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

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.

Disposition of the second messenger nitric oxide (NO) in mammalian tissues occurs through multiple pathways including dioxygenation by erythrocyte hemoglobin and red muscle myoglobin. Metabolism by a putative NO dioxygenase activity in non-striated tissues has also been postulated, but the exact nature of this activity is unknown. In the present study, we tested the hypothesis that cytoglobin, a newly discovered hexacoordinated globin, participates in cell-mediated NO consumption. Stable expression of small hairpin RNA targeting cytoglobin in fibroblasts resulted in decreased NO consumption and intracellular nitrate production. These cells were more sensitive to NO-induced inhibition of cell respiration and proliferation, which could be restored by re-expression of human cytoglobin. We also demonstrated cytoglobin expression in adventitial fibroblasts as well as vascular smooth muscle cells from various species including human and found that cytoglobin was expressed in the adventitia and media of intact rat aorta. These results indicate that cytoglobin contributes to cell-mediated NO dioxygenation and represents an important NO sink in the vascular wall. Nitric oxide (NO)2 plays a central role in the vasculature and regulates oxygen supply by relaxing smooth muscle and inhibiting mitochondrial respiration. NO is produced by nitric-oxide synthase and through mobilization of storage pools such as nitrite. Less is known on the mechanism of NO inactivation and removal in the vasculature, although it would be surprising that blood vessels rely on nonspecific chemical reactions to control its disposition.Blood removes NO through reaction with excess oxyhemoglobin in erythrocytes, but diffusional barriers such as the red blood cell-free layer near the vessel wall allow autocrine NO to occur in significant amounts in the vascular wall (1, 2). In striated muscles, oxymyoglobin is an NO scavenger, and studies using transgenic mice lacking myoglobin have demonstrated a role for oxymyoglobin in attenuating NO-mediated cardiac dysfunction (3, 4). In contrast, NO inactivation in non-striated tissues is poorly understood, and examination of NO consumption in various cells suggests mechanisms that differ in NO saturation, oxygen and cyanide sensitivity, and product formation (5-8). Specific pathways include increased partition of NO in cell membranes (7) and consumption of NO by cytochrome c oxidase (9 -11), NADPH oxidase (12), 15-lipoxygenase (13), prostaglandin-H synthase (14), and myeloperoxidase (15).Many of the above mechanisms implicate metalloproteins as modulators of NO bioavailability among which the role of oxyglobins such as oxyhemoglobin and oxymyoglobin have been already highlighted (16). One of the primary mechanisms is the dioxygenation of NO to form nitrate and the ferric (Fe(III)) form of the protein. The recent discovery of two new mammalian globins, cytoglobin and neuroglobin, would suggest that the reactions of NO with globins are not limited to red blood cells and striated muscles but could extend to other ...

Inflammation plays a critical role in promoting smooth muscle migration and proliferation during vascular diseases such as post-angioplasty restenosis and atherosclerosis. Another common feature of many vascular diseases is the contribution of reactive oxygen (ROS) and nitrogen (RNS) species to vascular injury. Primary sources of ROS and RNS in smooth muscle are several isoforms of NADPH oxidase (Nox) and the cytokine-regulated inducible nitric oxide (NO) synthase (iNOS). One important example of the interaction between NO and ROS is the reaction of NO with superoxide to yield peroxynitrite, which may contribute to the pathogenesis of hypertension. In this review, we discuss the literature that supports an alternate possibility: Nox-derived ROS modulate NO bioavailability by altering the expression of iNOS. We highlight data showing co-expression of iNOS and Nox in vascular smooth muscle and demonstrating the functional consequences of iNOS and Nox during vascular injury. We describe the relevant literature demonstrating that the mitogen activated protein kinases (MAP kinases) are important modulators of pro-inflammatory cytokinedependent expression of iNOS. A central hypothesis discussed is that ROS-dependent regulation of the serine/threonine kinase protein kinase Cδ (PKCδ) is essential to understanding how Nox may regulate signaling pathways leading to iNOS expression. Overall, the integration of non-phagocytic NADPHoxidase with cytokine signaling in general and in vascular smooth muscle in particular is poorly understood and merit further investigation.

This case report details a unique case of acute, reversible liver failure in a 12-year-old male with sickle cell anemia on chronic transfusion protocol and deferasirox chelation. There is substantial literature documenting deferasirox-induced renal injury, including Fanconi syndrome, but less documentation of hepatic toxicity and few reports of hepatic failure. The case highlights the importance of close monitoring of ferritin, bilirubin, and transaminases for patients on deferasirox.