Molecular mechanisms of decreased smooth muscle differentiation marker expression after vascular injury

Abstract

While it is well established that phenotypic modulation of vascular smooth muscle cells (VSMCs) contributes to the development and progression of vascular lesions, little is known regarding the molecular mechanisms of phenotypic modulation in vivo. Here we show that vascular injury reduces transcription of VSMC differentiation marker genes, and we identify cis regulatory elements that may mediate this decrease. Using a carotid wire-injury model in mice carrying transgenes for smooth muscle alpha-actin, smooth muscle myosin heavy chain, or a SM22alpha promoter-beta-gal reporter, we collected arteries 7 and 14 days after injury and assessed changes in endogenous protein and mRNA levels and in beta-gal activity. Endogenous levels for all markers were decreased 7 days after injury and returned to nearly control levels by 14 days. beta-gal staining in all lines followed a similar pattern, suggesting that transcriptional downregulation contributed to the injury-induced decreases. To begin to dissect this response, we mutated a putative G/C-rich repressor in the SM22alpha promoter transgene and found that this mutation significantly attenuated injury-induced downregulation. Hence, transcriptional downregulation contributes to injury-induced decreases in VSMC differentiation markers, an effect that may be partially mediated through a G/C-rich repressor element.

Figure 1

Western blot analysis of the SMMHC antibody used to assess changes in SMMHC protein levels after injury. Protein extracts from rat and mouse aorta, cultured VSMCs, and cultured endothelial cells were separated by SDS-PAGE, immobilized on a PVDF membrane and probed with a rabbit anti-chicken SMMHC antibody (17) as described in Methods. SM1 and/or SM2 isoforms of SMMHC were detected in protein extracts from mouse aorta (lane 1), rat aorta (lane 2), and cultured rat VSMCs (lane 3), but not cultured rat endothelial cells (lane 4).

Figure 2

Histological assessment of vessel morphology and immunohistochemical analysis of SMαA and SMMHC protein of mouse carotid arteries post injury. Mice were sacrificed 7 days or 14 days after injury, and carotids were collected and fixed in 10% formalin (see Methods). Tissue was processed for routine histology and hemotoxylin and eosin–stained (a–d). Sections were also stained using standard immunohistochemical techniques with antibodies specific to SMαA (mouse anti-human, Sigma Chemical Co.) (e–h) or SMMHC (a gift from U. Groschel-Stewart) (17) (i–l). Visualization was accomplished by diaminobenzidine. Arrows denote the internal elastic lamina, and the arrowheads denote the external elastic lamina.

Figure 3

In situ hybridization analyses of SMαA, SMMHC, and SM22α at 7 and 14 days after carotid injury. Mice were sacrificed 7 or 14 days after injury, and carotids were collected, fixed in 10% formalin, and processed for paraffin sectioning. Sections were treated as described in Methods, hybridized with ³⁵S-UTP labeled antisense riboprobes specific for SMαA (a–d), SMMHC (e–h), or SM22α (i–l) mRNA for 16 hours at 50°C, treated with RNaseA, and washed at 55°C in 1×SSC/0.1% SDS. Sections were then exposed to photographic emulsion for 3 weeks. Serial sections probed with sense riboprobes served as controls. There was little nonspecific binding of any sense probe (data not shown). Arrows denote the internal elastic lamina, and the arrowheads denote the external elastic lamina.

Figure 4

β-Galactosidase staining of mouse carotid arteries from SMαA (a–d), SMMHC-LacZ (e–h), SM22α-LacZ (i–l), and SM22αgc-LacZ (m–p) transgenic mice 7 or 14 days after injury or in uninjured controls. The left common carotid was injured as described in Methods, and the right carotid served as control. Carotids were harvested, stained for β-gal activity, processed for histology, and counterstained with eosin. Arrows denote the internal elastic lamina, and the arrowheads denote the external elastic lamina.

Figure 5

Basal expression of SM22α-lacZ and SM22αgc-LacZ transgenes at various embryonic time points and in adult aorta. Embryos and adult tissues were collected, stained for LacZ activity, and cleared as described previously (11). There was no qualitative differences in expression pattern due to the mutation of the G/C-rich region.

Figure 6

EMSA of SP1/SP3 binding to the SMMHC and SM22α G/C-rich element and effect of deletion mutant in SM22α. ³²P end-labeled oligonucleotides probes were as described in Methods. Incubation of radiolabeled probes with cultured VSMC nuclear extracts resulted in two distinct shift bands with either the SMMHC or SM22α G/C-rich elements (lanes 1 and 6). Antibodies to SP1 and SP3 supershifted the upper and lower bands, respectively (lanes 2, 4, and 5 and 7, 9, and 10, respectively). Human recombinant SP1 (Promega Corp., Madison, Wisconsin, USA) also bound both G/C-rich elements (lanes 3 and 8). The G/C deletion used to generate SM22gc-LacZ mice abrogated binding of SP1 and SP3 (lane 11) from nuclear extracts as well as recombinant SP1 (lane 12). Rec., recombinant.