Endothelial cell Orai1 is essential for endothelium-dependent contraction of mouse carotid arteries in normotensive and hypertensive mice.

Endothelium-dependent contraction (EDC) exists in blood vessels of normotensive animals, but is exaggerated in hypertension. An early signal in EDC is cytosolic Ca2+ rise in endothelial cells. In this study we investigated the functional role of Orai1, a major endothelial cell Ca2+ entry channel, in EDC. Hypertension model was established in WT mice by intake of L-NNA in the drinking water (0.5 g/L) for 4 weeks or osmotic pump delivery of Ang II (1.5 mg·kg-1·d-1) for 2 weeks. In TRPC5 KO mice, the concentration of L-NNA and Ang II were increased to 1 g/L or 2 mg·kg-1·d-1, respectively. Arterial segments were prepared from carotid arteries and aortas, and EDC was elicited by acetylcholine in the presence of Nω-nitro-L-arginine methyl ester. We showed that low concentration of acetylcholine (3-30 nM) initiated relaxation in phenylephrine-precontracted carotid arteries of both normotensive and hypertensive mice, while high concentration of acetylcholine (0.1-2 µM) induced contraction. Application of selective Orai1 inhibitors AnCoA4 (100 µM) or YM58483 (400 nM) had no effect on ACh-induced relaxation but markedly reduced acetylcholine-induced EDC. We found that EDC was increased in hypertensive mice compared with that of normotensive mice, which was associated with increased Orai1 expression in endothelial cells of hypertensive mice. Compared to TRPC5 and TRPV4, which were also involved in EDC, endothelial cell Orai1 had relatively greater contribution to EDC than either TRPC5 or TRPV4 alone. We identified COX-2, followed by PGF2α, PGD2 and PGE2 as the downstream signals of Orai1/TRPC5/TRPV4. In conclusion, Orai1 coordinates together with TRPC5 and TRPV4 in endothelial cells to regulate EDC responses. This study demonstrates a novel function of Orai1 in EDC in both normotensive and hypertensive mice, thus providing a general scheme about the control of EDC by Ca2+-permeable channels.

TRPM2 Promotes Atherosclerotic Progression in a Mouse Model of Atherosclerosis.

Atherosclerosis is a chronic inflammatory arterial disease characterized by build-up of atheromatous plaque, which narrows the lumen of arteries. Hypercholesterolemia and excessive oxidative stress in arterial walls are among the main causative factors of atherosclerosis. Transient receptor potential channel M2 (TRPM2) is a Ca2+-permeable cation channel activated by oxidative stress. However, the role of TRPM2 in atherosclerosis in animal models is not well studied. In the present study, with the use of adeno-associated virus (AAV)-PCSK9 and TRPM2 knockout (TRPM2-/-) mice, we determined the role of TRPM2 in hypercholesterolemia-induced atherosclerosis. Our results demonstrated that TRPM2 knockout reduced atherosclerotic plaque area in analysis of En face Oil Red O staining of both whole aortas and aortic-root thin sections. Furthermore, TRPM2 knockout reduced the expression of CD68, α-SMA, and PCNA in the plaque region, suggesting a role of TRPM2 in promoting macrophage infiltration and smooth-muscle cell migration into the lesion area. Moreover, TRPM2 knockout reduced the expression of ICAM-1, MCP-1, and TNFα and decreased the ROS level in the plaque region, suggesting a role of TRPM2 in enhancing monocyte adhesion and promoting vascular inflammation. In bone-marrow-derived macrophages and primary cultured arterial endothelial cells, TRPM2 knockout reduced the production of inflammatory cytokines/factors and decreased ROS production. In addition, a TRPM2 antagonist N-(p-amylcinnamoyl) anthranilic acid (ACA) was able to inhibit atherosclerotic development in an ApoE-/- mouse model of atherosclerosis. Taken together, the findings of our study demonstrated that TRPM2 contributes to the progression of hypercholesterolemia-induced atherosclerosis. Mechanistically, TRPM2 channels may provide an essential link that can connect ROS to Ca2+ and inflammation, consequently promoting atherosclerotic progression.

Endolysosomal ion channel MCOLN2 (Mucolipin-2) promotes prostate cancer progression via IL-1ß/NF-κB pathway.

BACKGROUND: Prostate cancer (Pca) is the most common cancer type among males worldwide. Dysregulation of Ca2+ signaling plays important roles during Pca progression. However, there is lack of information about the role of endolysosomal Ca2+ -permeable channels in Pca progression. METHODS: The expression pattern of MCOLN2 was studied by immunohistochemistry and western blot. Cell viability assay, transwell assay and in vivo tumorigenesis were performed to evaluate the functional role of MCOLN2. Downstream targets of MCOLN2 were investigated by cytokine array, enzyme-linked immunosorbent assay, Ca2+ release experiments and luciferase reporter assays. RESULTS: We report that MCOLN2 expression is significantly elevated in Pca tissues, and associated with poor prognosis. Overexpression of MCOLN2 promoted Pca cells proliferation, migration and invasion. Importantly, knockdown of MCOLN2 inhibited Pca xenograft tumor growth and bone lesion development in vivo. In addition, MCOLN2 promoted the production and release of IL-1ß. Moreover, luciferase reporter assay and western blot revealed that MCOLN2 promoted Pca development by regulating the IL-1ß/NF-κB pathway. CONCLUSION: In summary, MCOLN2 is crucially involved in Pca progression. Mechanistically, MCOLN2 regulates Pca progression via IL-1ß/NF-κB pathway. Our study highlights an intriguing possibility of targeting MCOLN2 as potential therapeutic strategy in Pca treatment.

Endothelial cell transient receptor potential channel C5 (TRPC5) is essential for endothelium-dependent contraction in mouse carotid arteries.

Augmented endothelium-dependent contractions (EDC) contributes to endothelial dysfunction and vascular disease progression. An early signal in EDC is cytosolic [Ca2+]i rise in endothelial cells, which stimulates the production of contractile prostanoids, leading to vascular contraction. In this study, the molecular identity of Ca2+-permeable channels in endothelial cells and its function were investigated. Vascular tension was measured by wire myograph. EDCs were elicited by acetylcholine (ACH) in the presence of NG-nitro-l-arginine methyl ester (L-NAME). [Ca2+]i was measured using a Ca2+-sensitive fluorescence dye. Enzyme Immunoassay (EIA) was used for prostaglandin measurement. Immunohistochemical staining found the expression of transient receptor potential channel C5 (TRPC5) in endothelial and smooth muscle cells of mouse carotid arteries. ACH-induced EDC in male mouse carotid arteries was found to be substantially reduced in TRPC5 knockout (KO) mice than in wild-type (WT) mice. TRPC5 inhibitors clemizole and ML204 also reduced the EDC. Furthermore, ACH-induced Ca2+ entry in endothelial cells was lower in TRPC5 KO mice than in WT mice. Moreover, the EDC was abolished by a cyclooxygenase-2 (COX-2) inhibitor NS-398, but not affected by a COX-1 inhibitor valeryl salicylate (VAS). Enzyme immunoassay results showed that TRPC5 stimulated the COX-2-linked production of prostaglandin F2α (PGF2α), prostaglandin E2 (PGE2), and prostaglandin D2 (PGD2). Exogeneous PGF2α, PGE2, and PGD2 could induce contractions in carotid arteries. Our present study demonstrated that TRPC5 in endothelial cells contributes to EDC by stimulating the production of COX-2-linked prostanoids. The finding extends our knowledge about EDC.

Author Correction: TRPC5 channels participate in pressure-sensing in aortic baroreceptors.

This corrects the article DOI: 10.1038/ncomms11947.

Gastrodin Inhibits Store-Operated Ca2+ Entry and Alleviates Cardiac Hypertrophy.

Cardiac hypertrophy is a major risk factor for heart failure, which are among the leading causes of human death. Gastrodin is a small molecule that has been used clinically to treat neurological and vascular diseases for many years without safety issues. In the present study, we examined protective effect of gastrodin against cardiac hypertrophy and explored the underlying mechanism. Phenylephrine and angiotensin II were used to induce cardiac hypertrophy in a mouse model and a cultured cardiomyocyte model. Gastrodin was found to alleviate the cardiac hypertrophy in both models. Mechanistically, gastrodin attenuated the store-operated Ca2+ entry (SOCE) by reducing the expression of STIM1 and Orai1, two key proteins in SOCE, in animal models as well as in cultured cardiomyocyte model. Furthermore, suppressing SOCE by RO2959, Orai1-siRNAs or STIM1-siRNAs markedly attenuated the phenylephrine-induced hypertrophy in cultured cardiomyocyte model. Together, these results showed that gastrodin inhibited cardiac hypertrophy and it also reduced the SOCE via its action on the expression of STIM1 and Orai1. Furthermore, suppression of SOCE could reduce the phenylephrine-induced cardiomyocyte hypertrophy, suggesting that SOCE-STIM1-Orai1 is located upstream of hypertrophy.

Aortic Baroreceptors Display Higher Mechanosensitivity than Carotid Baroreceptors.

Arterial baroreceptors are mechanical sensors that detect blood pressure changes. It has long been suggested that the two arterial baroreceptors, aortic and carotid baroreceptors, have different pressure sensitivities. However, there is no consensus as to which of the arterial baroreceptors are more sensitive to changes in blood pressure. In the present study, we employed independent methods to compare the pressure sensitivity of the two arterial baroreceptors. Firstly, pressure-activated action potential firing was measured by whole-cell current clamp with a high-speed pressure clamp system in primary cultured baroreceptor neurons. The results show that aortic depressor neurons possessed a higher percentage of mechano-sensitive neurons. Furthermore, aortic baroreceptor neurons show a lower pressure threshold than that of carotid baroreceptor neurons. Secondly, uniaxial stretching of baroreceptor neurons, that mimics the forces exerted on blood vessels, elicited a larger increase in intracellular Ca(2+) rise in aortic baroreceptor neurons than in carotid baroreceptor neurons. Thirdly, the pressure-induced action potential firing in the aortic depressor nerve recorded in vivo was also higher. The present study therefore provides for a basic physiological understanding on the pressure sensitivity of the two baroreceptor neurons and suggests that aortic baroreceptors have a higher pressure sensitivity than carotid baroreceptors.

TRPC5 channels participate in pressure-sensing in aortic baroreceptors.

Blood pressure is maintained within a normal physiological range by a sophisticated regulatory mechanism. Baroreceptors serve as a frontline sensor to detect the change in blood pressure. Nerve signals are then sent to the cardiovascular control centre in the brain in order to stimulate baroreflex responses. Here, we identify TRPC5 channels as a mechanical sensor in aortic baroreceptors. In Trpc5 knockout mice, the pressure-induced action potential firings in the afferent nerve and the baroreflex-mediated heart rate reduction are attenuated. Telemetric measurements of blood pressure demonstrate that Trpc5 knockout mice display severe daily blood pressure fluctuation. Our results suggest that TRPC5 channels represent a key pressure transducer in the baroreceptors and play an important role in maintaining blood pressure stability. Because baroreceptor dysfunction contributes to a variety of cardiovascular diseases including hypertension, heart failure and myocardial infarction, our findings may have important future clinical implications.

An upregulation in the expression of vanilloid transient potential channels 2 enhances hypotonicity-induced cytosolic Ca²âº rise in human induced pluripotent stem cell model of Hutchinson-Gillford Progeria.

Hutchinson-Gillford Progeria Syndrome (HGPS) is a fatal genetic disorder characterized by premature aging in multiple organs including the skin, musculoskeletal and cardiovascular systems. It is believed that an increased mechanosensitivity of HGPS cells is a causative factor for vascular cell death and vascular diseases in HGPS patients. However, the exact mechanism is unknown. Transient receptor potential (TRP) channels are cationic channels that can act as cellular sensors for mechanical stimuli. The aim of this present study was to examine the expression and functional role of TRP channels in human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from the patients with HGPS. The mRNA and protein expression of TRP channels in HGPS and control (IMR90) iPSC-ECs were examined by semi-quantitative RT-PCRs and immunoblots, respectively. Hypotonicity-induced cytosolic Ca²âº ([Ca²âº](i)) rise in iPSC-ECs was measured by confocal microscopy. RT-PCRs and immunoblots showed higher expressional levels of TRPV2 in iPSC-ECs from HGPS patients than those from normal individuals. In functional studies, hypotonicity induced a transient [Ca²âº](i) rise in iPSC-ECs from normal individuals but a sustained [Ca²âº](i) elevation in iPSC-ECs from HGPS patients. A nonselective TRPV inhibitor, ruthenium red (RuR, 20 µM), and a specific TRPV2 channel inhibitor, tranilast (100 µM), abolished the sustained phase of hypotonicity-induced [Ca²âº](i) rise in iPSC-ECs from HGPS patients, and also markedly attenuated the transient phase of the [Ca²âº](i) rise in these cells. Importantly, a short 10 min hypotonicity treatment caused a substantial increase in caspase 8 activity in iPSC-ECs from HGPS patients but not in cells from normal individuals. Tranilast could also inhibit the hypotonicity-induced increase in caspase 8 activity. Taken together, our data suggest that an up-regulation in TRPV2 expression causes a sustained [Ca²âº](i) elevation in HGPS-iPSC-ECs under hypotonicity, consequently resulting in apoptotic cell death. This mechanism may contribute to the pathogenesis of vascular diseases in HGPS patients.

Transient receptor potential channel TRPC5 is essential for P-glycoprotein induction in drug-resistant cancer cells.

An attractive strategy to overcome multidrug resistance in cancer chemotherapy is to suppress P-glycoprotein (P-gp), which is a pump overproduced in cancer cells to remove cytotoxic drugs from cells. In the present study, a Ca(2+)-permeable channel TRPC5 was found to be overproduced together with P-gp in adriamycin-resistant breast cancer cell line MCF-7/ADM. Suppressing TRPC5 activity/expression reduced the P-gp induction and caused a remarkable reversal of adriamycin resistance in MCF-7/ADM. In an athymic nude mouse model of adriamycin-resistant human breast tumor, suppressing TRPC5 decreased the growth of tumor xenografts. Nuclear factor of activated T cells isoform c3 (NFATc3) was the transcriptional factor that links the TRPC5 activity to P-gp production. Together, we demonstrated an essential role of TRPC5-NFATc3-P-gp signaling cascade in P-gp induction in drug-resistant cancer cells.