Oestrogen-dependent expression of the SM2 smooth muscle-type myosin isoform in rabbit myometrium.

Ovarectomized rabbits displayed a decreased SM1 to SM2 ratio of smooth muscle-type myosin heavy chain isoforms compared to unoperated, virgin females which was reversed after 17beta-oestradiol administration to a value similar to that of control animals. When this steroid was given to sexually immature animals or to adult virgin rabbits, SM2 expression was not induced, as also happened with proliferating myometrial smooth muscle cells grown in vitro. In growing rabbit, the 17beta-oestradiol administration induced the formation of the circular and the longitudinal muscle layers, characteristics of sexually competent females. The SM2 isoform was up-regulated during postnatal development and the SM1 to SM2 ratio changed during pregnancy and post-partum period but not with human gonadotropin treatment which increases the level of circulating progesterone. Immunofluorescence staining of adult myometrium with anti-SM2 antibody indicated that this isoform is localized to the longitudinal layer exclusively and, in contrast to the circular layer, its expression was independent of oestrogen level. Difference in oestrogen sensitivity between the two layers was also detected for the expression of the intermediate filament protein vimentin and the thin filament protein calponin. Changes of SM2 expression in the myometrium correlated with variations in the oestrogen receptor density as also confirmed by decreased SM2 content/oestrogen receptor density in the circular layer when ovarectomized females were treated with the oestrogen antagonist ICI 182,780. Our results indicate that: (1) a specific distribution of myosin heavy chain exists within rabbit myometrium, and (2) SM2 myosin expression in this smooth muscle is under oestrogen control.

Anipamil prevents intimal thickening in the aorta of hypertensive rabbits through changes in smooth muscle cell phenotype.

The aim of this study was to evaluate the effect of anipamil, a phenyalkylamine-derived Ca(2+)-antagonist, on aortic intimal thickening and smooth muscle cell (SMC) phenotype in 2K-1C hypertensive rabbits. Monoclonal antimyosin antibodies [SM-E7, NM-G2, and NM-F6, which respectively, recognize smooth muscle (SM), A-type-, and B-type-like nonmuscle (NM) myosin heavy chains (MyHC)] identify different aortic SMC types: adult (SM-E7-positive), postnatal (SM-E7- and NM-G2-positive), and fetal (SM-E7-, NM-G2-, and NM-F6-positive). Twenty-four hypertensive rabbits were studied 2.5 months (n = 12) and 4 months (n = 12) after clipping. Six animals from each group were given anipamil (40 mg orally, once daily) immediately after surgery. The remaining 2K-1C were given a daily oral placebo. Normotensive age-matched controls were also studied. Transverse cryosections of aorta were taken for computerized morphometry and immunocytochemical studies. Primary and secondary SMC cultures were used to define potential changes in cell phenotype after adding anipamil to the culture medium. In untreated 2K-1C, intimal thickening, mainly composed of postnatal-type SMC, was found by 2.5 months after clipping. Morphometric and immunofluorescence studies in anipamil-treated 2K-1C rabbits revealed absent or negligible intimal thickening and a decrease of postnatal-type SMC from the underlying media. In culture experiments, growth inhibition of SMC by anipamil was accompanied by the expression of SM-MyHC in all SMC, ie, the appearance of a more differentiated cell phenotype compared to control cultures. In conclusion, prevention of intimal thickening in anipamil-treated 2K-1C was achieved through selective reduction in the media of the postnatal-type SMC. This could be achieved by reducing NM-MyHC content or increasing synthesis of SM-MyHC expression. As blood pressure was not significantly lowered by anipamil treatment, a direct and specific antiproliferative action of this drug on medial SMC is likely to take place.

Expression of non-muscle myosin isoforms in rabbit myometrium is estrogen-dependent.

The putatative effects of different estrogen levels on the expression of non-muscle myosin isoforms in rabbit myometrium have been investigated using three monoclonal anti-platelet myosin heavy chain (MyHC) antibodies (NM-F6, NM-G2, and NM-A9). Western blotting analysis of proteolytic digests of human platelet actomyosin indicates that these antibodies are specific for three distinct epitopes. Comparative immunofluorescence tests on cultered human fibroblasts with polyclonal sequence-specific anti-MyHCA antibody suggest that the patterns of NM-F6, NM-.G2 and NM-A9, although similar, do not overlap with that of type-A MyHC. Distribution of NM myosin isoforms has been studied in indirect immunofluorescence assays using cryosections of tissues from rabbits at various stages of development, pregnancy, or from ovariectomized, 17beta-estradiol-treated ovariectomized, and human chorionic gonadotropin-treated animals. Non-muscle myosin antigenicity is still present in the myometrium when the female becomes sexually competent. The immunoreactivity of non-muscle myosin for NM-F6 is steroid-independent, since it does not change with pregnancy or ovariectomy, but that of NM-G2 is estrogen-dependent; the latter disappears during pregnancy and in ovariectomized animals treated with estradiol, whereas it is expressed in ovariectomized rabbits. Although non-muscle myosin immunoreactivity for NM-A9 is detectable under all the experimental conditions, it can assume different patterns of intracellular distribution in vitro (punctate vs filamentous), depending on culture conditions and the presence of estrogens.

Smooth muscle cell types at different aortic levels and in microvasculature of rabbits with renovascular hypertension.

BACKGROUND: The monoclonal antimyosin antibodies smooth muscle (SM)-E7, non-muscle (NM)-G2 and NM-F6 recognize smooth muscle myosin heavy chains, and A- and B-like non-muscle myosin heavy chains, respectively. On this basis, aortic smooth muscle cell types have been identified as adult (SM-E7-positive), postnatal (SM-E7- and NM-G2-positive) and fetal (SM-E7-, NM-G2- and NM-F6-positive). We have demonstrated previously that hypertrophy of the smooth muscle cell layer of the upper aorta in two-kidney, one clip hypertensive rabbits is achieved via a selective increase in postnatal-type smooth muscle cells. OBJECTIVE: To monitor the time-course change of postnatal-type smooth muscle cells along the entire aortic tree and to define the phenotypic characteristics of the microvasculature in the same rabbit model. MATERIALS AND METHODS: Hypertensive rabbits were killed 0.5, 1, 2.5, 4, 6 and 8 months after clipping. Normotensive age-matched rabbits served as controls. The entire aorta was frozen during perfusion at a constant pressure for morphometric and immunocytochemical studies. Transverse cryosections were taken 1 cm from the aortic valve (level A), immediately after the anonymous trunk (level B), immediately before the diaphragm (level C), and near the bifurcation (level D). Small vessels and arterioles were studied in psoas skeletal muscle and in left ventricular myocardium. RESULTS: On the whole, aortae from hypertensive rabbits displayed a striking increase in postnatal-type smooth muscle cells at all levels by 4 months of hypertension and a progressive decrease in the number of these cells to near the control value by 8 months of hypertension. A peculiar pattern of myosin heavy chain expression was found in the microvasculature. In control and in hypertensive rabbits, both at 4 and at 8 months, small vessels and arterioles were equally reactive with the three antimyosin heavy chain antibodies. This indicates a basic prevalence of fetal-type smooth muscle cells, which is little influenced by blood pressure. CONCLUSIONS: The present data elucidate some of the basic changes which the entire aortic segment and microvasculature undergo in the present experimental model.