Calponin

Vascular SMCs were isolated from second and third order mesenteric arteries of NT-littermates and S-P467L mice by the explant method [20] (link). Briefly, mesenteric beds were cleaned of adipose and connective tissue and placed in the culture dish and maintained with Dulbecco Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotics and maintained in an incubator at 37°C in a humidified 5% CO₂ atmosphere. After 72h, once VSMCs migrated from the edges of the explants, the mesenteric arteries were removed and discarded. Mesenteric SMCs exhibited the typical “hill and valley” growth morphology and were confirmed positive (>95%) for smooth muscle α-actin (Sigma) and calponin (Santa Cruz sc-28545) immunostaining, and negative for Pecam-1 (Abcam ab28364) band CD90/Thy1 (Santa Cruz sc9163). Cells at passage 3 maintain their contractile phenotype and were used in all experiments. At 80% confluence, the culture medium was replaced with serum-free medium for 24 hours to render the cells quiescent. Cells were then treated with Ang-II (0.01 µM) for 2, 5, 10, 30 and 60 minutes for determination of ERK1/2 activation and for 24, 48 and 72 hours for analysis of proliferation (as measured by PCNA expression). In some experiments mesenteric SMC were pre-incubated with losartan (1 µM) or Tempol (5 mM) for 30 min following treatment with Ang-II.

The cells were fixed in 4% paraformaldehyde for 15 min. Cells were incubated with primary anti-VEGFR2, VE-cadherin, PECAM-1, calponin, SMA-α, MHY-11, P2X7, P2Y1, P2Y2, and P2Y11 (Santa Cruz Biotechnology, CA, USA) diluted in a ratio of 1 : 100 in antibody dilution buffer containing 1% BSA and 0.2% Triton-X-100 in PBS at 4°C overnight. After rinsing with PBS 3 times, cells were stained with FITC-labeled anti-goat or rabbit antibody, respectively (1 : 100) (Southern Biotech, AL, USA), at RT for 60 min. Cell nuclei were stained with DAPI (Sigma, MO, USA), and the cell cytoskeleton was labeled using rhodamine (1 : 2,000) (Life Technologies, CA, USA). After washing with PBS, fluorescent signals were analyzed with an Axio Observer D1 fluorescence microscope (Carl Zeiss, Germany) or a FW300 confocal fluorescent microscope (Olympus, Japan), respectively.

After treatment with Ang-II, proteins were extracted from mesenteric SMC and separated by standard SDS-PAGE. Proteins (10 µg) were transferred to PVDF membranes and immunoblotted with the following primary antibodies: calponin (Santa Cruz sc-28545), ERK1/2 Cell Signaling 9102), phosphorylated ERK1/2 (Cell Signaling 9101), PCNA (Santa Cruz Sc56, Nox4 (Epitomics 3174-1), β-actin (Santa Cruz sc-130656), GAPDH (Santa Cruz: sc-32233), and their respective secondary antibodies. Proteins were detected using chemiluminescence (ECL Plus, Amersham Biosciences). Protein expression was normalized to β-actin or GAPDH.