artery blood pressure, along with glycerolipid metabolism abnormalities including decreased food intake and low body weight, hypoglycemia, hyperinsulinemia. Sunitinib administration also resulted in a significant increase in the levels of insulin autoantibody (IAA), cyclic adenosine monophosphate and free fatty acid in serum; whereas, sunitinib administration had no effects on serum glucagon levels. Sunitinib led to the decreased insulin sensitivity as determined by insulin tolerance test (ITT) and glucose tolerance test (GTT), reflecting insulin resistance occurred in sunitinib-treated rats. The results obtained from wire myograph assay in the mesenteric arteries show that endothelium-dependent relaxation, but not endothelium-independent relaxation, was impaired by sunitinib. Furthermore, western blot analysis revealed that the expressions levels of phosphorylated IRS-1, Pellino-1, AKT and eNOS were significantly attenuated by sunitinib in rat mesenteric artery tissues and in the sunitinib-treated primary cultured mesenteric artery endothelial cells. The levels of serum and endothelium-derived nitric oxide were also significantly decreased by sunitinib. Moreover, sunitinib-induced decrease in the expression levels of phosphorylated AKT and eNOS was further reduced by knocking down of Pellino-1 in MAECs. Our results suggest that sunitinib causes vascular dysfunction and hypertension, which are associated with insulin resistance- and Pellino-1-mediated inhibition of AKT/eNOS/NO signaling. Our results may provide a rational for preventing and/or treating sunitinib-induced endothelial dysfunction and hypertension.

Curcumin Induces Endothelium-Dependent Relaxation by Activating Endothelial TRPV4 Channels.

It is well-known that curcumin, as a plant substance, has vascular protective effects. TRPV4 (transient receptor potential vanilloid 4) is a highly Ca2+-selective channel in vascular endothelium. In our study, fluorescent Ca2+ imaging in mesenteric arterial endothelial cells (MAECs) and overexpressed TRPV4 human embryonic kidney (HEK293) cells showed that curcumin dose-dependently stimulated Ca2+ influx. Whole-cell patch clamp proved that curcumin stimulated the TRPV4-mediated currents in TRPV4-HEK293 cells. The TRPV4-specific blocker HC067047 markedly decreased the whole-cell current. Molecular modeling and docking showed that the binding site of curcumin and TRPV4 was mainly in the amino acid sequence LYS340-LEU349 of TRPV4 protein. Furthermore, curcumin dose-dependently induced the endothelium-dependent vessel dilatation in small mesenteric arteries. Therefore, our results demonstrated that curcumin stimulates Ca2+ entry in endothelial cells and improves endothelium-dependent vessel relaxation by activating TRPV4 channels. Moreover, we identified the specific binding sites of curcumin and TRPV4, thereby highlighting its potential therapeutic target of cardiovascular diseases.

Morin induces endothelium-dependent relaxation by activating TRPV4 channels in rat mesenteric arteries

Morin, a natural flavonol, has been reported to have beneficial pharmacological effects. Although its vascular protective effects have been studied, little is known about its effects on the mesenteric artery and the underlying mechanisms. Transient receptor potential vanilloid type 4 (TRPV4) channels are one of the most important Ca2+-permeable cation channels in vascular endothelial cells and play an important role in regulating rat mesenteric vascular tone. In the present study, the myogenic effects of morin were investigated using wire and pressure myography in the isolated mesenteric artery. Morin induced endothelium-dependent relaxation of isolated rat mesenteric arteries in a concentration-dependent manner. In addition, morin stimulated relaxation by activating TRPV4-mediated Ca2+ influx without affecting the nitric oxide (NO), hydrogen peroxide (H2O2), cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) pathways. In primary cultured rat mesenteric artery endothelial cells and over-expressing TRPV4 HEK 293 cells, the TRPV4 inhibitor HC067047 significantly reduced the morin-induced increase in intracellular Ca2+ concentration. Furthermore, in rats with hypertension induced by NꞶ-nitro-L-arginine methyl ester (L-NAME), oral administration of morin (50 mg/kg/day) decreased systolic blood pressure. In L-NAME-induced hypertensive rats, morin significantly improved the relaxation response of the arteries to acetylcholine. Thus, we demonstrated that morin induces endothelium-dependent relaxation in the rat mesenteric artery by acting on TRPV4 channels to mediate Ca2+ influx and attenuate blood pressure in L-NAME-induced hypertension, thereby highlighting the potential of morin in the treatment of hypertension.

Stimulation of Muscarinic Receptors Activate Acid Sensing Ion Channel 1 in Isolated Mesenteric Artery Endothelial Tubes

Acid sensing ion channel 1 (ASIC1) belongs to the amiloride‐sensitive degenerin/epithelial sodium channel superfamily and is permeable to both Na+ and Ca2+. ASIC1 is expressed in vascular smooth muscle and/or endothelial cells of various vascular beds and is activated following stimulation of G protein‐coupled receptors (GPCR). In the pulmonary circulation, ASIC1 contributes to GPCR‐induced vasoconstriction and vascular smooth muscle Ca2+ influx via a store‐operated Ca2+ entry (SOCE) mechanism. In small mesenteric arteries, we have recently shown that ASIC1 contributes to acetylcholine (ACh)‐induced vasodilation and endothelial cell Ca2+ entry, however ASIC1‐mediated Ca2+ influx is not SOCE‐dependent in mesenteric artery endothelial cells. Thus, further investigation is needed to understand the mechanism by which ASIC1 is activated by GPCR in small mesenteric artery endothelial cells. Previous studies show ASIC1 is activated by arachidonic acid, therefore we hypothesized that acetylcholine stimulates ASIC1 in mesenteric artery endothelial cells through activation of phospholipase A2 (PLA2) and release of arachidonic acid. To test this hypothesis, we examined endothelial Ca2+ influx via Mn2+ quenching of fura‐2 fluorescence in freshly isolated mesenteric artery endothelial tubes. The PLA inhibitor methyl arachidonyl fluorophosphonate (MAF; 500 nM) attenuated the Ca2+ entry response to ACh, confirming that PLA2‐signaling contributes to the ACh‐induced Ca2+ entry in mesenteric artery endothelial cells. In addition, the selective inhibitor of ASIC1, psalmotoxin 1 (PcTX1; 20 nM), attenuated arachidonic acid‐induced Ca2+‐influx in mesenteric artery endothelial tubes. Although these studies suggest stimulation of muscarinic receptors activate ASIC1 through a PLA2‐arachidonic acid signaling mechanism in mesenteric endothelium, further studies are needed to identify the downstream effector of ASIC1.Support or Funding InformationSupported by National Heart, Lung, and Blood Institute Grants R01 HL‐111084 (to N.L. Jernigan), T32 HL007736 (to T.C. Resta), and American Heart Association Grant 18TPA34110281 (to N.L. Jernigan).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Heteromeric TRPV4/TRPC1 channels mediate calcium-sensing receptor-induced relaxations and nitric oxide production in mesenteric arteries: comparative study using wild-type and TRPC1−/- mice

ABSTRACTWe have previously provided pharmacological evidence that stimulation of calcium-sensing receptors (CaSR) induces endothelium-dependent relaxations of rabbit mesenteric arteries through activation of heteromeric TRPV4/TRPC1 channels and nitric oxide (NO) production. The present study further investigates the role of heteromeric TRPV4/TRPC1 channels in these CaSR-induced vascular responses by comparing responses in mesenteric arteries from wild-type (WT) and TRPC1-/- mice. In WT mice, stimulation of CaSR induced endothelium-dependent relaxations of pre-contracted tone and NO generation in endothelial cells (ECs), which were inhibited by the TRPV4 channel blocker RN1734 and the TRPC1 blocking antibody T1E3. In addition, TRPV4 and TRPC1 proteins were colocalised at, or close to, the plasma membrane of endothelial cells (ECs) from WT mice. In contrast, in TRPC1-/- mice, CaSR-mediated vasorelaxations and NO generation were greatly reduced, unaffected by T1E3, but blocked by RN1734. In addition, the TRPV4 agonist GSK1016790A (GSK) induced endothelium-dependent vasorelaxations which were blocked by RN1734 and T1E3 in WT mice, but only by RN1734 in TRPC1-/- mice. Moreover, GSK activated cation channel activity with a 6pS conductance in WT ECs but with a 52 pS conductance in TRPC1-/- ECs. These results indicate that stimulation of CaSR activates heteromeric TRPV4/TRPC1 channels and NO production in ECs, which are responsible for endothelium-dependent vasorelaxations. This study also suggests that heteromeric TRPV4-TRPC1 channels may form the predominant TRPV4-containing channels in mouse mesenteric artery ECs. Together, our data further implicates CaSR-induced pathways and heteromeric TRPV4/TRPC1 channels in the regulation of vascular tone.

TRPV4 is involved in irisin-induced endothelium-dependent vasodilation

Irisin, an exercise-induced myokine, induces conversion of white into brown adipocytes, promoting mitochondrial biogenesis and energy expenditure. Irisin has a vascular protective effect on endothelial function in animals, including humans. Defects in irisin signaling pathways result in endothelial dysfunction in obesity and diabetes. However, the mechanisms underlying the effects of irisin on endothelial function have not been elucidated. Transient receptor potential vanilloid subtype 4 (TRPV4) channels are one of the most important Ca2+-permeable cation channels in vascular endothelial cells. In this study, we hypothesized that irisin may induce endothelium-dependent vasodilation by activating Ca2+ influx into endothelial cells via TRPV4 channels. In primary cultured rat mesenteric artery endothelial cells, irisin caused an increase in [Ca2+]i due to extracellular Ca2+ influx rather than release from Ca2+ stores. Moreover, irisin-induced increases in [Ca2+]i were completely abolished by a TRPV4 inhibitor. In addition, irisin induced endothelium-dependent vasodilation of rat mesenteric arteries. However, irisin had no effect on endothelium-independent vasodilation. Furthermore, irisin-induced vasodilation was fully abolished in the presence of a TRPV4 inhibitor, indicating the involvement of TRPV4 channels in endothelium-dependent vasodilation. This study provides the first evidence that irisin-induced endothelium-dependent vasodilation is related to the stimulation of extracellular Ca2+ influx via TRPV4 channels in rat mesenteric arteries.

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.

Transient Receptor Potential Melastatin 4 (TRPM4) Contributes to High Salt Diet-Mediated Early-Stage Endothelial Injury

Background/Aims: The present study investigated whether the transient receptor potential melastatin 4 (TRPM4) channel plays a role in high salt diet (HSD)-induced endothelial injuries. Methods: Western blotting and immunofluorescence were used to examine TRPM4 expression in the mesenteric endothelium of Dahl salt-sensitive (SS) rats fed a HSD. The MTT, TUNEL, and transwell assays were used to evaluate the cell viability, cell apoptosis, and cell migration, respectively, of human umbilical vein endothelial cells (HUVECs). Enzyme-linked immunosorbent assays were used to determine the concentrations of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion protein 1 (VCAM-1), and E-selectin. Carboxy-H2DCFDA, a membrane-permeable reactive oxygen species (ROS)-sensitive fluorescent probe, was used to detect intracellular ROS levels. Results: TRPM4 was mainly expressed near the plasma membrane of mesenteric artery endothelial cells, and its expression level increased in SS hypertensive rats fed a HSD. Its protein expression was significantly upregulated upon treatment with exogenous hydrogen peroxide (H₂O₂) and aldosterone in cultured HUVECs. Cell viability decreased upon treatment with both agents in a concentration-dependent manner, which could be partially reversed by 9-phenanthrol, a specific TRPM4 inhibitor. Exogenous H₂O₂ induced apoptosis, enhanced cell migration, and increased the release of adhesion molecules, including ICAM-1, VCAM-1, and E-selectin, all of which were significantly attenuated upon treatment with 9-phenanthrol. Aldosterone and H₂O₂ induced the accumulation of intracellular ROS, which was significantly inhibited by 9-phenanthrol, suggesting that oxidative stress is one of the mechanisms underlying aldosterone-induced endothelial injury. Conclusions: Given the fact that oxidative stress and high levels of circulating aldosterone are present in hypertensive patients, we suggest that the upregulation of TRPM4 in the vascular endothelium may be involved in endothelial injuries caused by these stimuli.

Nobiletin Relaxes Isolated Mesenteric Arteries by Activating the Endothelial Ca2+-eNOS Pathway in Rats

Aims: Nobiletin, a natural polymethoxylated flavonoid compound, has various beneficial properties, such as anticancer, anti-inflammatory, and antioxidant effects, and it also improves memory. Therefore, we investigated the effects and mechanisms of nobiletin on rat isolated mesenteric arteries (MAs). Methods: We examined vasodilation induced by nobiletin on rat isolated MA rings with a tension study, the expression and activity of endothelial nitric oxide synthase (eNOS) with Western blotting, NO production with DAF- FMDA fluorescence, and intracellular Ca²⁺ concentration ([Ca²⁺]i) with Fluo-3AM. Results and Conclusion: Our study showed that nobiletin elicited a dose-dependent vasodilation in phenylephrine precontracted MA rings, but it did not elicit the same response in pulmonary artery rings. Vasodilation was related to endothelium and activated partly by eNOS. Vasodilation was inhibited by denudation of the endothelium or inhibition of eNOS activity. Nobiletin increased NO production by promoting the phosphorylation of eNOS at Ser-1177 without changing the level of eNOS expression in rat mesenteric artery endothelial cells (RMAECs). Nobiletin increased the concentration of endothelial [Ca²⁺]i, which enhances eNOS activity in RMAECs. In summary, vasodilation induced by nobiletin was dependent on the endothelium and partly on eNOS activation, which is mediated by high endothelial [Ca²⁺]i. Results suggest nobiletin may offer a therapeutic benefit and could potentially be used as a vasodilator for the treatment of hypertension.

Increasing TRPV4 expression restores flow-induced dilation impaired in mesenteric arteries with aging.

The flow-stimulated intracellular Ca2+ concentration ([Ca2+]i) rise in endothelial cells is an important early event leading to flow-induced blood vessel dilation. Transient receptor potential vanilloid subtype 4 (TRPV4), a Ca2+-permeable cation channel, facilitates the flow-stimulated [Ca2+]i rise. To determine whether TRPV4 is involved in age-related flow-induced blood vessel dilation impairment, we measured blood vessel diameter and nitric oxide (NO) levels and performed Ca2+ imaging, immunoblotting, and immunostaining assays in rats. We found that the flow-induced and TRPV4 activator 4α-PDD-induced dilation of mesenteric arteries from aged rats were significantly decreased compared with those from young rats. The flow- or 4α-PDD-induced [Ca2+]i rise was also markedly reduced in primary cultured mesenteric artery endothelial cells (MAECs) from aged rats. Immunoblotting and immunostaining results showed an age-related decrease of TRPV4 expression levels in MAECs. Additionally, the 4α-PDD-induced NO production was significantly reduced in aged MAECs. Compared with lentiviral GFP-treated aged rats, lentiviral vector delivery of TRPV4 increased TRPV4 expression level in aged MAECs and restored the flow- and 4α-PDD-induced vessel dilation in aged mesenteric arteries. We concluded that impaired TRPV4-mediated Ca2+ signaling causes endothelial dysfunction and that TRPV4 is a potential target for clinical treatment of age-related vascular system diseases.

Eugenol dilates mesenteric arteries and reduces systemic BP by activating endothelial cell TRPV4 channels.

Eugenol, a vanilloid molecule found in some dietary plants, relaxes vasculature in part via an endothelium-dependent process; however, the mechanisms involved are unclear. Here, we investigated the endothelial cell-mediated mechanism by which eugenol modulates rat mesenteric artery contractility and systemic BP. The isometric tension of rat mesenteric arteries (size 200-300 μm) was measured using wire myography; non-selective cation currents (ICat ) were recorded in endothelial cells using patch clamp electrophysiology. Mean arterial pressure (MAP) and heart rate (HR) were determined in anaesthetized rats. Eugenol relaxed endothelium-intact arteries in a concentration-dependent manner and this effect was attenuated by endothelium denudation. L-NAME, a NOS inhibitor, a combination of TRAM-34 and apamin, selective blockers of intermediate and small conductance Ca(2+) -activated K(+) channels, respectively, and HC-067047, a TRPV4 channel inhibitor, but not indomethacin, a COX inhibitor, reduced eugenol-induced relaxation in endothelium-intact arteries. Eugenol activated HC-067047-sensitive ICat in mesenteric artery endothelial cells. Short interfering RNA (siRNA)-mediated TRPV4 knockdown abolished eugenol-induced ICat activation. An i.v. injection of eugenol caused an immediate, transient reduction in both MAP and HR, which was followed by prolonged, sustained hypotension in anaesthetized rats. This sustained hypotension was blocked by HC-067047. Eugenol activates TRPV4 channels in mesenteric artery endothelial cells, leading to vasorelaxation, and reduces systemic BP in vivo. Eugenol may be therapeutically useful as an antihypertensive agent and is a viable molecular candidate from which to develop second-generation TRPV4 channel activators that reduce BP.

The GPR55 agonist lysophosphatidylinositol relaxes rat mesenteric resistance artery and induces Ca(2+) release in rat mesenteric artery endothelial cells.

Lysophosphatidylinositol (LPI), a lipid signalling molecule, activates GPR55 and elevates intracellular Ca(2+). Here, we examine the actions of LPI in the rat resistance mesenteric artery and Ca(2+) responses in endothelial cells isolated from the artery. Vascular responses were studied using wire myographs. Single-cell fluorescence imaging was performed using a MetaFluor system. Hypotensive effects of LPI were assessed using a Biopac system. In isolated arteries, LPI-induced vasorelaxation was concentration- and endothelium-dependent and inhibited by CID 16020046, a GPR55 antagonist. The CB1 receptor antagonist AM 251 had no effect, whereas rimonabant and O-1918 significantly potentiated LPI responses. Vasorelaxation was reduced by charybdotoxin and iberiotoxin, alone or combined. LPI decreased systemic arterial pressure. GPR55 is expressed in rat mesenteric artery. LPI caused biphasic elevations of endothelial cell intracellular Ca(2+). Pretreatment with thapsigargin or 2-aminoethoxydiphenyl borate abolished both phases. The PLC inhibitor U73122 attenuated the initial phase and enhanced the second phase, whereas the Rho-associated kinase inhibitor Y-27632 abolished the late phase but not the early phase. LPI is an endothelium-dependent vasodilator in the rat small mesenteric artery and a hypotensive agent. The vascular response involves activation of Ca(2+)-sensitive K(+) channels and is not mediated by CB1 receptors, but unexpectedly enhanced by antagonists of the 'endothelial anandamide' receptor. In endothelial cells, LPI utilizes PLC-IP3 and perhaps ROCK-RhoA pathways to elevate intracellular Ca(2+). Overall, these findings support an endothelial site of action for LPI and suggest a possible role for GPR55 in vasculature.

Attenuated store-operated Ca2+ entry in mesenteric artery endothelial cells of aged rat

Objective To observe attenuated store-operated Ca2+ entry in superior mesenteric artery endothelial cells of aged rat. Methods The pressure myograph was applied to measure the vasodilation of superior mesenteric artery induced by bradykinin (BK) in aged versus non-elder rats. Primarily cultured MAECs were treated by thapsigargin (TG) and BK to deplete intracellular Ca2+ stores for comparing SOCE. SOCE of MAECs was detected by calcium imaging technology and was compared between aged and non-elder rats. The expression levels of Orai 1 and stromal interaction factor 1 (STIM 1) were observed by immunofluorescence method. Results Compared with the non-elder rats (70.0±4.1%), BK-induced vasodilation in aged rats (27.0±4.9)% was declined by (60.7±4.3)%. SOCE induced by BK and TG in primarily cultured MAECs was attenuated by 37.6% and 39.2% in aged rats (1.71±0.18 and 1.06±0.03) respectively as compared with non-elder rats (2.72±0.39 and 1.75±0.06). The expressions of SOCE's components Orai 1 and STIM 1 in MAECs were decreased obviously in aged rats than in non-elder rats. Conclusions SOCE of MAECs and SOCE-induced vasodilation are significantly attenuated in aged rats. The decreased expression level of Orai 1 and STIM 1 may contribute to this alteration. Key words: Mesenteric artery superior; Endothelial cells; Calcium