Orai1-mediated I (CRAC) is essential for neointima formation after vascular injury

Abstract

Rationale: The molecular correlate of the calcium release-activated calcium current (I(CRAC)), 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 I(CRAC) 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-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.

Figure 1. Effect of shOrai1 and shSTIM1 lentiviruses on SOCE and I_CRAC

A. Cultured VSMCs were infected with either shLuciferase, shOrai1 or shSTIM1 lentiviruses and infected cells are visualized by GFP fluorescence. Phase pictures are shown to gauge the infection efficiency. Note the reduced cell number in shOrai1 and shSTIM1 conditions compared to shLuciferase control. B. quantitative PCR shows that shOrai1 and shSTIM1 lentiviruses specifically and efficiently reduced their target mRNA (normalized to rpl32 mRNA) 5 days post infection. ShOrai1and shSTIM1 infection of VSMCs caused a small (not statistically significant) increase in STIM1 and Orai1 mRNA respectively. C. Western blotting shows that shOrai1 and shSTIM1 lentiviruses caused efficient protein knockdown of their respective proteins 5 days post infection. D. Statistical analysis of Orai1 and STIM1 band densitometry normalized to β-actin is shown. E. shOrai1 lentiviral infection essentially abrogated SOCE activated by thapsigargin (2μM) compared to shLuciferase lentivirus control in VSMCs; statistical analysis from 3 independent experiments totaling 21 cells is depicted (control shluciferase, 3 experiments, n=23). F. shSTIM1 lentiviral infection also caused substantial reduction in SOCE activated by thapsigargin (2μM) compared to shLuciferase lentivirus control in VSMCs; statistical analysis from 3 independent experiments totaling 19 cells is depicted (control shluciferase, 3 experiments, n=24). G-I. Whole cell patch clamp recordings of I_CRAC activated in VSMCs by dialysis of 20mM BAPTA through the patch pipette and showing substantial decrease of Ca²⁺ I_CRAC currents (at -100mV) recorded in 20mM external Ca²⁺ and Na⁺ I_CRAC currents recorded in divalent free (DVF) external solutions, from shOrai1- (n=4) and shSTIM1-infected VSMCs (n=5) compared to shLuciferase-infected VSMCs (n=3). A DVF pulse was applied immediately after break-in to gauge leak current, which was subsequently subtracted from the DVF current obtained after store depletion. J. I/V relationships of Na⁺ I_CRAC from VSMCs infected with shLuciferase, shOrai1 or shSTIM1 lentiviruses were taken where indicated by the color-coded asterisks. K. Statistical analyses on Ca²⁺ I_CRAC and Na⁺ I_CRAC currents taken at -100mV from several independent VSMCs infected with shLuciferase, shOrai1 or shSTIM1 lentiviruses.

Figure 2. Orai1/STIM1 proteins and balloon carotid injury in rats

A. H&E staining of balloon-injured left carotid artery sections showing increased neointima as early as 7 day after injury compared to sham-operated arteries (intact). Neointima formation peaks at 14 days post injury and is still prominent 21 day post-injury. B. IF staining with specific antibodies shows increased protein expression of Orai1 and STIM1in neointima of left injured carotid arteries 7 day post injury in comparison with right non-injured control carotids. IF staining also show increased expression of the proliferative marker Ki67 and decreased expression of VSMC contractile marker myosin heavy chain (MHC) in neointimal layers of balloon-injured left carotid artery sections compared to right non-injured control carotids. C. Western blotting on medial and neointimal VSMC from balloon-injured left carotid arteries compared with the medial layer from sham-operated arteries showing increased protein expression of Orai1, STIM1 and the proliferative marker PCNA in balloon-injured left carotid arteries 14 day post-injury. A, adventitia; M, media; NI, Neointima; L, leumen.

Figure 3. Orai1/STIM1 ShRNA lentiviral infection, VSMC proliferation and protein expression

A. ShLuciferase, shOrai1 and shSTIM1 lentiviruses efficiently infected carotid vessels as evidenced with expression of GFP in medial and neointimal protein extracts from left carotid arteries that are balloon-injured (14 day after injury) and infected with viral particles; right non-infected carotids show no GFP expression. ShOrai1 inhibited upregulation of Orai1 protein as well as that of STIM1 and CamKIIδ2, 14 day post-injury. Similarly, shSTIM1 inhibited upregulation of STIM1 protein as well as that of Orai1 and CamKIIδ2. ShOrai1 and shSTIM1 inhibited VSMC proliferation as evidenced by reduced protein expression of PCNA in medial and neointimal protein extracts from balloon-injured left carotids treated with shOrai1 and shSTIM1 lentiviruses compared to injured left carotids treated with shLuciferase lentiviruses. B. Statistical analyses on Orai1, STIM1, CamKIIδ2 and PCNA protein expression data on extracts of medial and neointimal VSMC from ballon-injured left carotids and treated with shLuciferase, shOrai1 or shSTIM1 lentiviruses. Data represent densitometry on protein bands with average ± SEM from 6 rats per condition, determined using Image J and normalized to βactin expression. C-D. IF using specific anti-Orai1 (C) and anti-STIM1 (D) antibodies on left carotid sections (14 day after injury) injured and treated with shLuciferase, shOrai1 or shSTIM1 lentiviruses (bottom) and corresponding right non-injured non-treated control carotids (top).

Figure 4. ShOrai1 and shSTIM1 lentiviral infection after balloon-injury inhibited neointima formation

A. H&E staining on left carotid artery sections from either sham-operated (intact) or balloon-injured rats treated with shLuciferase (shLuc), shOrai1 or shSTIM1 lentiviruses 14 day post-injury and lentiviral treatment. B,C. Statistical analyses on medial and neointimal areas (mm²; B) and media/neointima (M/N) ratios (C) from 5-7 independent rats per condition determined using the Image J software from left injured and treated with lentiviruses and right non injured non-treated controls. Neointimal regions are highlighted in green.

Figure 5. Orai1 and STIM1 knockdown prevents NFAT nuclear translocation and activity

A. VSMCs were transfected with a plasmid encoding NFAT-GFP after transfection with either siRNA against Orai1 (siO1), STIM1 (siS1) or non-targeting siRNA control (siNT). NFAT-GFP nuclear translocation in response to thapsigargin (2μM for 15 minutes) was monitored under fluorescence microscope. B. Data was quantified using Image J and efficiency of NFAT-GFP translocation was represented as a ratio of GFP fluorescence in nucleus/cytosol. C. Luciferase reporter assays with and without 2μM thapsigargin treatment of VSMCs transfected with siOrai1 (siO1), siSTIM1 (siS1) or non-targeting siRNA (siNT).

Figure 6. Orai1/STIM1 proteins are upregulated in carotid arteries from mice subjected to ligation

IHC staining with specific antibodies against Orai1 and STIM1 in ligation-injured carotids from mice shows a marked upregulation of Orai1 and STIM1 proteins in media and neointima from ligation-injured carotids 21 days post-ligation compared to their respective control vessels. Contiguous sections from the same vessels were also stained with anti-SMα-actin antibody as a VSMC marker. Dilutions used for primary antibodies are: anti-Orai1, 1:400; anti-STIM1, 1:2000 (inset section dilution was 1:1200 for control vessel); anti- SMα-actin, 1:800.