Restoration of prostacyclin synthase in vascular smooth muscle cells after aspirin treatment: regulation by epidermal growth factor

Prostacyclin is a potent vasodilator and inhibitor of platelet aggregation and plays an important role in maintenance of vascular homeostasis. Aspirin irreversibly inactivates prostacyclin synthetase by acetylating the enzyme. Recovery of the enzyme following inactivation by aspirin was studied in rat aorta smooth muscle cells in tissue culture. Confluent cultures superfused with [14C]arachidonic acid, synthesized prostacyclin (PGI2) together with prostaglandins E2, D2, and F2 alpha. Brief treatment with physiological levels of aspirin (0.2 mM) completely inactivated prostacyclin synthesis. Following aspirin removal and addition of fresh growth medium, PGI2 synthesis recovered rapidly with a T 1/2 of only 30-40 min, compared to a doubling time of 24-30 hr for the cells. Recovery of PGE2, PGD2, and PGF2a synthesis paralleled that of PGI2, confirming that cyclooxygenase rather than endoperoxide-prostacyclin isomerase was the labile component. Recovery of PGE2 synthesis after aspirin was blocked by cycloheximide but not by actinomycin D. Recovery of aspirin-inactivated cells required a non-dialyzable component present in serum. All samples tested, including fetal bovine, new-born calf, human, and guinea pig, showed the activity. Fresh serum also induced a cycloheximide-sensitive 2- to 3-fold increase in cyclooxygenase levels in resting confluent cells within 1 to 2 hr. Serum factor was also required to restore PG synthesis after aspirin-inactivation in other cells, including 3T3 mouse fibroblasts, SV40-3T3 and K-Balb 3T3 transformed mouse fibroblasts, NRK rat kidney cells, and REF-9 rat embryonic fibroblasts. The activity was thermolabile, and was completely removed from the medium by growing cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

The vascular S1P gradient-cellular sources and biological significance

Sphingosine 1-phosphate (S1P), a product of sphingomyelin metabolism, is enriched in the circulatory system whereas it is estimated to be much lower in interstitial fluids of tissues. This concentration gradient, termed the vascular S1P gradient appears to form as a result of substrate availability and the action of metabolic enzymes. S1P levels in blood and lymph are estimated to be in the muM range. In the immune system, the S1P gradient is needed as a spatial cue for lymphocyte and hematopoietic cell trafficking. During inflammatory reactions in which enhanced vascular permeability occurs, a burst of S1P becomes available to its receptors in the extravascular compartment, which likely contributes to the tissue reactions. Thus, the presence of the vascular S1P gradient is thought to contribute to physiological and pathological conditions. From an evolutionary perspective, S1P receptors may have co-evolved with the advent of a closed vascular system and the trafficking paradigms for hematopoietic cells to navigate in and out of the vascular system.     

COX-2 inhibitors and genetic background reduce mammary tumorigenesis in cyclooxygenase-2 transgenic mice

Cyclooxygenase-2 (COX-2) overexpression is a widely recognized feature of human breast cancer and inhibitors of the enzyme have antitumor effects in a subset of tumor settings. Previously, we demonstrated that direct overexpression of COX-2 under control of the mammary-specific MMTV promoter/enhancer, was itself oncogenic and lead to the induction of mammary tumors in multiparous, outbred CD1 mice. In the present study, we provide evidence that COX-2 dependent tumor progression can also be studied in FVB/N, an inbred strain widely used for analysis of breast cancer progression. In these mice, the human COX-2 transgene was strongly induced during pregnancy/lactation and mammary tumors developed after multiple pregnancies. However, crossing the COX-2 FVB/N mice with the C57BL6 strain resulted in loss of the mammary tumorigenic phenotype despite the fact that the human COX-2 gene was induced. Treatment of the COX-2 transgenic mice in the FVB/N strain with celecoxib (1600 ppm), a COX-2 selective inhibitor, resulted significant reduction in tumor size and multiplicity when compared to transgenic mice fed with control chow. SC-560 (20 ppm), a COX-1 selective inhibitor did not have significant effect on tumorigenesis. These studies suggest that FVB/N is a susceptible mouse strain well suited to the study of COX-2 mediated tumor progression and may provide a tool for the identification of interacting genes and therapeutic treatments for this clinically important target.     

Platelet-derived growth factor (PDGF)-induced chemotaxis does not require the G protein-coupled receptor S1P1 in murine embryonic fibroblasts and vascular smooth muscle cells

Sphingosine 1-phosphate (S1P), a bioactive lipid mediator, signals via G protein-coupled receptors (GPCR). The prototypical S1P receptor, S1P1 (also known as EDG-1), a Gi-linked receptor, is critical for vascular maturation during development. Recent work suggested that platelet-derived growth factor (PDGF)-induced cell migration required the S1P1 receptor, representing a novel mechanism for cross-talk between receptor tyrosine kinases and GPCRs. Since both S1P and PDGF are implicated in vascular smooth muscle cell (VSMC) pathobiology and development, we investigated this issue in rat VSMC and in embryonic fibroblasts derived from S1P1 null mice. Our data suggest that the S1P1 receptor is critical for S1P-induced, Gi-dependent migration but not for PDGF-BB-induced, receptor tyrosine kinase-dependent chemotaxis in VSMC. In addition, lack of S1P1 receptor in mouse embryonic fibroblasts did not significantly affect PDGF-induced cell migration. These data question the generality of the concept that S1P1 GPCR is a critical downstream component of PDGF-induced chemotaxis.     

Early weaning among Japanese women in Hawaii

This study examined factors associated with early weaning among Japanese women in comparison with Caucasian women in Hawaii. A mailed survey was sent to all women who delivered an infant in Hawaii between January 1st 1989 and March 31st 1989. Only Japanese (N=228) and Caucasian women (N=439) were included in this study. All Japanese breast-feeding women in the study introduced formula at some point in time. Cox multivariate regression analysis was conducted in two separate models of Japanese and Caucasian women, respectively. Higher maternal age and working part-time among Japanese women, and multiparity, college education, rural island and long-term Hawaii residence among Caucasian women were protective against early weaning. Introduction of formula was strongly associated with early weaning for both groups, the association being stronger for Japanese women. The early practice of partial breast-feeding may play an important role for early weaning among Japanese women in Hawaii.     

Sphingosine 1-phosphate activates Akt, nitric oxide production, and chemotaxis through a Gi protein/phosphoinositide 3-kinase pathway in endothelial cells

Sphingosine 1-phosphate (SPP) binds to members of the endothelial differentiation gene family (EDG) of receptors and leads to diverse signaling events including cell survival, growth, migration and differentiation. However, the mechanisms of how SPP activates these proangiogenic pathways are poorly understood. Here we show that SPP signals through the EDG-1 receptor to the heterotrimeric G protein G(i), leading to activation of the serine/threonine kinase Akt and phosphorylation of the Akt substrate, endothelial nitric-oxide synthase (eNOS). Inhibition of G(i) signaling, and phosphoinositide 3-kinase (PI 3-kinase) activity resulted in a decrease in SPP-induced endothelial cell chemotaxis. SPP also stimulates eNOS phosphorylation and NO release and these effects are also attenuated by inhibition of G(i) signaling, PI 3-kinase, and Akt. However, inhibition of NO production did not influence SPP-induced chemotaxis but effectively blocked the chemotactic actions of vascular endothelial growth factor. Thus, SPP signals through G(i) and PI 3-kinase leading to Akt activation and eNOS phosphorylation.     

Sphingosine 1-phosphate-induced endothelial cell migration requires the expression of EDG-1 and EDG-3 receptors and Rho-dependent activation of alpha vbeta3- and beta1-containing integrins

Sphingosine 1-phosphate (SPP), a platelet-derived bioactive lysophospholipid, is a regulator of angiogenesis. However, molecular mechanisms involved in SPP-induced angiogenic responses are not fully defined. Here we report the molecular mechanisms involved in SPP-induced human umbilical vein endothelial cell (HUVEC) adhesion and migration. SPP-induced HUVEC migration is potently inhibited by antisense phosphothioate oligonucleotides against EDG-1 as well as EDG-3 receptors. In addition, C3 exotoxin blocked SPP-induced cell attachment, spreading and migration on fibronectin-, vitronectin- and Matrigel-coated surfaces, suggesting that endothelial differentiation gene receptor signaling via the Rho pathway is critical for SPP-induced cell migration. Indeed, SPP induced Rho activation in an adherence-independent manner, whereas Rac activation was dispensible for cell attachment and focal contact formation. Interestingly, both EDG-1 and -3 receptors were required for Rho activation. Since integrins are critical for cell adhesion, migration, and angiogenesis, we examined the effects of blocking antibodies against alpha(v)beta(3), beta(1), or beta(3) integrins. SPP induced Rho-dependent integrin clustering into focal contact sites, which was essential for cell adhesion, spreading and migration. Blockage of alpha(v)beta(3)- or beta(1)-containing integrins inhibited SPP-induced HUVEC migration. Together our results suggest that endothelial differentiation gene receptor-mediated Rho signaling is required for the activation of integrin alpha(v)beta(3) as well as beta(1)-containing integrins, leading to the formation of initial focal contacts and endothelial cell migration.     

Cytotoxic T cells specifically induce Fas on target cells, thereby facilitating exocytosis-independent induction of apoptosis

Cytotoxic T (Tc) cells deficient in perforin lyse Fas-negative targets after lengthy incubation periods. This process is independent of granzymes, and killing occurs via the Fas pathway for the following reasons. Interaction of perforin-deficient Tc cells with Fas-negative targets leads to an up-regulation of Fas that is dependent on Ag recognition, de novo synthesis, and transport of proteins to the target cell surface. Treatment of effectors with brefeldin A, but not with the exocytosis inhibitor concanamycin, inhibited this process. Lysis of targets is inhibited by anti-Fas Abs, soluble mouse Fas-Fc, and the caspase-cascade inhibitor, crm-A. Targets from Fas-mutant lpr mice are refractory to lysis, and Tc cells from mice deficient in Fas- and perforin-mediated lysis do not lyse Fas-negative targets. The possible relevance of this exocytosis-independent cytolytic process in the regulation of T cell activity and control of pathogens is discussed.