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Background: Unbalanced vascular smooth muscle cell (VSMC) proliferation, migration, and apoptosis contribute to vascular disorders such as atherosclerosis, restenosis, and pulmonary hypertension. The effect of extracellular acidosis (EA) on VSMC homeostasis is incompletely understood but we previously reported that EA increases heme oxygenase-1 (HO-1) expression in VSMCs. Since HO-1 regulates VSMC proliferation and apoptosis we sought to define the role of HO-1 in VSMC responses to EA. Methods: Mouse aortic smooth muscle cells (MASMCs) were isolated from wild-type and HO-1-null mice. Cell proliferation and migration assays were done in a physiologic pH (7.4) or EA (pH 6.8). VSMC apoptosis in response to hydrogen peroxide was assessed by JC-1 staining, caspase-3 cleavage, annexin V, and Hoechst staining. Results: Wild-type MASMCs showed decreased proliferation and migration at pH 6.8 compared to pH 7.4. This observation was also true in HO-1-null MASMCs. Although wild-type and HO-1-null cells showed differences in the mode and kinetics of cell death, both genotypes exhibited increased susceptibility to hydrogen peroxide-induced apoptosis at pH 6.8 compared to 7.4. Conclusions: EA inhibits VSMC proliferation and migration and increases susceptibility to oxidant-induced apoptosis. These effects of acidosis on VSMC homeostasis are independent of HO-1.

ETS family proteins play a role in immune responses. A unique member of this family, Elk-3, is a transcriptional repressor that regulates the expression of HO-1. Elk-3 is very sensitive to the effects of inflammatory mediators and is down-regulated by bacterial endotoxin (LPS). In the present study, exposure of mouse macrophages to Escherichia coli LPS resulted in decreased, full-length, and splice-variant isoforms of Elk-3. We isolated the Elk-3 promoter and demonstrated that LPS also decreased promoter activity. The Elk-3 promoter contains GC-rich regions that are putative binding sites for zinc-finger transcription factors, such as Sp1 and KLFs. Mutation of the GC-rich region from bp –613 to –603 blunted LPS-induced down-regulation of the Elk-3 promoter. Similar to the LPS response, coexpression of KLF4 led to repression of Elk-3 promoter activity, whereas coexpression of Sp1 increased activity. ChIP assays revealed that KLF4 binding to the Elk-3 promoter was increased by LPS exposure, and Sp1 binding was decreased. Thus, down-regulation of Elk-3 by bacterial LPS is regulated, in part, by the transcriptional repressor KLF4. Overexpression of Elk-3, in the presence of E. coli bacteria, resulted in decreased macrophage phagocytosis. To determine whether limited expression of HO-1 may contribute to this response, we exposed HO-1-deficient bone marrow-derived macrophages to E. coli and found a comparable reduction in bacterial phagocytosis. These data suggest that down-regulation of Elk-3 and the subsequent induction of HO-1 are important for macrophage function during the inflammatory response to infection.