David S. Loose-Mitchell

Prevention of arterial thrombosis by adenovirus-mediated transfer of cyclooxygenase gene.

BACKGROUND Prostacyclin is an important vasoprotective molecule. It inhibits platelet aggregation, monocyte interaction with endothelium, and smooth muscle cell lipid accumulation. Vascular cyclooxygenase-1 (COX-1) is the rate-limiting step in prostacyclin synthesis. The objective of this study was to determine whether adenovirus-mediated transfer of COX-1 could restore COX-1 activity, augment prostacyclin synthesis, and prevent thrombus formation in a porcine carotid angioplasty model. METHODS AND RESULTS Human COX-1 cDNA driven by a cytomegalovirus promoter was constructed into a replication-defective adenovirus 5 vector by homologous recombination. Recombinant adenovirus without a foreign gene (Ad-RR) and buffer were included as controls. Recombinant Ad-LacZ was used for marking the transfected cells in vivo. In the in vitro experiments, cultured human endothelial cells (ECs) and porcine arterial smooth muscle cells (SMCs) were incubated with Ad-COX-1 for 2 hours and 6-keto-PGF(1 alpha) level and the transgene expression were determined 72 hours after infection. In the in vivo experiments, recombinant adenoviruses were directly instilled into angioplasty-injured porcine carotid arteries for 30 minutes. Cyclic flow changes were monitored for 10 days and thrombus formation was examined histologically thereafter. Transgene expression and prostaglandin I2 (PGI2) synthesis by the injured arteries were determined. Cultured ECs infected with Ad-COX-1 produced a fivefold to eightfold increase in PGI2, and the transgene expression in cultured porcine SMCs was demonstrated by Northern analysis. Direct administration of Ad-COX-1 at a dose of 3 x 10(10) pfu completely inhibited carotid cyclic flow changes and thrombus formation accompanied by a fourfold increase in PGI2 synthesis by the injured arteries 10 days after infection, whereas Ad-COX-1 at a lower dose, 5 x 10(9) pfu, had no antithrombotic effects when compared with Ad-RR vector and buffer controls. CONCLUSIONS Adenovirus-mediated transfer of COX-1 to angioplasty-injured carotid arteries was efficacious in augmenting PGI2 synthesis and was associated with an inhibition of thrombosis when a relatively high titer of adenovirus was instilled.

Estrogen regulates expression of the jun family of protooncogenes in the uterus

Treatment of immature female rats with estradiol increases uterine levels of c-jun and jun-B mRNAs approx. 10-fold. This effect is specific for estrogenic steroids. The induction of jun transcripts is blocked by actinomycin D but not puromycin, suggesting that the hormonal effect is due at least in part to transcriptional activation. The hormone effect is rapid and peak levels of jun mRNAs are seen within 3 h after treatment. Inductions of jun and fos transcripts in the uterus by estradiol exhibit similar dose response curves (maximum responses at 4 micrograms/kg). Estradiol also elevates uterine levels of jun-D, and this induction is insensitive to puromycin. In vivo treatment with the phorbol ester TPA rapidly elevates uterine levels of fos, jun, and myc transcripts, indicating that expression of these protooncogenes is under non-estrogenic as well as estrogenic regulation in this target tissue. These results suggest that multiple members of the jun and fos protooncogene families may play a role in amplifying the uterine response to estrogens.

Interactions between estrogen and EGF in uterine growth and function.

The rat uterus contains specific, high-affinity EGF receptors which possess a tyrosine kinase activity. As demonstrated autoradiographically, these receptors are present in the epithelial, stromal and myometrial cells of the uterus. Estrogen treatment in vivo produces a 2-3-fold increase in EGF receptor levels in the immature rat, the immature mouse and the ovariectomized adult rat; furthermore, EGF receptor levels vary throughout the estrus cycle in concert with levels of occupied nuclear estrogen receptor. This estrogen-induced increase in EGF receptor is preceded by an increase in the level of EGF receptor mRNA as judged by Northern blot analysis. In general, there is a good correlation between estrogen-induced DNA synthesis and EGF receptor levels in the uterus, although in certain situations EGF receptor levels are elevated without a subsequent increase in DNA synthesis. These observations suggest that an increase in tissue EGF receptor levels is important in estrogen-induced uterine growth, but that this increase in receptor levels alone is not sufficient to stimulate DNA synthesis. In addition to its possible role in tissue growth, we have shown very recently that EGF causes contraction of myometrial smooth muscle in a completely in vitro organ bath system. The qualitative nature of this contractile response is distinct from that produced by other classical uterotonic agents. The physiological significance of this uterine response to EGF remains to be elucidated.

Inhibition of endothelial cell prostaglandin H synthase gene expression by naproxen

Naproxen is a potent anti-inflammatory drug whose action is attributed to inhibition of prostaglandin biosynthesis. In view of our recent discovery that aspirin and sodium salicylate are capable of reducing cellular levels of prostaglandin H (PGH) synthase mRNA, we have evaluated the effect of naproxen on PGH synthase protein and mRNA levels in cultured human umbilical vein endothelial cells (HUVEC). PGH synthase mRNA levels were quantified by a competitive polymerase chain reaction (PCR) assay; protein was assessed by Western blotting. Naproxen decreased the PGH synthase protein level in HUVEC in a concentration-dependent manner. It abolished entirely the 70 kDa PGH synthase subunit at 5 micrograms/ml. It appears more effective in blocking interleukin-1 inducible PGH synthase levels. Naproxen also inhibited the synthase mRNA level in a concentration-dependent manner; levels were reduced by 33% at 5 micrograms/ml and 60% at 30 micrograms/ml naproxen. These results indicate that naproxen, like the salicylates, inhibits PGH synthase levels in cultured endothelial cells either by inhibiting transcription of the PGH synthase gene or by destabilizing its messenger RNA.

Cholesterol metabolism in hypercholesterolemia-resistant rabbits.

Normal rabbits typically respond to a diet high in cholesterol with a large increase in the concentration of plasma cholesterol. We have previously described the breeding and partial characterization of a variant rabbit which does not respond to a high cholesterol diet with changes in plasma cholesterol concentration. In the present report we have characterized three components involved in cholesterol homeostasis: the B/E (LDL) receptor, 3-hydroxy-3-methylglutaryl coenzyme A reductase activity (HMG-CoA reductase, EC 1.1.1.34) and acyl-coenzyme A: cholesterol acyltransferase activity (ACAT, EC 2.3.1.26) in the livers of the hypercholesterolemia-resistant rabbits. Using normal cholesterol-fed rabbit [125I] beta-VLDL as a ligand, liver membranes prepared from resistant rabbits fed a low-cholesterol diet had 70% higher binding capacity than membranes from normal rabbits fed the same diet. Similar experiments demonstrated that the resistant rabbits had a 240% higher B/E receptor binding capacity compared to normal animals when liver membranes were prepared from animals fed a 0.25% cholesterol-enriched diet. No difference in the binding affinity of [125I]beta-VLDL was detected in membranes prepared from normal or resistant animals. When fed a low-cholesterol diet, the resistant rabbits had approximately 2-fold higher hepatic HMG-CoA reductase activity (97.4 +/- 3.5 pmol product/mg/min in resistant animals compared to 45 +/- 1.1 pmol product/min/mg in normal animals). The difference was exaggerated in animals fed the 0.25% cholesterol-enriched diet, 73.3 +/- 5.5 vs 2.4 +/- 0.56 pmol product/min/mg for resistant and normal membranes respectively. The basal activity of ACAT in hepatic membranes was significantly lower in the resistant rabbits compared to normal rabbits (138 +/- 11 vs 268 +/- 19 pmol cholesteryl ester/min/mg in resistant and normal rabbits respectively); when fed a 0.25% cholesterol-enriched diet, the enzyme was induced 6-fold in normal animals but was increased only 2-fold in the resistant animal. These biochemical data suggested that the resistant rabbit maintained low intracellular cholesterol even when fed a cholesterol-enriched diet. Direct measurement of cellular cholesterol and cholesteryl esters demonstrated that the concentration of these lipids was significantly lower in the resistant animal than in normal animals with the largest differences found in the cytoplasmic rather than the membrane compartment. These studies demonstrate that the resistant rabbit manifests several quantitative differences in cholesterol metabolism and in the regulation of cholesterol metabolism; but these studies do not directly explain the underlying cause of the resistance to hypercholesterolemia in the resistant rabbit.

Estrogen Regulation of Protooncogene Expression

Our basic approach to the problem of hormonal carcinogenesis has been to investigate the mechanisms by which physiologically relevant levels of estrogens regulate the growth and function of normal target tissues. This information is required to understand the mechanisms of carcinogenesis in hormone target tissues, the role of the hormone in these processes, and rational approaches to the prevent,ion and treatment of major human diseases such as prostate, endometrial, and breast cancer. The experimental system we have used for our studies is the immature rat uterus.

Growth Factor, Oncogene, and Steroidal Interactions in the Regulation of Uterine Growth and Function

Historically, most studies of uterine growth and function in the non-pregnant uterus focused on the hormones estradiol and progesterone. In some cases, other hormones such as growth hormone (Grattarola and Li, 1959), triclothymine (T3, Gardner et al., 1978: Kirkland et al., 1981 a) insulin (Kirkland et al., 1981 b; Fowler et al., 1963) and glucocorticoids (Szego and Roberts, 1953; Campbell, 1978) were also studied, but the principal emphasis remained on the two ovarian hormones noted above. These studies emphasized the structure, function, and regulation of steroid receptors; nuclear interactions of receptors; and biological responses resulting from hormonal stimulation of the tissue (Jensen and DeSombre, 1972; Gorski and Gannon, 1976). During this time, scant attention was given to possible interactions between steroids and polypeptide growth factors in the regulation of uterine growth and function.

Regulation of EGF Receptors and Nuclear Protooncogenes by Estrogen

Estrogens stimulate the growth of both normal target tissues and estrogen sensitive tumor cells. Recent studies from a number of laboratories have raised the possibility that these growth responses may involve an interplay between the steroid, peptide growth factors, and nuclear protooncogenes. For example, estrogens stimulate secretion of IGF and EGF-like peptides in MCF-7 human breast cancer cells, and these peptides can support MCF-7 cell growth in the absence of the steroid (Dickson et al., 1986a, b). Similarly, in vivo, estrogens regulate uterine EGF (Gonzales et al.; DiAugustine et al.), EGF receptors (Mukku et al., 1985a), IGF (Murphy et al., 1988; Murphy et al., 1987a) and several nuclear protooncogenes (Travers et al.; Murphy et al., 1987b; Loose-Mitchell et al.; Weisz et al.). Furthermore, it has been reported that antibodies to EGF can diminish estrogen induced uterine growth in organ cultures (McLachlan et al.). Consequently, our laboratories have studied the in vivo induction of the nuclear protooncogene c-fos (Loose-Mitchell et al.) and the EGF receptor (Lingham et al.; Mukku et al., 1985a) by estrogens.

Estrogen Regulation of Uterine Epidermal Growth Factor Receptor and Nuclear Proto-Oncogenes

The effects of the ovarian steroids on growth of the reproductive tract have been recognized for many years. Receptors for estrogen and progesterone are present in such tissues as the uterus, and it seems clear that these sex steroids stimulate a variety of responses, including growth.

Estrogen Regulation of Uterine Proliferation: How Many ERRs Are Required?

Estrogen-stimulated endometrial proliferation is an important preparatory event for implantation and is thought to be initiated by estrogen receptor (E-R)-mediated changes in gene expression. Many laboratories have thus focused on the biochemical nature of E-R interactions and how this complex interacts with the consensus estrogen response element (ERE) originally identified in the vitellogenin genes of birds and amphibians.