Differential Effect of 17-ß-Estradiol on Smooth Muscle Cell and Aortic Explant MMP2¹

Objective

The present investigation tested the hypothesis that intrinsic gender-related differences exist in rat aortic smooth muscle cell matrix metalloproteinase 2 (MMP2).

Methods

This investigation comprised 3 sets of experiments. Experiment I: Adult male and female rat aortic smooth muscle cells (RASMCs) at passages 4-8 were stimulated in serum-free media for 48 h with interleukin(IL)1ß at doses encountered in human abdominal aortic aneurysms (2 ng/mL). Messenger RNA was extracted from the RASMCs, and gene expression of MMP2 and tissue inhibitor of metalloproteinase 2 (TIMP2), a major MMP2 inhibitor, was measured by real-time polymerase chain reaction. MMP2 protein levels in conditioned media were measured by Western blotting, and MMP2 and TIMP2 activity quantified by standard and reverse gelatin zymography. Experiment II: Male and female RASMCs were incubated for 48 h in Dulbecco's modified Eagler's medium containing IL-1β and 17-β-estradiol at doses from 1×10⁻¹⁰ to 1×10⁻⁶ molar. MMP2 activity in the conditioned media was then determined. Experiment III: Male rats underwent sustained 17-β-estradiol exposure for 21 d using extended-release, subcutaneously implanted pellets prior to sacrifice and aortic explantation. Aortas from males, females, and estradiol-treated males were stimulated with IL-1β for 48-h, and MMP2 activity in the conditioned media was determined.

Results

Experiment I: MMP2 gene expression was 3-fold higher in male compared with female IL-1β stimulated RASMCs (P<0.0001). MMP2:TIMP2 gene expression ratio was 7.5-fold greater in male versus female RASMCs. MMP2 protein levels were 3-fold higher (2.68 versus 0.96 o.d./mg total protein, P=0.003) in male versus female RASMCs. Gelatinolytic activity was more than 6-fold higher (15,010 versus 2,472 o.d./mg total protein, P=0.002) in male versus female RASMCs. Experiment II: MMP2 activity in male and female RASMCs was not altered by a wide range of 17-β-estradiol concentrations. Experiment III: When pretreated with 17-β-estradiol, MMP2 activity in the media of male rat whole-aortic explants decreased 2-fold (P=0.002). This post-17-β-estradiol treatment male level was not different than baseline female aortic explant MMP2 levels.

Conclusions

MMP2 is higher in male RASMCs compared to female RASMCs. Exogenous 17-β-estradiol did not alter MMP2 activity in vitro, but in vivo 17-β-estradiol exposure greatly decreased male aortic MMP2 production to levels seen in the female aorta. Gender differences in MMP2 are speculated to be associated with phenotypic differences in human abdominal aortic aneurysm formation.

Key Words: MMP, aorta, smooth muscle cells, estrogen, hormones