Khalid Matrougui

STIM1 and Orai1 mediate CRAC channel activity and are essential for human glioblastoma invasion

The Ca2+ sensor stromal interacting molecule 1 (STIM1) and the Ca2+ channel Orai1 mediate the ubiquitous store-operated Ca2+ entry (SOCE) pathway activated by depletion of internal Ca2+ stores and mediated through the highly Ca2+-selective, Ca2+ release-activated Ca2+ (CRAC) current. Furthermore, STIM1 and Orai1, along with Orai3, encode store-independent Ca2+ currents regulated by either arachidonate or its metabolite, leukotriene C4. Orai channels are emerging as important contributors to numerous cell functions, including proliferation, migration, differentiation, and apoptosis. Recent studies suggest critical involvement of STIM/Orai proteins in controlling the development of several cancers, including malignancies of the breast, prostate, and cervix. Here, we quantitatively compared the magnitude of SOCE and the expression levels of STIM1 and Orai1 in non-malignant human primary astrocytes (HPA) and in primary human cell lines established from surgical samples of the brain tumor glioblastoma multiforme (GBM). Using Ca2+ imaging, patch-clamp electrophysiology, pharmacological reagents, and gene silencing, we established that in GBM cells, SOCE and CRAC are mediated by STIM1 and Orai1. We further found that GBM cells show upregulation of SOCE and increased Orai1 levels compared to HPA. The functional significance of SOCE was evaluated by studying the effects of STIM1 and Orai1 knockdown on cell proliferation and invasion. Utilizing Matrigel assays, we demonstrated that in GBM, but not in HPA, downregulation of STIM1 and Orai1 caused a dramatic decrease in cell invasion. In contrast, the effects of STIM1 and Orai1 knockdown on GBM cell proliferation were marginal. Overall, these results demonstrate that STIM1 and Orai1 encode SOCE and CRAC currents and control invasion of GBM cells. Our work further supports the potential use of channels contributed by Orai isoforms as therapeutic targets in cancer.

Orai1-Mediated ICRAC Is Essential for Neointima Formation After Vascular Injury

Rationale: The molecular correlate of the calcium release-activated calcium current (ICRAC), the channel protein Orai1, is upregulated in proliferative vascular smooth muscle cells (VSMC). However, the role of Orai1 in vascular disease remains largely unknown. Objective: The goal of this study was to determine the role of Orai1 in neointima formation after balloon injury of rat carotid arteries and its potential upregulation in a mouse model of VSMC remodeling. Methods and Results: Lentiviral particles encoding short-hairpin RNA (shRNA) targeting either Orai1 (shOrai1) or STIM1 (shSTIM1) caused knockdown of their respective target mRNA and proteins and abrogated store-operated calcium entry and ICRAC in VSMC; control shRNA was targeted to luciferase (shLuciferase). Balloon injury of rat carotid arteries upregulated protein expression of Orai1, STIM1, and calcium–calmodulin kinase IIdelta2 (CamKII&dgr;2); increased proliferation assessed by Ki67 and PCNA and decreased protein expression of myosin heavy chain in medial and neointimal VSMC. Incubation of the injured vessel with shOrai1 prevented Orai1, STIM1, and CamKII&dgr;2 upregulation in the media and neointima; inhibited cell proliferation and markedly reduced neointima formation 14 days post injury; similar results were obtained with shSTIM1. VSMC Orai1 and STIM1 knockdown inhibited nuclear factor for activated T-cell (NFAT) nuclear translocation and activity. Furthermore, Orai1 and STIM1 were upregulated in mice carotid arteries subjected to ligation. Conclusions: Orai1 is upregulated in VSMC during vascular injury and is required for NFAT activity, VSMC proliferation, and neointima formation following balloon injury of rat carotids. Orai1 provides a novel target for control of VSMC remodeling during vascular injury or disease.

Orai3 is an estrogen receptor α-regulated Ca2+ channel that promotes tumorigenesis

Store‐operated Ca2+ entry (SOCE) encoded by Orai1 proteins is a ubiquitous Ca2+‐selective conductance involved in cellular proliferation and migration. We recently described up‐regulation of Orai3 channels that selectively mediate SOCE in estrogen receptor α‐expressing (ERα+) breast cancer cells. However, the connection between ERα and Orai3 and the role of Orai3 in tumorigenesis remain unknown. Here, we show that ERα knockdown decreases Orai3 mRNA (by ~63%) and protein (by ~44%) with no effect on Orai1. ERα knockdown decreases Orai3‐mediated SOCE (by ~43%) and the corresponding Ca2+ release‐activated Ca2+ (CRAC) current (by ~42%) in ERα+ MCF7 cells. The abrogation of SOCE in MCF7 cells on ERα knockdown can be rescued by ectopic expression of Orai3. ERα activation increased Orai3 expression and SOCE in MCF7 cells. Epidermal growth factor (EGF) and thrombin stimulate Ca2+ influx into MCF7 cells through Orai3. Orai3 knockdown inhibited SOCE‐dependent phosphorylation of extracellular signal‐regulated kinase (ERK1/2; by ~44%) and focal adhesion kinase (FAK; by ~46%) as well as transcriptional activity of nuclear factor for activated T cells (NFAT; by ~49%). Significantly, Orai3 knockdown selectively decreased anchorage‐independent growth (by ~58%) and Matrigel invasion (by ~44%) of ERα+ MCF7 cells with no effect on ERα– MDA‐MB231 cells. Moreover, Orai3 knockdown inhibited ERα+ cell tumorigenesis in immunodeficient mice (~66% reduction in tumor volume). These data establish Orai3 as an ERα‐regulated channel and a potential selective therapeutic target for ERα+ breast cancers.—Motiani, R. K., Zhang, X., Harmon, K. E., Keller, R. S., Matrougui, K., Bennett, J. A., Trebak, M. Orai3 is an estrogen receptor α‐regulated Ca2+ channel that promotes tumorigenesis. FASEB J. 27, 63–75 (2013). www.fasebj.org

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

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

Modified multipotent stromal cells with epidermal growth factor restore vasculogenesis and blood flow in ischemic hind-limb of type II diabetic mice

Diabetes is increasing in the world and causes severe cardiovascular complications. Diabetes-induced limb ischemia leads to foot amputation and therapeutic remedies are urgently needed. Here we report that local injection of mesenchymal stem cells (MSCs) prestimulated with epidermal growth factor (EGF) restored blood flow and vasculogenesis in the ischemic hind-limb of type II diabetic (db−/db−) mice. Bone marrow cells from db−/db− mice are altered as evidenced by increased oxidative stress and reduced Akt and adhesion molecules when compared with control (db−/db+). Femoral artery ligation-induced ischemia was performed in the hind-limb of db−/db− and db−/db+ mice for 28 days. Enhanced green fluorescent protein (EGFP)-MSCs stimulated±exogenous EGF for 24u2009h were injected locally into the ischemic muscle. Blood flow measured with MoorLDI-Laser and microangiography assessed with X-ray showed 100% recovery in db−/db+ compared to 50% recovery in db−/db− mice. Interestingly, db−/db− mice had 60 and 96% blood flow recovery and 61 and 98% of vasculogenesis when treated with MSCs alone or MSCs modified with EGF, respectively. Western blot analysis of hind-limb muscles revealed an increase in Akt and vascular endothelial growth factor receptor phosphorylation and hypoxia-inducible factor) expression in db−/db− mice injected with MSCs or MSCs+EGF compared to db−/db− mice. Fluorescent microscopic images show that EGFP-MSCs differentiate into new microvessels. Adhesion and migration of MSCs on cultured endothelial cells were ICAM1-, VCAM1- and Akt-dependent mechanism and elevated when MSCs were prestimulated with EGF compared with nonstimulated MSCs. Our novel study data provide evidence that in type II diabetes, stimulated MSCs with EGF enhance the recovery of blood flow and angiogenesis.

Mechanism of endoplasmic reticulum stress-induced vascular endothelial dysfunction.

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

STIM1 controls endothelial barrier function independently of Orai1 and Ca2+ entry.

The calcium sensor STIM1 disrupts the endothelial barrier by coupling the thrombin receptor to the actin cytoskeleton. Breaking the Endothelial Barrier Thrombin is an endogenous ligand that induces vasoconstriction and can also disrupt the barrier formed by blood vessel endothelial cells, which leads to increased vascular permeability and leakage of plasma into the tissue. Using the thrombin-induced decrease in transendothelial resistance in two types of cultured endothelial cells as a model of barrier disruption, Shinde et al. found that the calcium-responsive protein STIM1 coupled the thrombin receptor to activation of the guanosine triphosphatase RhoA and rearrangement of the actin cytoskeleton, which contribute to loss of cell-cell contact. Surprisingly, this role did not involve various cation channels that are targets of STIM1. How STIM1 couples the thrombin receptor to RhoA remains an open question. Endothelial barrier function is critical for tissue fluid homeostasis, and its disruption contributes to various pathologies, including inflammation and sepsis. Thrombin is an endogenous agonist that impairs endothelial barrier function. We showed that the thrombin-induced decrease in transendothelial electric resistance of cultured human endothelial cells required the endoplasmic reticulum–localized, calcium-sensing protein stromal interacting molecule 1 (STIM1), but was independent of Ca2+ entry across the plasma membrane and the Ca2+ release–activated Ca2+ channel protein Orai1, which is the target of STIM1 in the store-operated calcium entry pathway. We found that STIM1 coupled the thrombin receptor to activation of the guanosine triphosphatase RhoA, stimulation of myosin light chain phosphorylation, formation of actin stress fibers, and loss of cell-cell adhesion. Thus, STIM1 functions in pathways that are dependent on and independent of Ca2+ entry.

High-fat diet induces lung remodeling in ApoE-deficient mice: an association with an increase in circulatory and lung inflammatory factors.

Hypercholesterolemia is increasingly considered the basis for not only cardiovascular pathologies but also several complications affecting other organs such as lungs. In this study, we examined the effect of hypercholesterolemia on lung integrity using a mouse model (ApoE−/−) of high-fat (HF) diet-induced atherosclerosis. A 12-week HF diet regimen induced systemic production of TNF-α, IFN-γ, GMC-SF, RANTES, IL-1α, IL-2 and IL-12 with TNF-α as the predominant cytokine in ApoE−/− mice. Concomitantly, TNF-α, IFN-γ and MIP-1α were detected in brochoalveolar lavage (BAL) fluids of these mice, coinciding with lung inflammation consisting primarily of monocytes/macrophages. Such lung inflammation correlated with marked collagen deposition and an increase in matrix metalloproteinase-9 activity in ApoE−/−mice without mucus production. Although TGF-β1 was undetectable in the BAL fluid of ApoE−/− mice on HF diet, it showed a much wider tissue distribution compared with that of control animals. Direct exposure of smooth muscle cells to oxidized-LDL, in vitro, induced a time-dependent expression of TNF-α. Direct intratracheal TNF-α-administration induced a lung inflammation pattern in wild-type mice that was strikingly similar to that induced by HF diet in ApoE−/− mice. TNF-α administration induced expression of several factors known to be critically involved in lung remodeling, such as MCP-1, IL-1β, TGF-β1, adhesion molecules, collagen type-I and TNF-α itself in the lungs of treated mice. These results suggest that hypercholesterolemia may promote chronic inflammatory conditions in lungs that are conducive to lung remodeling potentially through TNF-α-mediated processes.

Emerging role of G protein-coupled receptors in microvascular myogenic tone

Blood flow autoregulation results from the ability of resistance arteries to reduce or increase their diameters in response to changes in intravascular pressure. The mechanism by which arteries maintain a constant blood flow to organs over a range of pressures relies on this myogenic response, which defines the intrinsic property of the smooth muscle to contract in response to stretch. The resistance to flow created by myogenic tone (MT) prevents tissue damage and allows the maintenance of a constant perfusion, despite fluctuations in arterial pressure. Interventions targeting MT may provide a more rational therapeutic approach in vascular disorders, such as hypertension, vasospasm, chronic heart failure, or diabetes. Despite its early description by Bayliss in 1902, the cellular and molecular mechanisms underlying MT remain poorly understood. We now appreciate that MT requires a complex mechanotransduction converting a physical stimulus (pressure) into a biological response (change in vessel diameter). Although smooth muscle cell depolarization and a rise in intracellular calcium concentration are recognized as cornerstones of the myogenic response, the role of wall strain-induced formation of vasoactive mediators is less well established. The vascular system expresses a large variety of Class 1 G protein-coupled receptors (GPCR) activated by an eclectic range of chemical entities, including peptides, lipids, nucleotides, and amines. These messengers can function in blood vessels as vasoconstrictors. This review focuses on locally generated GPCR agonists and their proposed contributions to MT. Their interplay with pivotal G(q-11) and G(12-13) protein signalling is also discussed.

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

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