Chronic antiprogestin therapy produces a stable atrophic endometrium with decreased fibroblast growth factor: a 1-year primate study on contraception and amenorrhea.

OBJECTIVE: To describe the efficacy of mifepristone in the prevention of menstrual bleeding and ovulation, with similar observations in comparison groups. DESIGN: Prospective experimental study. Thirty-two cynomolgus monkeys were divided equally into four treatment groups (n = 8). Treatment lasted for 1 year. INTERVENTION(S): Group I received GnRH-agonist (GnRH-a) and in-sequence mifepristone, group II received mifepristone only, group III received GnRH-a only, and group IV received vehicle control. MAIN OUTCOME MEASURE(S): Serum estradiol and progesterone, menstrual bleeding, endometrial thickness, and endometrial expression of basic fibroblast growth factor (bFGF) as determined by immunohistochemistry. RESULT(S): Weekly progesterone determinations showed that mifepristone-treated monkeys seldom ovulated (6 ovulations in 8 years), compared with the controls (100 ovulations in 8 years), while maintaining early to midfollicular levels of circulating serum estradiol. The GnRH-a-only group also rarely ovulated, but was chronically and severely hypoestrogenic. The mifepristone-only group showed scant menstrual bleeding (5 days in 8 years) as compared with the menstrual frequency in control animals (422 days in 8 years). Endometrial proliferation, as determined by biopsy, was similarly minimal for both the GnRH-a and mifepristone groups, and statistically less than in control monkeys. Both the mifepristone and GnRH-a treatments suppressed endometrial gland expression of the angiogenesis polypeptide bFGF. CONCLUSION(S): Chronic mifepristone induced anovulation along with virtual amenorrhea, which suggests the worth of this novel hormonal contraceptive.

Pericyte growth and contractile phenotype: modulation by endothelial-synthesized matrix and comparison with aortic smooth muscle.

We compared the effects of endothelial-synthesized matrix and purified matrix molecules on pericyte (PC) and aortic smooth muscle cell (SMC) growth, heparin sensitivity, and contractile phenotype in vitro. When PC are plated on endothelial-synthesized (EC) matrix, cell number is, on average, 3.1-fold higher than identical populations grown on plastic. Under the same conditions, SMC proliferation is stimulated 1.6-fold. Purified matrix molecules, such as collagen type IV (COLL) or fibronectin (FN), both major components of the EC matrix, stimulate PC/SMC growth 1.2-1.7-fold. Heparin (100 micrograms/ml), which inhibits the growth of early passage SMC by 60%, inhibits PC growth approximately 50%, when cells were plated on plastic. However, PC plated on EC matrix in the presence of heparin (100 micrograms/ml) grow as well as parallel cultures grown on plastic (in the absence of heparin). Concomitant with matrix-stimulated proliferation, we observed a marked reduction in PC containing alpha vascular smooth muscle actin (alpha VSMA), as seen by immunofluorescence using affinity-purified antibodies (173/615 positive pericytes on DOC matrix (28%) vs. 221/285 (77%) positive on glass). SMC respond similarly. Whereas alpha VSMA protein is markedly altered when PC and SMC are cultured on EC matrix, similar reductions in mRNA are not observed. However, Northern blotting does reveal that PC contain 17-30 times the steady-state levels of alpha VSMA mRNA compared to SMC. When SMC and PC cultures on plastic are treated with heparin, the steady-state levels of vascular smooth muscle actin mRNA increase 5 and 1.5 fold, respectively. Similarly, heparin treatment of PC grown on plastic induces a 1.8 fold increase in nonmuscle actin mRNA. These heparin-induced alterations in isoactin mRNA levels are not seen when PC are cultured on EC matrix. We also observed reductions in alpha VSMA and beta actin mRNA levels when PC are plated on FN, where they maintain a ratio of 13:1 (alpha:beta). Similar ratios are found in SMC present in rat and bovine aortae in vivo. These steady-state isoactin mRNA ratios are slightly different from those seen in cultured PC (8-10:1; alpha:beta). These results suggest that selective synthesis and remodelling of the endothelial basal lamina may signal alterations in pericyte growth and contractile phenotype during normal vascular morphogenesis, angiogenesis, or during the microvascular remodelling that accompanies hypertensive onset.

Beta actin and its mRNA are localized at the plasma membrane and the regions of moving cytoplasm during the cellular response to injury.

Previous work in our laboratory has shown that microvascular pericytes sort muscle and nonmuscle actin isoforms into discrete cytoplasmic domains (Herman, I. M., and P. A. D'Amore. 1985. J. Cell Biol. 101:43-52; DeNofrio, D.T.C. Hoock, and I. M. Herman. J. Cell. Biol. 109:191-202). Specifically, muscle (alpha-smooth) actin is present on the stress fibers while nonmuscle actins (beta and gamma) are located on stress fibers and in regions of moving cytoplasm (e.g., ruffles, lamellae). To determine the form and function of beta actin in microvascular pericytes and endothelial cells recovering from injury, we prepared isoform-specific antibodies and cDNA probes for immunolocalization, Western and Northern blotting, as well as in situ hybridization. Anti-beta actin IgG was prepared by adsorption and release of beta actin-specific IgG from electrophoretically purified pericyte beta actin bound to nitrocellulose paper. Anti-beta actin IgGs prepared by this affinity selection procedure showed exclusive binding to beta actin present in crude cell lysates containing all three actin isoforms. For controls, we localized beta actin as a bright rim of staining beneath the erythrocyte plasma membrane. Anti-beta actin IgG, absorbed with beta actin bound to nitrocellulose, failed to stain erythrocytes. Simultaneous localization of beta actin with the entire F-actin pool was performed on microvascular pericytes or endothelial cells and 3T3 fibroblasts recovering from injury using anti-beta actin IgG in combination with fluorescent phalloidin. Results of these experiments revealed that pericyte beta actin is localized beneath the plasma membrane in association with filopods, pseudopods, and fan lamellae. Additionally, we observed bright focal fluorescence within fan lamellae and in association with the ends of stress fibers that are preferentially associated with the ventral plasmalemma. Whereas fluorescent phalloidin staining along the stress fibers is continuous, anti-beta actin IgG localization is discontinuous. When injured endothelial and 3T3 cells were stained through wound closure, we localized beta actin only in motile cytoplasm at the wound edge. Staining disappeared as cells became quiescent upon monolayer restoration. Appearance of beta actin at the wound edge correlated with a two- to threefold increase in steady-state levels of beta actin mRNA, which rose within 15-60 min after injury and returned to noninjury levels during monolayer restoration. In situ hybridization revealed that transcripts encoding beta actin were localized at the wound edge in association with the repositioned protein. Results of these experiments indicate that beta actin and its encoded mRNA are polarized at the membrane-cytoskeletal interface within regions of moving cytoplasm.

Characterization of microvascular cell cultures from normotensive and hypertensive rat brains: pericyte-endothelial cell interactions in vitro.

We used specific markers and fluorescence microscopy to identify and characterize cerebrovascular cells. Cultures were derived from brain microvessels isolated from normotensive (Wistar Kyoto, WKY) and spontaneously hypertensive (SHR) rat brains prior to, coincident with and following the onset of chronic hypertension. Endothelial cells were characterized using di-acyl LDL and non-muscle isoactin-specific antibodies. Cerebrovascular pericytes were identified with the anti-muscle and non-muscle actin antibody staining. Using this combination of cell culture and fluorescence localization, we have been able to demonstrate that brain pericytes are tightly associated with the endothelial cells of the hypertensive-prone and hypertensive cell cultures, but not with the normotensive endothelial cultures. While the endothelial-pericyte ratio in the hypertensive-prone microvascular cultures was between 5:1 and 10:1, the number of pericytes associated with the hypertensive rat brain cultures increased two to five times (2:1-1:1). Muscle and non-muscle actin antibody staining localized the spindle-shaped pericytes of the hypertensive microvascular colonies. Pericytes were found overlaying and encircling the endothelial cells. Normotensive pericytes were not endothelial-associated. Whereas the hypertensive pericyte is devoid of stress fibers, the normotensive pericyte is a larger, spread-out cell possessing numerous stress fibers rich in muscle and non-muscle actin. These results provide the first evidence that the etiology and inception of cerebrovascular disease may be pericyte-related and suggest that pericyte contraction could play a pivotal role in regulating the flow of blood within the brain microcirculation.