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To study mechanisms controlling growth and phenotype in human vascular smooth muscle cells, we established culture conditions under which these cells proliferate rapidly and achieve life-spans of 50-60 population doublings. In medium containing heparin and heparin-binding growth factors, growth rate and life-span of human vascular smooth muscle cells increased more than 50% relative to cultures with neither supplement, and more than 20% compared to cultures supplemented only with heparin-binding growth factors. In contrast to observations made in rat vascular smooth muscle cells, smooth muscle-specific alpha-actin in the human cells was expressed only in the presence of heparin and colocalized with beta/gamma nonmuscle actins in stress fibers, not in adhesion plaques. Heparin, in the presence of heparin-binding growth factors, also caused more than 170% stimulation of tracer glucosamine incorporation into hyaluronic acid and a 7.5-fold increase in hyaluronic acid accumulation. In comparison, total sulfate incorporation into sulfated glycosaminoglycans increased by less than 40%. In light of our previous findings that heparin suppresses collagen gene expression, we conclude that heparin induces human vascular smooth muscle cells exposed to heparin-binding growth factors to remodel their extracellular matrix by altering the relative rates of hyaluronic acid (HA) and collagen synthesis. The resulting hyaluronic-acid-rich, collagen-poor matrix may enhance infiltration of CD44/hyaluronate-receptor-bearing T-lymphocytes and monocytes into the vascular wall, an early event in atherogenesis.

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Vascular smooth muscle cells produce and respond to interleukin-1, a cytokine which modifies inflammation-associated vascular activities including the synthesis of extracellular matrix proteins. We have established vascular smooth muscle cells culture conditions in which heparin, in the presence of endothelial cell growth supplement, promotes cell proliferation and inhibits interleukin-1 and matrix protein expression. To test whether interleukin-1 mediates growth and matrix modulation by heparin/endothelial cell growth supplement, vascular smooth muscle cells were transfected with an Epstein-Barr virus-derived expression vector designed to express interleukin-1 antisense transcripts. RNase protection and ELISA assays demonstrated a complete block of interleukin-1 transcription and protein synthesis. Northern blot analysis also showed that interleukin-1 antisense decreased the expression of matrix genes such as type I collagen, fibronectin, and decorin similar to downregulation after heparin/endothelial cell growth supplement treatment. In contrast, the expression of versican was not affected, indicating a selective suppression of matrix proteins. In addition, interleukin-1 antisense significantly prolonged the life span of vascular smooth muscle cells in culture. Our data suggest that heparin/endothelial cell growth supplement induces matrix remodeling and controls growth and senescence of vascular smooth muscle cells through down-regulation of interleukin-1.

The autonomic outflow and sensory structures in the ovary and accessory reproductive organs of the hamster are described by means of specific fluorescence and enzyme histochemical techniques for the demonstration of catecholamine and acetylcholinesterase (AChE), respectively. Sympathetic nerves accompany branches of the major blood vessels in the mesentery of the ovary, oviduct and tubal uterine horn and invest the vascular bed in each of these organs. Vasomotor fibers predominate in the ovary and oviduct, though occasional adrenergic axons supply thecal and interstitial tissues in the ovary and the longitudinal smooth muscle of the oviduct. Fluorescent myomotor axons run in the suspensory ligament and outer myometrial layer of the uterus, but most of the numerous sympathetic and AChE-fibers in the tubal third of the horn supply the intramural and submucosal vascular plexuses. A limited electron microscopic study of the central spiral (preplacental) arteries of the endometrium indicates that the surrounding terminal AChE-fibers are identical to the fluorescent and granular vesicle-bearing adrenergic axons which form neuromuscular junctions with these vessels. Based on the discovery of specialized sensory endings in the walls of the large collecting veins which drain the hamster uterus, a mechanism is proposed to account for the regulation of blood flow through maternal placental vessels which are devoid of an arteriolar neuromuscular apparatus.

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Terrestrial mammals range over six orders of magnitude in size, while average cellular, or specific metabolic rate (sMR) varies only ~5-fold, constrained by the strict matching of protein synthesis to cell size. Protein synthesis, the single most costly metabolic activity, accounts for 20% of ATP turnover, and the speed of translation can be increased only at the expense of increased energy dissipation due to misreading errors and stochastic translation noise. We hypothesized that global microRNA activity evolved under the same constraints assMRand would therefore exhibit a similar pattern of variation. This expectation was met by the number of microRNA families: based on the manually curated microRNA gene database MirGeneDB, the acquisition of microRNA families (mirFam) accelerated two-fold in late-branching mammals with body temperatures (Tb) > 36oC, relative to early-branching mammals (Tb36oC), independent of phylogenetic distance. WhensMRis fit tomirFamby maximum likelihood, the variation is homoscedastic, allowing us to compare models for the evolution ofsMRin relation tomirFam. WithmirFamas the predictor, an Ornstein-Uhlenbeck (OU) process of stabilizing selection accounted better for the biphasic evolution ofsMRthan did the corresponding Brownian motion model, and was optimized whensMRwas scaled toM0.75. OU simulations with an adaptive shift at the divergence of Boreoeutheria predicted 95% of the variation insMR. We infer that the advent of placentation led to the emergence of an adaptive optimum characterized by higher body temperatures, faster metabolic rates, and heightened global microRNA activity.

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