Platelet-neutrophil-smooth muscle cell interactions: lipoxygenase-derived mono- and dihydroxy acids activate cholesteryl ester hydrolysis by the cyclic AMP dependent protein kinase cascade

Fluid-phase interactions between hematologic cells and those of the vessel wall were studied in order to define a role for lipoxygenase products as cell signals in the control of vascular cholesterol metabolism. A functional parameter for hydroxy acids in this system has not been previously demonstrated. We report herein for the first time a biochemical effect of lipoxygenase-derived eicosanoids in the modulation of cholesterol metabolism in smooth muscle cells. Products of platelet-neutrophil interactions served as cell signals in vitro to modulate cholesterol metabolism. We demonstrate that 12-HETE, 12,20-DiHETE, and 12-HETE-1,20-dioic acid activate both lysosomal and cytoplasmic cholesteryl ester (CE) hydrolytic activities, although no effect was observed on CE synthetic (ACAT) activity. The platelet lipoxygenase product, 12-HETE, was the most effective stimulator of CE hydrolysis in the smooth muscle cell, and its conversion to 12,20-DiHETE and the dioic acid derivative by the neutrophils was not necessary for the activation of CE hydrolase. A 2-fold enhancement on CE hydrolysis occurred and was independent of any "cross-activation" by hydroxy acids on production of cyclooxygenase or other lipoxygenase products. The activation of cytoplasmic CE hydrolysis had a lesser cofactor dependence on bile salts in the presence of 12-HETE. This suggested a reduced requirement for surface-active agents in an enzyme-substrate interaction where enzymes are hydrolyzing insoluble lipid substrates. Moreover, 12-HETE induced an additive effect with several lipolytic hormones in the activation of CE catabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of arterial cells: III. Stathmokinetic analyses of smooth muscle cells cocultured with endothelial cells

Arterial endothelial cells (EC) or their conditioned medium (ECCM) can alter the proliferation of cocultured arterial smooth muscle cells (SMC). Previously, we have shown, as have others, that EC regulate the growth of cocultured SMC depending on the density of both cell types. To ascertain the rate of cell-cycle traverse in preconfluent arterial SMC cocultured with arterial EC or ECCM (derived from preconfluent EC), we have conducted a series of stathmokinetic experiments using flow cytometry to determine where specific changes may occur in the cell cycle. Results of our experiments indicate for the first time that ECCM stimulates the proliferation of preconfluent SMC by significantly shortening the residence times in the G1 and S phases of the cell cycle. The predominant relative effect occurs within the early G1 (G1A) compartment where pretreatment with ECCM shortens the residence time by approximately 55%. Furthermore, we have observed that preincubation of serum-free ECCM with antiplatelet-derived growth factor (PDGF) antibody abolishes any mitogenic effect on SMC. This suggests that EC secrete PDGF-like molecules which enhance the proliferation rate of preconfluent, cocultured SMC. These findings support the hypothesis that arterial EC may secrete mitogens which stimulate arterial SMC proliferation in the vascular wall.     

Chronic sialodacryoadenitis virus (SDAV) infection in athymic rats.

Sialodacryoadenitis virus (SDAV) was detected in athymic rats subcutaneously implanted with human tumor cell lines. Clinical signs included sneezing, dyspnea, weight loss and death. Necropsy revealed both upper and lower respiratory tract disease from which Staphylococcus aureus, Pasteurella pneumotropica and Pseudomonas aeruginosa were recovered. Histopathological changes consisted of suppurative rhinitis and bronchopneumonia. Lesions characteristic of SDAV infection were found in lacrimal and salivary glands, and viral antigens were detected in the salivary glands and respiratory tract by immunohistochemistry. Submaxillary salivary gland. Harderian gland and lung homogenates from affected athymic rats were inoculated intranasally into euthymic rats as a rat antibody production test. All euthymic rats seroconverted to SDAV. Seroconversion to SDAV was demonstrated in consecutive pairs of sentinel euthymic rats housed for 6 months with infected athymic rats. Inoculation of supernatants of the original tumor cell lines into euthymic rats did not result in seroconversion. The source of the virus was not determined. In this study, spontaneously acquired SDAV infection persisted for at least 6 months in athymic rats.     

Regulation of neutral cholesteryl esterase in arterial smooth muscle cells: stimulation by agonists of adenylate cyclase and cyclic AMP-dependent protein kinase

Cultured arterial smooth muscle cells have been found to contain an activatable neutral cholesteryl esterase (EC 3.1.1.13). This enzyme is similar to that previously described in adipose tissue, adrenal cortex, and aortic homogenates. Although both the lysosomal (acid) and cytoplasmic (neutral) cholesteryl esterases were activated two- to threefold by the addition of 100 microM dibutyryl cyclic AMP, only neutral cholesteryl esterase was responsive to 100 microM dibutyryl cyclic AMP, 10 mM MgATP, and 50 micrograms/ml exogenous protein kinase when added together. Protein kinase inhibitor (10 micrograms/ml) reversed the action of cyclic AMP-dependent protein kinase; deactivation of neutral cholesteryl esterase was also shown to occur with 50 micrograms/ml phosphoprotein phosphatase. In addition, 0.2 microM prostacyclin, 50 microM forskolin, and an agonist of the beta-adrenergic receptor, 5 microM isoproterenol, significantly stimulated intracellular cyclic AMP accumulation and activated cholesteryl esterase in arterial smooth muscle cells. The data indicate that neutral cholesteryl esterase in arterial smooth muscle cells can be modulated by a phosphorylation-dephosphorylation system involving the cyclic AMP-dependent protein kinase-phosphoprotein phosphatase. Regulation of cholesteryl esterase by this mechanism may affect lipid accumulation in these arterial cells.     

Metabolic activity of cholesteryl esters in aortic smooth muscle cells is altered by prostaglandins I2 and E2

Among the biochemical processes associated with the atherogenic process are increased aortic cholesteryl ester (CE) accumulation and altered prostaglandin (PG) production. The precise physiological role of PG, particularly prostacyclin (PGI2), in the control of CE metabolism in intact aortic smooth muscle cells remains to be fully elucidated. We report here that cytosolic neutral cholesteryl ester hydrolytic activity (NCEH) in intact cultured aortic smooth muscle cells is significantly increased by 75-250 nM PGI2 at the end of a 2-hr incubation period. The effect was mediated by increased intracellular cAMP levels since the effect of PGI2 on NCEH activity was abolished in the presence of an inhibitor of adenylate cyclase activity, viz., dideoxyadenosine (DDA0. Although the addition of 20-100 microM dibutyryl cAMP (Bt2cAMP) and 50-100 microM sodium arachidonate also increased NCEH activity twofold, 6-keto PGF1 alpha, PGE1, and PGE2 did not increase the activity of this enzyme. In contrast to these findings, 75-250 nM PGE2 significantly inhibited CE synthetic activity (ACAT) approximately 60%. Arachidonate or Bt2cAMP did not affect ACAT activity. This decrease in ACAT activity induced by PGE2 does not appear to be mediated by cAMP. Taken together, these findings suggest that PGI2, a well known potent vasodilator and inhibitor of platelet aggregation, and PGE2 may have an important regulatory role in aortic CE metabolism.     

Viral activation of the coagulation cascade: molecular interactions at the surface of infected endothelial cells

Herpesviral infection of endothelial cells (ECs) induces arterial injury. We now demonstrate that such infection promoted enhanced monocyte-endothelial adhesion. Enhanced adhesion was blocked by monoclonal antibodies to the viral-encoded cell surface glycoprotein gC but not by antibodies to gD or gE. Adhesion was also blocked by treating ECs with specific thrombin inhibitors or by growing cells in prothrombin-depleted serum. We found that gC bound and promoted activation of factor X on infected ECs, thereby contributing to thrombin generation. Factor X also bound to transfected L cells that were induced to express gC. Cross-linking and immunoprecipitation studies demonstrated factor X-gC complex formation on the surface of these cells. We suggest that gC-dependent thrombin generation by herpes-infected endothelium may be an important mediator of vascular pathology during viral infection.