Files in this Data Supplement:

• Figure I - (MS PowerPoint file) (481 KB). Representative Western blot analyses with β-actin as a loading control of human cultured umbilical vein endothelial cells (HUVEC) which were exposed to cyclic stretch (15% elongation at 0.5 Hz) for 18 hours or exposed to cyclic stretch followed by a 3-hour recovery phase show that CD40 protein is initially down-regulated by cyclic stretch but returns towards the basal level if the biomechanical stimulus ceases.
• Figure II - (MS PowerPoint file) (1367 KB). Analysis of cell culture supernatants for cytokine expression. (A) Supernatants of three experiments with each experimental set-up (solo endothelial cells (EC) and EC/human smooth muscle cell (SMC) co-cultures under static conditions or exposed to 18 hours of cyclic stretch) were pooled and analyzed by using a cytokine protein array. Relative concentrations of 76 cytokines were determined and the increase or decrease upon cyclic stretch was calculated. TGF-β1 is only slightly increased in stretch stimulated direct co-cultures (arrow). (B) Semi-quantitative RT-PCR analyses (statistical summary with the CD40/RPL32 ratio under control conditions set to 100% and representative gels shown beneath) revealed a modest increase in TGF-β1 expression in stretch stimulated EC/SMC co-cultures which did not gain statistical significance though (n.s., not significant).
• Figure III - (MS PowerPoint file) (5638 KB). Analysis of CD40 protein abundance and mRNA expression in stretch stimulated HUVEC co-cultured with human SMC (HSMC) separated by a defined space. (A) HUVEC (right hand side) were co-cultured with HSMC (left hand side) at an initial distance of 2 mm (red fluorescence, endothelial cell specific CD31 staining; blue fluorescence, Hoechst 33298 nuclear staining; scale bar: 50 μm). CD40 protein abundance was analyzed by Western blot (B,C) with the CD40/CD31 ratio under control conditions set to 100% and representative blots shown beneath and CD40 mRNA levels by semi-quantitative RT-PCR (D,E) under static conditions or following 18 hours exposure to cyclic stretch in the absence (B,D) or presence (C,E) of a neutralizing anti-human TGF-β1 antibody at a concentration of 2 μg/mL (** p < 0.01; n.s., not significant). The data indicate that exposure to cyclic stretch does not cause an increase in endothelial cell CD40 expression when the co-culture conditions prevent a close interaction between the two cell types while blockade of TGF-β1 activity in the co-culture setup mimics the inhibitory effect of cyclic stretch on CD40 protein abundance in solo cultured HUVEC.
• Figure IV - (MS PowerPoint file) (217 KB). Analysis of CD40 protein abundance and Alk-1 mRNA expression in paracrine (distant) co-cultures of HUVEC and HSMC. Addition of soluble Alk-1 (2 μg/mL) does not affect endothelial cell CD40 protein abundance in this co-culture set-up upon exposure to 18 hours of cyclic stretch (A, statistical summary; n.s., not significant; representative blots are shown beneath). Likewise, paracrine (distant) co-culture conditions do not facilitate stretch dependent up-regulation of endothelial cell Alk-1 mRNA levels (B, statistical summary; n.s., not significant; representative gels are shown beneath).
• Figure V - (MS PowerPoint file) (324 KB). Analysis of TGF-β1 induced changes in CD40 protein abundance in solo cultured HUVEC. (A) Only high concentrations of TGF-β1 (8 and 16 ng/mL) up-regulate CD40 protein abundance in cells under static conditions (representative Western blot analysis with β-actin as a loading control). (B) While low concentrations of TGF-β1 (2 ng/mL, 4-hour exposure) again do not cause a rise in CD40 protein in quiescent cells (left panel), this is significantly up-regulated in cells that have been exposed to cyclic stretch for 24 hours prior to the addition of TGF-β1 (right panel), suggesting that cyclic stretch enhances the endothelial cell responsiveness towards the cytokine. Statistical summary of the Western blot analyses (representative blots shown beneath) with the CD40/β-actin ratio under control conditions and following 24-hour exposure o cyclic stretch set to 100%t (* p < 0.05; n.s., not significant).