Glucocorticoid uncoupling of antiogensin II-dependent phospholipase C activation in rat vascular smooth muscle cells.

Vascular tone is maintained by both angiotensin II (Ang II) and glucocorticoids, but the effect of glucocorticoids on Ang II function in vascular smooth muscle cells (VSMC) is unclear. To determine the direct influence of glucocorticoids on VSMC Ang II receptor function, the effects of dexamethasone on Ang II receptor binding, Ang II-induced phospholipase C (PLC) activation, and Ang II-dependent cell growth were studied in cultured rat VSMC. Dexamethasone caused concentration- and time-dependent increases in Ang II binding which were prevented by glucocorticoid receptor inhibition with RU 38486. Dexamethasone-induced enhancement of Ang II binding resulted from increased AT1 receptors, as indicated by Northern blot analysis and competitive binding assays. Despite causing increased Ang II receptor number, dexamethasone preincubation prevented Ang II-induced PLC activation, as indicated by phosphatidylinositol 4,5-bisphosphate degradation and inositol trisphosphate formation. When PLC activity was directly measured in VSMC soluble and membrane fractions, Ang II receptor activation caused decreased soluble and increased membrane PLC activity, consistent with the interpretation that Ang II caused cytosol-to-membrane PLC translocation. The effect of Ang II on PLC translocation was prevented by dexamethasone preincubation. Finally, prolonged incubation with dexamethasone and Ang II had additive effects on VSMC hypertrophy. In conclusion, glucocorticoids directly altered Ang II function in VSMC by causing increased Ang II receptor number, Ang II receptor/PLC uncoupling, and enhanced Ang II-dependent hypertrophy.

Increased platelet-derived growth factor production and intimal thickening during healing of Dacron grafts in a canine model.

PURPOSE: Growth factor production by endothelial cells on grafts may play a role in the development of intimal hyperplasia and subsequent graft failure. METHODS: To study the relationship between platelet-derived growth factor production and graft healing, 26 beagles underwent placement of 20 cm long, 6 mm internal diameter, knitted Dacron thoracoabdominal grafts, either seeded with autologous endothelial cells (n = 14) or unseeded controls (n = 12). The grafts and adjacent arteries were removed 4 or 20 weeks after implantation for measurement of platelet-derived growth factor production in organ culture, endothelial cell coverage, and intimal thickness. RESULTS: Midgraft platelet-derived growth factor production by seeded graft segments increased from 41 +/- 6 to 148 +/- 27 pg/cm2/72 hr (p < 0.002) between 4 and 20 weeks. This was accompanied by a significant increase in inner-capsule thickness. Platelet-derived growth factor production by control graft segments also increased from 58 +/- 21 to 163 +/- 42 pg (p < 0.05) and was similar to that of seeded grafts despite more rapid endothelialization of seeded grafts. The increase in growth factor production by Dacron grafts was greater than that of the expanded polytetrafluoroethylene grafts studied previously despite similar endothelial cell coverage. CONCLUSION: This increase corresponded with the rapid appearance of smooth muscle cells in the pseudointima of Dacron grafts, suggesting that these cells may be responsible for the observed increase in platelet-derived growth factor production.

Elevated platelet-derived growth factor production by aortic grafts implanted on a long-term basis in a canine model.

An endothelial cell lining in a prosthetic vascular graft has been shown to decrease graft thrombogenicity. However, previous studies in our laboratory demonstrated that grafts seeded with endothelial cells produced platelet-derived growth factor, a potent smooth muscle cell mitogen that may promote intimal hyperplasia. This study was undertaken to assess temporal changes in platelet-derived growth factor production by grafts seeded with endothelial cells and unseeded grafts and adjacent arteries. Adult beagles underwent placement of 20 to 22 cm long, 8 mm inner diameter, expanded polytetrafluoroethylene thoracoabdominal aortic grafts that were either seeded with autologous jugular vein endothelial cells or were unseeded controls. Grafts and adjacent arteries were removed at times up to 2 years after implantation. The tissue was studied in organ culture and platelet-derived growth factor production was measured after 72 hours with use of a radioreceptor assay. Platelet-derived growth factor production by endothelialized grafts increased significantly over the period studied, especially at the anastomoses, whereas that by arterial segments did not change significantly. The increase in platelet-derived growth factor production was greater in the distal than the proximal anastomotic segment suggesting a possible explanation for the clinical finding of more severe intimal hyperplasia at the distal anastomosis.

Effect of complement and arachidonic acid pathway inhibition on white blood cell count and deposition on vascular grafts.

To determine the role of complement and arachidonic acid metabolites in the decrease in peripheral white blood cell count (pWBC) observed with graft implantation, Dacron aortic grafts were implanted in control rabbits (Group I, n = 13), or rabbits pretreated with cobra venom factor (80 U/kg) to deplete complement (Group II, n = 13), indomethacin (2.5 mg/kg) to inhibit cyclooxygenase (Group III, n = 7), or diethylcarbamazine (DEC, 90 mg/kg) to inhibit leukotriene synthesis (Group IV, n = 7). pWBC was measured 15 min and 1 hr after graft implantation. After graft removal, the WBC count on grafts (gWBC) was determined by light microscopy (LM) and scanning electron microscopy (SEM). One hr after graft implantation, pWBC decreased significantly in Groups I-IV to 46%, 52%, 40%, and 45% of preoperative pWBC, respectively. There was no significant difference among the groups. LM revealed gWBC per 63x field of 8.0, 12.3, 5.8, and 6.8 in Groups I-IV, respectively. Similarly, SEM showed gWBC per 2000x field of 2.5, 5.6, 0.7, and 1.5 in Groups I-IV, respectively. SEM gWBC was significantly greater in Group II than I (p less than 0.01), and significantly less in Group III than I (p less than 0.05). Results suggested that complement and arachidonic acid pathways alone do not affect the fall in pWBC, but may influence gWBC.

Accelerated rates of collagen synthesis in atherosclerotic arteries quantified in vivo.

Rates of arterial collagen and noncollagen protein synthesis were quantified in vivo in rabbits maintained for 4 months on a control diet or the same diet supplemented with 2% peanut oil and 0.25% cholesterol. Thoracic aortas from animals fed the atherogenic diet exhibited raised lesions covering 75% to 100% of the surface. The dry delipidated weight and collagen content of these arterial segments both were significantly increased. The rates of protein synthesis were determined in rabbits given a bolus intravenous injection of 3H-L-proline (1.0 mCi/kg) and unlabelled proline (7 mmol/kg) to attain steady-state levels of specific radioactivity of free proline in plasma and tissues. Plasma proline specific activity decreased only 20% over a 5-hour period and was similar to free proline in arterial tissue, skin, and lung. Collagen synthesis rates (ng/mg dry delipidated weight per hour) were increased 10-fold in the intima plus inner media of atherosclerotic thoracic aortas compared with controls. Rates of collagen synthesis were also increased in the abdominal aortas, whereas protein synthesis in lung and skin was unaffected by diet. Increased rates of collagen synthesis in atherosclerotic arteries significantly exceeded the increases in noncollagen protein synthesis. In addition, collagen synthesis rates in vivo were 12 to 20 times greater than previously measured in vitro. These results demonstrate for the first time in vivo that collagen accumulation in the developing atherosclerotic plaque is in part due to accelerated rates of collagen synthesis by intimal smooth muscle cells.