Endothelial cells are exposed to an acidotic environment in a variety of pathological and physiological conditions. However, the effect of acidosis on endothelial cell function is still largely unknown, and it was evaluated in the present study. Bovine aortic endothelial cells (BAECs) were grown in bicarbonate buffer equilibrated either with 20% CO(2) (pH 7.0, acidosis) or 5% CO(2) (pH 7.4, control). Acidosis inhibited BAEC proliferation in 10% FCS, whereas by day 7 in serum-free medium, cell number was 3-fold higher in acidotic cells than in control cells. Serum deprivation enhanced BAEC apoptosis, and apoptotic cell death was markedly inhibited by acidosis. Additionally, acidosis inhibited FCS-stimulated migration in a modified Boyden chamber assay and FCS-stimulated differentiation into capillary-like structures on reconstituted basement membrane proteins. Conditioned media from BAECs cultured for 48 hours either at pH 7.0 or pH 7.4 enhanced BAEC proliferation and migration at pH 7.4, and both effects were more marked with conditioned medium from BAECs grown in acidotic than in control conditions. Acidosis enhanced vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) mRNA expression as well as bFGF secretion, and a blocking bFGF antibody inhibited enhanced BAEC migration in response to conditioned medium from acidotic cells. These results show that acidosis protects endothelial cells from apoptosis and inhibits their proangiogenic behavior despite enhanced VEGF and bFGF mRNA expression and bFGF secretion.

AimsHMGB1 injection into the mouse heart, acutely after myocardial infarction (MI), improves left ventricular (LV) function and prevents remodeling. Here, we examined the effect of HMGB1 in chronically failing hearts.Methods and ResultsAdult C57 BL16 female mice underwent coronary artery ligation; three weeks later 200 ng HMGB1 or denatured HMGB1 (control) were injected in the peri-infarcted region of mouse failing hearts. Four weeks after treatment, both echocardiography and hemodynamics demonstrated a significant improvement in LV function in HMGB1-treated mice. Further, HMGB1-treated mice exhibited a ∼23% reduction in LV volume, a ∼48% increase in infarcted wall thickness and a ∼14% reduction in collagen deposition. HMGB1 induced cardiac regeneration and, within the infarcted region, it was found a ∼2-fold increase in c-kit+ cell number, a ∼13-fold increase in newly formed myocytes and a ∼2-fold increase in arteriole length density. HMGB1 also enhanced MMP2 and MMP9 activity and decreased TIMP-3 levels. Importantly, miR-206 expression 3 days after HMGB1 treatment was 4-5-fold higher than in control hearts and 20–25 fold higher that in sham operated hearts. HMGB1 ability to increase miR-206 was confirmed in vitro, in cardiac fibroblasts. TIMP3 was identified as a potential miR-206 target by TargetScan prediction analysis; further, in cultured cardiac fibroblasts, miR-206 gain- and loss-of-function studies and luciferase reporter assays showed that TIMP3 is a direct target of miR-206.ConclusionsHMGB1 injected into chronically failing hearts enhanced LV function and attenuated LV remodelling; these effects were associated with cardiac regeneration, increased collagenolytic activity, miR-206 overexpression and miR-206 -mediated inhibition of TIMP-3.

Background-After endovascular injury, smooth muscle cells (SMCs) may be exposed to hemodynamic shear stress (SS), and these forces modulate neointima accumulation. The effect of SS on SMC migration and invasion is unknown, and it was examined in the present study. Methods and Results-Bovine aortic SMCs were exposed to laminar SS of 12 dyne/cm 2 for 3 (SS3) or 15 (SS15) hours; control (C3 and C15) SMCs were kept under static conditions. Platelet-derived growth factor (PDGF)-BB-directed SMC migration and invasion were evaluated by a modified Boyden chamber assay with filters coated with either gelatin or reconstituted basement membrane proteins (Matrigel), respectively. SS15 inhibited both SMC migration and invasion (PϽ0.0001). There was no significant difference between SS3 and C3 cells. Media conditioned with SS15 cells exhibited a reduction in matrix metalloprotease-2 (MMP-2) by zymography and Western analysis. Northern blot analysis revealed no effect of SS15 on MMP-2 mRNA. In contrast, SS15 decreased MMP-2 activator and membrane-type MMP (MT-MMP or MMP-14) mRNA and protein. Furthermore, SS15 decreased PDGF receptor-␤ (PDGF-R␤) mRNA and protein (PϽ0.05), and the SS-dependent decrease in PDGF-BB-directed cell migration was rescued by overexpressing PDGF-R␤. Conclusions-SS