Stimulation of ORAI1 expression, store-operated Ca2+ entry, and osteogenic signaling by high glucose exposure of human aortic smooth muscle cells.

Diabetes and chronic kidney disease (CKD) both trigger vascular osteogenic signaling and calcification leading to early death by cardiovascular events. Osteogenic signaling involves upregulation of the transcription factors CBFA1, MSX2, and SOX9, as well as alkaline phosphatase (ALP), an enzyme fostering calcification by degrading the calcification inhibitor pyrophosphate. In CKD, osteogenic signaling is triggered by hyperphosphatemia, which upregulates the serum and glucocorticoid-inducible kinase SGK1, a strong stimulator of the Ca2+-channel ORAI1. The channel is activated by STIM1 and accomplishes store-operated Ca2+-entry (SOCE). The present study explored whether exposure of human aortic smooth muscle cells (HAoSMCs) to high extracellular glucose concentrations similarly upregulates ORAI1 and/or STIM1 expression, SOCE, and osteogenic signaling. To this end, HAoSMCs were exposed to high extracellular glucose concentrations (15 mM, 24 h) without or with additional exposure to the phosphate donor ß-glycerophosphate. Transcript levels were estimated using qRT-PCR, protein abundance using Western blotting, ALP activity using a colorimetric assay kit, calcium deposits utilizing Alizarin red staining, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and SOCE from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). As a result, glucose enhanced the transcript levels of SGK1 and ORAI1, ORAI2, and STIM2, protein abundance of ORAI1, SOCE, the transcript levels of CBFA1, MSX2, SOX9, and ALPL, as well as calcium deposits. Moreover, glucose significantly augmented the stimulating effect of ß-glycerophosphate on transcript levels of SGK1 and ORAI1, SOCE, the transcript levels of osteogenic markers, as well as calcium deposits. ORAI1 inhibitor MRS1845 (10 µM) significantly blunted the glucose-induced upregulation of the CBFA1 and MSX2 transcript levels. In conclusion, the hyperglycemia of diabetes stimulates expression of SGK1 and ORAI1, thus, augmenting store-operated Ca2+-entry and osteogenic signaling in HAoSMCs.

Reversal of phosphate-induced ORAI1 expression, store-operated Ca2+ entry and osteogenic signaling by MgCl2 in human aortic smooth muscle cells.

In chronic kidney disease, renal phosphate retention leads to hyperphosphatemia with subsequent vascular osteogenic signaling and calcification. Osteogenic signaling involves up-regulation of the transcription factors CBFA1, MSX2, and SOX9, as well as alkaline phosphatase (ALP), an enzyme stimulating calcification by degrading the calcification inhibitor pyrophosphate. Stimulation of osteogenic signaling and calcification by phosphate donor ß-glycerophosphate in human aortic smooth muscle cells (HAoSMCs) is attenuated by MgCl2, an effect mimicked by Ca2+-sensing receptor agonist GdCl3. Most recent observations revealed that the effect of ß-glycerophosphate on osteogenic signaling requires ORAI1, a Ca2+-channel accomplishing store-operated Ca2+-entry (SOCE), which is stimulated by Ca2+-sensor STIM1. The present study explored whether ORAI1 and/or STIM1 expression and, thus, SOCE and osteogenic signaling in HAoSMCs are sensitive to MgCl2 and/or GdCl3. To this end, transcript levels were estimated using q-RT-PCR, protein abundance with western blotting, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and SOCE from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1  µM). As a result, 24 h exposure to ß-glycerophosphate (2 mM) significantly enhanced transcript levels of ORAI1 and STIM1 as well as SOCE, effects significantly blunted or virtually abrogated by 1.5 mM MgCl2 and by 50  µM GdCl3. In conclusion, MgCl2 and GdCl3 are powerful inhibitors of ORAI1 and STIM1 expression and store-operated Ca2+-entry, effects affecting osteogenic signalling in vascular smooth muscle cells.

High-Throughput RNAi Screening Identifies a Role for the Osteopontin Pathway in Proliferation and Migration of Human Aortic Smooth Muscle Cells.

PURPOSE: Understanding of the mechanisms of vascular smooth muscle cells (VSMCs) phenotypic regulation is critically important to identify novel candidates for future therapeutic intervention. While HTS approaches have recently been used to identify novel regulators in many cell lines, such as cancer cells and hematopoietic stem cells, no studies have so far systematically investigated the effect of gene inactivation on VSMCs with respect to cell survival and growth response. METHODS AND RESULTS: 257 out of 2000 genes tested resulted in an inhibition of cell proliferation in HaoSMCs. After pathway analysis, 38 significant genes were selected for further study. 23 genes were confirmed to inhibit proliferation, and 13 genes found to induce apoptosis in the synthetic phenotype. 11 genes led to an aberrant nuclear phenotype indicating a central role in cell mitosis. 4 genes affected the cell migration in synthetic HaoSMCs. Using computational biological network analysis, 11 genes were identified to have an indirect or direct interaction with the Osteopontin pathway. For 10 of those genes, levels of proteins downstream of the Osteopontin pathway were found to be down-regulated, using RNAi methodology. CONCLUSIONS: A phenotypic high-throughput siRNA screen could be applied to identify genes relevant for the cell biology of HaoSMCs. Novel genes were identified which play a role in proliferation, apoptosis, mitosis and migration of HaoSMCs. These may represent potential drug candidates in the future.

Vasopressin-stimulated ORAI1 expression and store-operated Ca2+ entry in aortic smooth muscle cells.

Vascular calcification may result from stimulation of osteogenic signalling with upregulation of the transcription factors CBFA1, MSX2 and SOX9, as well as alkaline phosphatase (ALPL), which degrades and thus inactivates the calcification inhibitor pyrophosphate. Osteogenic signalling further involves upregulation of the Ca2+-channel ORAI1. The channel is activated by STIM1 and then accomplishes store-operated Ca2+ entry. ORAI1 and STIM1 are upregulated by the serum & glucocorticoid inducible kinase 1 (SGK1) which is critically important for osteogenic signalling. Stimulators of vascular calcification include vasopressin. The present study explored whether exposure of human aortic smooth muscle cells (HAoSMCs) to vasopressin upregulates ORAI1 and/or STIM1 expression, store-operated Ca2+ entry and osteogenic signalling. To this end, HAoSMCs were exposed to vasopressin (100 nM, 24 h) without or with additional exposure to ORAI1 blocker MRS1845 (10 µM) or SGK1 inhibitor GSK-650394 (1 µM). Transcript levels were measured using q-RT-PCR, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and store-operated Ca2+ entry from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). As a result, vasopressin enhanced the transcript levels of ORAI1 and STIM1, store-operated Ca2+ entry, as well as the transcript levels of CBFA1, MSX2, SOX9 and ALPL. The effect of vasopressin on store-operated Ca2+ entry as well as on transcript levels of CBFA1, MSX2, SOX9 and ALPL was virtually abrogated by MRS1845 and GSK-650394. In conclusion, vasopressin stimulates expression of ORAI1/STIM1, thus augmenting store-operated Ca2+ entry and osteogenic signalling. In HAoSMCs, vasopressin (VP) upregulates Ca2+ channel ORAI1 and its activator STIM1. VP upregulates store-operated Ca2+ entry (SOCE) and osteogenic signalling (OS). VP-induced SOCE, OS and Ca2+-deposition are disrupted by ORAI1 inhibitor MRS1845. VP-induced SOCE, OS and Ca2+-deposition are disrupted by SGK1 blocker GSK-650394. KEY MESSAGES: • In HAoSMCs, vasopressin (VP) upregulates Ca2+ channel ORAI1 and its activator STIM1. • VP upregulates store-operated Ca2+ entry (SOCE) and osteogenic signalling (OS). • VP-induced SOCE, OS and Ca2+-deposition are disrupted by ORAI1 inhibitor MRS1845. • VP-induced SOCE, OS and Ca2+-deposition are disrupted by SGK1 blocker GSK-650394.

Phosphate-induced ORAI1 expression and store-operated Ca2+ entry in aortic smooth muscle cells.

Compromised renal phosphate elimination in chronic kidney disease (CKD) leads to hyperphosphatemia, which in turn triggers osteo-/chondrogenic signaling in vascular smooth muscle cells (VSMCs) and vascular calcification. Osteo-/chondrogenic transdifferentiation of VSMCs leads to upregulation of the transcription factors MSX2, CBFA1, and SOX9 as well as tissue-nonspecific alkaline phosphatase (ALPL) which fosters calcification by degrading the calcification inhibitor pyrophosphate. Osteo-/chondrogenic signaling in VSMCs involves the serum- and glucocorticoid-inducible kinase SGK1. As shown in other cell types, SGK1 is a powerful stimulator of ORAI1, a Ca2+-channel accomplishing store-operated Ca2+-entry (SOCE). ORAI1 is stimulated following intracellular store depletion by the Ca2+ sensor STIM1. The present study explored whether phosphate regulates ORAI1 and/or STIM1 expression and, thus, SOCE in VSMCs. To this end, primary human aortic smooth muscle cells (HAoSMCs) were exposed to the phosphate donor ß-glycerophosphate. Transcript levels were estimated by qRT-PCR, protein abundance by western blotting, ALPL activity by colorimetry, calcification by alizarin red S staining, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and SOCE from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin. As a result, ß-glycerophosphate treatment increased ORAI1 and STIM1 transcript levels and protein abundance as well as SOCE in HAoSMCs. Additional treatment with ORAI1 inhibitor MRS1845 or SGK1 inhibitor GSK650394 virtually disrupted the effects of ß-glycerophosphate on SOCE. Moreover, the ß-glycerophosphate-induced MSX2, CBFA1, SOX9, and ALPL mRNA expression and activity in HAoSMCs were suppressed in the presence of the ORAI1 inhibitor and upon ORAI1 silencing. In conclusion, enhanced phosphate upregulates ORAI1 and STIM1 expression and store-operated Ca2+-entry, which participate in the orchestration of osteo-/chondrogenic signaling of VSMCs. KEY MESSAGES: • In aortic SMC, phosphate donor ß-glycerophosphate upregulates Ca2+ channel ORAI1. • In aortic SMC, ß-glycerophosphate upregulates ORAI1-activator STIM1. • In aortic SMC, ß-glycerophosphate upregulates store-operated Ca2+-entry (SOCE). • The effect of ß-glycerophosphate on SOCE is disrupted by ORAI1 inhibitor MRS1845. • Stimulation of osteogenic signaling is disrupted by MRS1845 and ORAI1 silencing.

A novel 2,3-diphenyl-4H-pyrido[1,2-a]pyrimidin-4-one derivative inhibits endothelial cell dysfunction and smooth muscle cell proliferation/activation.

Hyper-proliferation and migration of vascular smooth muscle cells and endothelial cell dysfunction are central events in the development of neo-intimal lesions. Pursuing our interest in the synthesis of bioisosters of flavonoids, we studied in depth a novel synthetic 2,3-diphenyl-4H-pyrido[1,2-a]pyrimidin-4-one derivative, examining its effects in vitro on induced-cell proliferation and activation in human aortic smooth muscle cells (HAoSMCs) and in human umbilical vein endothelial cells (HUVECs). Compared with two well known flavonoids, apigenin and quercetin, the novel compound, 2-(3,4-dimethoxyphenyl)-3-phenyl-4H-pyrido[1,2-a]pyrimidin-4-one, 3, was not toxic for HUVECs, even at high concentrations and for long incubation times, while the two flavonoids were not tolerated, even at concentrations as low as 10 µmol/L. Compound 3 inhibited selectively, and in a concentration-dependent manner, the proliferation of HAoSMCs but not that of HUVECs. In HUVECs, it inhibited the cytokine-induced vascular cell adhesion molecule-1 expression, but not the cyclooxygenase-2 (COX-2) expression. Instead, in HAoSMC, it inhibited the induction of COX-2 expression and the relative release of prostaglandin E2. In addition, it inhibited the transcription of the matrix metalloproteinase-9 and its activity. Thanks to its multiple and tissue-specific function, 2-(3,4-dimethoxyphenyl)-3-phenyl-4H-pyrido[1,2-a]pyrimidin-4-one might replace or assist the action of current drugs eluted by coronary stents, in order to promote a functional repair of damaged wall.