Tissue Homogenization and Gelatin Zymography. Snap-frozen MCAs were homogenized, and gelatin zymography was performed on them as described previously (Harris et al., 2005). Gelatinolytic activity check FAQ was assessed by densitometric analysis (Gel-Pro version 3.1; Media Cybernetics, Carlsbad, CA). Tissue inhibitor of metalloproteinase-2 (TIMP-2) levels were measured by enzyme-linked immunosorbent assay (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK). Immunoblotting. MCAs were homogenized in modified radioimmunoprecipitation assay buffer (50 mM Tris-HCl, 1% Nonidet P-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 ��g/ml aprotinin, 1 ��g/ml leupeptin, 1 ��g/ml pepstatin, 1 mM sodium orthovanadate, and 1 mM sodium fluoride) and sonicated at room temperature for 8- to 10-s bursts.

Samples were placed on ice between sonications. Total protein was measured using the Bradford method (Bio-Rad Laboratories, Hercules, CA). Vascular extracts (20 ��g) were separated on 10% SDS gels and transferred to a nitrocellulose membrane in Tris-glycine transfer buffer supplemented with 20% methanol. The immunoblots were blocked for 1 h in 5% bovine serum albumin diluted in 0.2 M Tris-base, 1.4 M NaCl, 0.1% Tween 20, and 0.02% NaN3. MMP-2 and MMP-13 antibodies were from Calbiochem (Cambridge, MA). Collagen type-1 levels were quantified by slot-blot analysis using an antibody from BD Biosciences Transduction Laboratories (San Jose, CA).

To determine the effect of diabetes and bosentan treatment on ET receptor expression, in a separate set of vehicle- and bosentan-treated control and diabetic animals, one MCA was frozen intact, and in the other MCA endothelium was denuded by passing an air bubble after the vessel was mounted on a pressurized arteriograph (Miller et al., 2001). To identify VSM ET receptors (vETs) and endothelial ET receptors (eETs), endothelium-intact (vETA, vETB, and eETB) and endothelium-denuded (vETA and vETB) MCAs were used in immunoblotting experiments using antibodies for ETA and ETB receptors as recommended by the manufacturer (Alomone Labs, Jerusalem, Israel). Specificity of the bands was confirmed by using increasing Batimastat concentrations of competing peptide for each antibody. eETB was calculated as the difference in band intensities of intact and denuded vessels. In all immunoblotting experiments, bands were visualized using ChemiGlow and images were captured using Alpha Imager from Alpha Innotech (San Leandro, CA). All blots were stripped and reprobed with anti-actin antibody to ensure equal protein loading. Statistical Analysis. A rank transformation was applied to the data before analysis to address issues of non-normality and heterogeneity of variance (Conover and Iman, 1981).