Abstract-Recent breakthroughs in the store-operated calcium (Ca 2ϩ ) entry (SOCE) pathway have identified Stim1 as the endoplasmic reticulum Ca 2ϩ sensor and Orai1 as the pore forming subunit of the highly Ca 2ϩ -selective CRAC channel expressed in hematopoietic cells. Previous studies, however, have suggested that endothelial cell (EC) SOCE is mediated by the nonselective canonical transient receptor potential channel (TRPC) family, TRPC1 or TRPC4. Here, we show that passive store depletion by thapsigargin or receptor activation by either thrombin or the vascular endothelial growth factor activates the same pathway in primary ECs with classical SOCE pharmacological features. ECs possess the archetypical Ca 2ϩ release-activated Ca 2ϩ current (I CRAC ), albeit of a very small amplitude. Using a maneuver that amplifies currents in divalent-free bath solutions, we show that EC CRAC has similar characteristics to that recorded from rat basophilic leukemia cells, namely a similar time course of activation, sensitivity to 2-aminoethoxydiphenyl borate, and low concentrations of lanthanides, and large Na ϩ currents displaying the typical depotentiation. RNA silencing of either Stim1 or Orai1 essentially abolished SOCE and I CRAC in ECs, which were rescued by ectopic expression of either Stim1 or Orai1, respectively. Surprisingly, knockdown of either TRPC1 or TRPC4 proteins had no effect on SOCE and I CRAC . Ectopic expression of Stim1 in ECs increased their I CRAC to a size comparable to that in rat basophilic leukemia cells. Knockdown of Stim1, Stim2, or Orai1 inhibited EC proliferation and caused cell cycle arrest at S and G2/M phase, although Orai1 knockdown was more efficient than that of Stim proteins. These results are first to our knowledge to establish the requirement of Stim1/Orai1 in the endothelial SOCE pathway. ATPase inhibitors such as thapsigargin activates SOCE. 1,2 Under physiological conditions SOCE is initiated by inositol 1,4,5 triphosphate (IP 3 )-mediated depletion of ER Ca 2ϩ in response to a plethora of stimuli acting through phospholipase C-coupled receptors. The best characterized SOC current is the Ca 2ϩ release-activated Ca 2ϩ current (I CRAC ), first recorded in rat basophilic leukemia (RBL) mast cells, 3 and later described in other cell types. 4 SOCE is necessary for the replenishment of ER Ca 2ϩ content and is a key regulator of many Ca 2ϩ -dependent physiological processes. 4 Recently, high-throughput RNA silencing (siRNA) screens by several laboratories have identified 2 molecules, Stim1 and Orai1, as key components of the I CRAC pathway in mast cells, lymphocytes, and HEK293 cells. 5-8 On ER Ca 2ϩ depletion, Ca 2ϩ -sensing Stim1 proteins translocate to close proximity of the plasma membrane, where they aggregate into multiple puncta. Strikingly, Orai1 molecules also translocate to the same Stim1-containing structures on store depletion, where they open to mediate Ca 2ϩ influx. 9 -12 In endothelial cells (ECs), SOCE in response to passive store depletion was reported for several EC ...

The identity of store-operated calcium (Ca(2+)) entry (SOCE) channels in vascular smooth muscle cells (VSMCs) remains a highly contentious issue. Whereas previous studies have suggested that SOCE in VSMCs is mediated by the nonselective transient receptor potential canonical (TRPC) 1 protein, the identification of STIM1 and Orai1 as essential components of I(CRAC), a highly Ca(2+)-selective SOCE current in leukocytes, has challenged that view. Here we show that cultured proliferative migratory VSMCs isolated from rat aorta (called "synthetic") display SOCE with classic features, namely inhibition by 2-aminoethoxydiphenyl borate, ML-9, and low concentrations of lanthanides. On store depletion, synthetic VSMCs and A7r5 cells display currents with characteristics of I(CRAC). Protein knockdown of either STIM1 or Orai1 in synthetic VSMCs greatly reduced SOCE, whereas Orai2, Orai3, TRPC1, TRPC4, and TRPC6 knockdown had no effect. Orai1 knockdown reduced I(CRAC) in synthetic VSMCs and A7r5 cells. Synthetic VSMCs showed up-regulated STIM1/Orai1 proteins and SOCE compared with quiescent freshly isolated VSMC. Knockdown of STIM1 and Orai1 inhibited synthetic VSMC proliferation and migration, whereas STIM2, Orai2, and Orai3 knockdown had no effect. To our knowledge, these results are the first to show I(CRAC) in VSMCs and resolve a long-standing controversy by identifying CRAC as the elusive VSMC SOCE channel important for proliferation and migration.

Calcium (Ca 2+ ) is a highly versatile second messenger that controls vascular smooth muscle cell (VSMC) contraction, proliferation, and migration. By means of Ca 2+ permeable channels, Ca 2+ pumps and channels conducting other ions such as potassium and chloride, VSMC keep intracellular Ca 2+ levels under tight control. In healthy quiescent contractile VSMC, two important components of the Ca 2+ signaling pathways that regulate VSMC contraction are the plasma membrane voltageoperated Ca 2+ channel of the high voltage-activated type (L-type) and the sarcoplasmic reticulum Ca 2+ release channel, Ryanodine Receptor (RyR). Injury to the vessel wall is accompanied by VSMC phenotype switch from a contractile quiescent to a proliferative motile phenotype (synthetic phenotype) and by alteration of many components of VSMC Ca 2+ signaling pathways. Specifically, this switch that culminates in a VSMC phenotype reminiscent of a non-excitable cell is characterized by loss of L-type channels expression and increased expression of the low voltage-activated (T-type) Ca 2+ channels and the canonical transient receptor potential (TRPC) channels. The expression levels of intracellular Ca 2+ release channels, pumps and Ca 2+ -activated proteins are also altered: the proliferative VSMC lose the RyR3 and the sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase isoform 2a pump and reciprocally regulate isoforms of the ca 2+ /calmodulin-dependent protein kinase II. This review focuses on the changes in expression of Ca 2+ signaling proteins associated with VSMC proliferation both in vitro and in vivo. The physiological implications of the altered expression of these Ca 2+ signaling molecules, their contribution to VSMC dysfunction during vascular disease and their potential as targets for drug therapy will be discussed.

Jourd'heuil D, Trebak M. Essential role for STIM1/Orai1-mediated calcium influx in PDGF-induced smooth muscle migration.

Rationale Through largely unknown mechanisms, Ca2+ signaling plays important roles in vascular smooth muscle cell (VSMC) remodeling. Orai1-encoded store-operated Ca2+ entry (SOCE) has recently emerged as an important player in VSMC remodeling. However, the role of the exclusively mammalian Orai3 protein in native VSMC Ca2+ entry pathways, its upregulation during VSMC remodeling and its contribution to neointima formation remain unknown. Objective The goal of this study was to determine the agonist-evoked Ca2+ entry pathway contributed by Orai3; Orai3 potential upregulation and role during neointima formation after balloon-injury of rat carotid arteries. Methods and Results Ca2+ imaging and patch clamp recordings showed that while the platelet-derived growth factor (PDGF) activates the canonical Ca2+ release-activated Ca2+ (CRAC) channels via store depletion in VSMC, the pathophysiological agonist thrombin activates a distinct Ca2+-selective channel contributed by Orai1, Orai3 and STIM1 in the same cells. Unexpectedly, Ca2+ store depletion is not required for activation of Orai1/3 channel by thrombin. Rather, the signal for Orai1/3 channel activation is cytosolic leukotrieneC4 produced downstream thrombin receptor stimulation through the catalytic activity of leukotrieneC4 synthase. Importantly, Orai3 is upregulated in an animal model of VSMC neointimal remodeling and in vivo Orai3 knockdown inhibits neointima formation. Conclusions These results demonstrate that distinct native Ca2+-selective Orai channels are activated by different agonists/pathways and uncover a mechanism whereby leukotrieneC4 acts through hitherto unknown intracrine mode to elicit store-independent Ca2+ signaling that promotes vascular occlusive disease. Orai3 and Orai3-containing channels provide novel targets for control of VSMC remodeling during vascular injury or disease.

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 (CamKIIδ2); 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 CamKIIδ2 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-cells (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.

We recently showed, in primary vascular smooth muscle cells (VSMCs), that the platelet-derived growth factor activates canonical store-operated Ca 2؉ entry and Ca 2؉ release-activated Ca 2؉ currents encoded by Orai1 and STIM1 genes. However, thrombin activates store-independent Ca 2؉ selective channels contributed by both Orai3 and Orai1. These store-independent Orai3/Orai1 channels are gated by cytosolic leukotriene C 4 (LTC 4 ) and require STIM1 downstream LTC 4 action. However, the source of LTC 4 and the signaling mechanisms of STIM1 in the activation of this LTC 4 -regulated Ca 2؉ (LRC) channel are unknown. Here, we show that upon thrombin stimulation, LTC 4 is produced through the sequential activities of phospholipase C, diacylglycerol lipase, 5-lipo-oxygenease, and leukotriene C 4 synthase. We show that the endoplasmic reticulum-resident STIM1 is necessary and sufficient for LRC channel activation by thrombin. STIM1 does not form sustained puncta and does not colocalize with Orai1 either under basal conditions or in response to thrombin. However, STIM1 is precoupled to Orai3 and Orai3/Orai1 channels under basal conditions as shown using Forster resonance energy transfer (FRET) imaging. The second coiled-coil domain of STIM1 is required for coupling to either Orai3 or Orai3/Orai1 channels and for LRC channel activation. We conclude that STIM1 employs distinct mechanisms in the activation of store-dependent and store-independent Ca 2؉ entry pathways.

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