A repressor protein, Mnt, is a novel negative regulator of vascular smooth muscle cell hypertrophy by angiotensin II and neointimal hyperplasia by arterial injury

Abstract

Objective: The Max-interacting protein Mnt is a transcriptional repressor that can antagonize the transcriptional and proliferation-related activities of Myc. Here, we tested the hypothesis that Mnt is a negative regulator of pathological vascular remodeling.

Methods: Adenovirus encoding Mnt or control GFP was infected to cultured rat vascular smooth muscle cells (VSMC) and carotid arteries after a balloon angioplasty.

Results: In VSMC, adenoviral gene transfer of Mnt suppressed angiotensin II-induced protein expression of early growth response protein-1 (Egr1) and its promoter activation. Mnt adenovirus did not interfere with upstream signaling of angiotensin II. Angiotensin II-induced protein accumulation in VSMC was inhibited by Mnt adenovirus. Mnt adenovirus also inhibited platelet-derived growth factor-induced VSMC proliferation. Moreover, Mnt adenovirus prevented neointima formation in response to arterial injury. The adenoviral Mnt gene transfer also prevented Egr1 induction in neointima.

Conclusion: These data identify Mnt as a previously unrecognized negative regulator of pathological vascular remodeling.

Fig. 1

Effects of Mnt gene transfer by adenovirus on Ang II signals in VSMC. VSMC were infected with adenovirus expressing Mnt or control GFP at 100 moi for 2 days. A, The cells were stimulated with 100 nM Ang II for 1 hour. Cell lysates were analyzed by immunoblotting with antibodies as indicated. The densitometry analysis of the data is from quadruplicated experiments (mean ± SEM, * p <0.05 compared with stimulated control). Representative blots are shown. B, After the adenoviral infection, cells were cotransfected with the egr-1 promoter luciferase construct and control vector (SEAP). Serum-deprived cells were incubated with 100 nM Ang II for 24 hours. Bars represent % induction in normalized luciferase activity from quadruplicated experiments (mean ± SEM, * p <0.05 compared with stimulated control). C, The cells were stimulated with 100 nM Ang II for 10 min (ERK-p, ERK2 and Mnt) or 2 min (EGFR-p and EGFR). Cell lysates were analyzed by immunoblotting with antibodies as indicated. Representative blots are shown from the quadruplicated experiments.

Fig. 2

Mnt gene transfer inhibited hypertrophy and proliferation of VSMC in vitro and neointimal hyperplasia in vivo. A, Subconfluent VSMC infected with adenovirus (100 moi) encoding Mnt or the control GFP vector were stimulated with Ang II for 3 days and cellular protein contents were determined as a marker of hypertrophy from quadruplicated experiments (mean ± SEM, * p <0.05 compared with stimulated control). B, VSMC were infected with adenovirus (100 moi) encoding Mnt or control GFP vector for 2 days. The cells were then stimulated with 25 ng/mL PDGF-BB for 3 days and cell numbers were determined by a Coulter counter from quadruplicated experiments. Data are mean±SEM. *p < 0.05 compared to the stimulated control. C, Mnt adenovirus inhibited arterial neointima formation after balloon injury. Representative sections (x40 magnification) are shown. 14 days after the injury, common carotid artery was stained and the area of neointima and media were quantified. Data are mean ± SEM of sections from 4 rats. *p<0.05 compared to the GFP adenovirus-infected control. D, Arterial sections obtained on day 14 after injury were stained with Egr1 antibody (x200 magnification). Representative sections (x200 magnification) are shown.