Coronary smooth muscle differentiation from proepicardial cells requires rhoA-mediated actin reorganization and p160 rho-kinase activity.

We recently reported that the first detectable expression of SMC-specific proteins during coronary smooth muscle cell (CoSMC) differentiation from isolated proepicardial cells was restricted to cells undergoing epithelial-to-mesenchymal transformation (EMT). The objectives of this study were to examine more closely the relation between actin cytoskeletal rearrangements and serum response factor (SRF)-dependent transcription, and to specifically test whether rhoA-GTPase signaling is required for CoSMC differentiation. We report here that PDGF-BB stimulates EMT and promotes SRF-dependent expression of SMC marker genes calponin, SM22alpha, and SMgamma(actin) (SMgammaA) in proepicardial cells. C3 exoenzyme or rhoGDI, inhibitors of rhoA signaling, blocked PDGF-BB-induced EMT, prevented actin reorganization into stress fibers, and inhibited CoSMC differentiation. Incubation with the selective p160 rho-kinase (p160RhoK) inhibitor Y27632 (RKI) blocked EMT, prevented the appearance of calponin and SMgammaA-positive cells, and abolished expression and nuclear localization of SRF. To test the role of RhoK signaling for CoSMC differentiation in vivo, quail proepicardial organs (PEOs) were pretreated with RKI or vehicle and then grafted into age-matched host chick embryos to produce a chimeric epicardium. The ability of grafted cells to participate in coronary vessel formation was monitored by staining with antibodies for quail cell nuclear antigen and SMC marker proteins. Proepicardial cells pretreated with RKI failed to form CoSMCs in vivo. Time course studies traced this deficiency to a failure of epicardial-derived mesenchymal cells to migrate into or survive within the myocardium. In summary, these data point to important roles for rhoA-RhoK signaling in molecular pathways controlling cytoskeletal reorganization, SRF-dependent transcription, and cell survival that are required to produce CoSMCs from proepicardial cells.

A role for serum response factor in coronary smooth muscle differentiation from proepicardial cells.

Coronary artery smooth muscle (SM) cells originate from proepicardial cells that migrate over the surface of the heart, undergo epithelial to mesenchymal transformation and invade the subepicardial and cardiac matrix. Prior to contact with the heart, proepicardial cells exhibit no expression of smooth muscle markers including SMalphaactin, SM22alpha, calponin, SMgammaactin or SM-myosin heavy chain detectable by RT-PCR or by immunostaining. To identify factors required for coronary smooth muscle differentiation, we excised proepicardial cells from Hamburger-Hamilton stage-17 quail embryos and examined them ex vivo. Proepicardial cells initially formed an epithelial colony that was uniformly positive for cytokeratin, an epicardial marker. Transcripts for flk-1, Nkx 2.5, GATA4 or smooth muscle markers were undetectable, indicating an absence of endothelial, myocardial or preformed smooth muscle cells. By 24 hours, cytokeratin-positive cells became SMalphaactin-positive. Moreover, serum response factor, undetectable in freshly isolated proepicardial cells, became strongly expressed in virtually all epicardial cells. By 72 hours, a subset of epicardial cells exhibited a rearrangement of cytoskeletal actin, focal adhesion formation and acquisition of a motile phenotype. Coordinately with mesenchymal transformation, calponin, SM22alpha and SMgammaactin became expressed. By 5-10 days, SM-myosin heavy chain mRNA was found, by which time nearly all cells had become mesenchymal. RT-PCR showed that large increases in serum response factor expression coincide with smooth muscle differentiation in vitro. Two different dominant-negative serum response factor constructs prevented the appearance of calponin-, SM22alpha- and SMgammaactin-positive cells. By contrast, dominant-negative serum response factor did not block mesenchymal transformation nor significantly reduce the number of cytokeratin-positive cells. These results indicate that the stepwise differentiation of coronary smooth muscle cells from proepicardial cells requires transcriptionally active serum response factor.

Intralesion injection of basic fibroblast growth factor alters glial reactivity to neural trauma.

A traumatic injury to the rat brain is known to induce astrocyte proliferation and hypertrophy leading to astrogliosis. In addition, trauma also induces microglial proliferation in the brain. Since basic fibroblast growth factor (bFGF) is believed to play a role in mediating glial responses to brain injury, we examined the effects of bFGF administration on astrocyte and microglial proliferation and astrocyte hypertrophy resulting from a traumatic injury to the rat brain. Intracerebroventricular infusion of bFGF for 2 days prior to and following injury failed to alter glial reactivity. In contrast, a single intralesion injection of bFGF immediately after injury increased total cell division 2 and 5 days later, with an exclusive effect on glial fibrillary acidic protein-negative glia which consisted mostly of cells of macrophage-microglial phenotype. In addition, bFGF also enhanced injury-induced astrocyte hypertrophy. These results support a role for bFGF in macrophage-microglia proliferation and astrocyte hypertrophy following brain injury. They also suggest that alteration of injury responses of nonneuronal cells of the brain may be a potential side effect of intracerebral administration of bFGF.

Adipose tissue lipolysis in vitro: a predictor of diet-induced obesity in female rats.

Wide ranges in weight and fat gain in response to high-fat diets have been reported in rats. This study measured epinephrine-stimulated lipolysis in adipose tissue of normal weight, 3-mo-old, female Sprague-Dawley rats as a metabolic predictor of "efficient gain" before feeding them a high-fat (84% kcal) diet for 11 wk. Subcutaneous (inguinal) adipose tissue (1-1.5 g) was excised, and the glycerol release was measured after incubation with 0, 10(-7), 10(-5), and 10(-3) M epinephrine. Differences in gain efficiency explained much of the variation in lipid (r = 0.78) and weight (r = 0.83) gain. Low glycerol release by 10(-5) and 10(-3) M epinephrine was significantly correlated to high final fat accumulation. Low and high quartiles based on glycerol release were not different in numbers of adipocytes or food intake but were significantly different in gain efficiency (P = 0.011), final weight (P = 0.036), carcass lipid (P = 0.033), and carcass lean mass (P = 0.017). In conclusion, female Sprague-Dawley rats are preferentially predisposed to efficiency of adipose accumulation on a high-fat diet. This tendency is negatively correlated in the preobese state to the lipolytic response to epinephrine of subcutaneous adipose tissue in vitro.

Effect of fish oil supplementation and exercise on serum lipids and aerobic fitness.

The effects of fish oil supplementation and exercise were investigated in healthy, previously sedentary males, ages 19-34. Thirty-two subjects were assigned to four groups: control (C), fish (F), exercise (E), fish and exercise (FE). The fish groups consumed 4 g.d-1 of omega-3 fatty acids. The exercise groups performed aerobic exercise for one hour three per week. The study was conducted for 10 weeks with pre and post values obtained for cholesterol, triglycerides, HDL-cholesterol, LDL-cholesterol, maximal oxygen consumption (VO2max), ventilatory anaerobic threshold (VAT), percent body fat, and dietary composition of macronutrients and polyunsaturated to saturated fat (P:S) ratio. No significant differences were noted between groups for any of the blood lipid values, percent body fat or dietary variables. VO2max and VAT did exhibit significant changes among groups. VO2max was greater for the exercise groups (E, FE) as compared to the control group (p less than 0.05). E, but not FE, was significantly greater than F. VAT was significantly greater in F, E, and FE as compared to controls, however the control's VAT decreased slightly. The slight improvement, although statistically non-significant, in VO2max and VAT by the F group requires further study. This data indicates an improvement in aerobic metabolism from aerobic exercise, alone or in combination with fish oil, compared to controls.