Effects of L-749,329, an ET(A)/ET(B) endothelin receptor antagonist, in a porcine coronary artery injury model of vascular restenosis.

BACKGROUND: Previous studies in animal models of angioplasty have suggested a role in neointimal hyperplasia for endothelins (ETs), potent vasoconstricting peptides that also exert growth-promoting effects. The present studies were undertaken to test the hypothesis that endothelin receptor blockade can reduce neointimal thickening in injured porcine coronary arteries. METHODS AND RESULTS: An ET(A)/ET(B) antagonist, L-749,329, was evaluated as an inhibitor of intimal thickening in a porcine balloon/stent model of coronary artery injury. L-749,329 competitively inhibited [(125)I]ET-1 binding to porcine ET(A) (IC(50) approximately 0.3 nmol/L) or ET(B) (IC(50) approximately 20 nmol/L) receptors and inhibited ET-1-stimulated signaling in cell culture. In anesthetized pigs, big ET-1-stimulated increases in systemic blood pressure were totally inhibited after intravenous infusion of L-749,329 (>/=0.2 mg. kg(-1). h(-1)). In vascular injury studies, pigs were treated with vehicle or L-749,329 (1 mg. kg(-1). h(-1)) beginning 2 days before and continuing 28 days after experimental angioplasty. Left anterior descending, left circumflex, and/or right coronary arteries were injured by inflation of an angioplasty balloon wrapped with a coiled metallic stent. After 28 days, mean neointimal thickness in the L-749,329-treated group was reduced by 9.0% compared with vehicle-treated controls, but this effect was not statistically significant (P=0.13). CONCLUSIONS: Blockade of endothelin receptors for 28 days with only a mixed ET(A)/ET(B) receptor antagonist is insufficient to substantially inhibit intimal hyperplasia after balloon/stent coronary artery injury in the pig, in contrast to results with a selective ET(A) antagonist. The effects of selective or mixed ET(A)/ET(B) antagonists in diseased vessels remain to be determined in this model.

Smooth muscle cells isolated from the neointima after vascular injury exhibit altered responses to platelet-derived growth factor and other stimuli.

A variety of evidence suggests that vascular smooth muscle cells (SMC) exhibit a more immature phenotype when stimulated by injury to replicate in the adult. One growth characteristic common to immature (embryonic, fetal, and neonatal) SMC is a markedly reduced responsiveness to platelet-derived growth factor (PDGF) and other mitogenic stimuli. Here we demonstrate that SMC isolated from the 14-day neointima of experimentally injured carotid arteries exhibit a similar growth phenotype. The proliferative responses of neointimal cells to the BB homodimer of PDGF, which interacts with both forms of the PDGF receptor, were up to twenty-fold less (as assessed by BrdU immunocytochemistry) than that of adult control tunica media cells over a wide range of PDGF concentrations. Paradoxically, these cells expressed abundant mRNA for the alpha- and beta-subunits of the PDGF receptor (by RT-PCR) and expressed abundant PDGF receptor protein (by Western blotting). Addition of PDGF-BB to neointimal SMC induced significant autophosphorylation of the PDGF receptor, suggesting that the PDGF receptors were fully functional. The chemotactic responses of neointimal SMC to PDGF, in in vitro migration assays, were identical to that of control medial cells. The data further establish the existence of vascular SMC phenotypes characterized by a refractoriness to growth stimulation by specific mitogens, and provide further evidence for the reiteration of developmentally regulated programs following vascular injury in vivo.

Effects of subtype-selective and balanced angiotensin II receptor antagonists in a porcine coronary artery model of vascular restenosis.

BACKGROUND: Numerous studies have demonstrated the ability of angiotensin II (Ang II) receptor antagonists and angiotensin-converting enzyme (ACE) inhibitors to inhibit intimal hyperplasia after balloon dilation of noncoronary arteries in small-animal models, suggesting an important role for Ang II in the response to injury. Although ACE inhibitors have not been similarly effective in nonhuman coronary models or in human restenosis trials, questions remain regarding the efficacy ACE inhibitors against tissue ACE and the contributions of ACE-independent pathways of Ang II generation. Unlike ACE inhibitors, Ang II receptor antagonists have the potential to inhibit responses to Ang II independent of its biosynthetic origin. METHODS AND RESULTS: In separate studies, three Ang II receptor antagonists, including AT1 selective (L-158,809), balanced AT1/AT2 (L-163,082), and AT2 selective (L-164,282) agents, were evaluated for their ability to inhibit vascular intimal thickening in a porcine coronary artery model of vascular injury. Preliminary studies in a rat carotid artery model revealed that constant infusion of L-158,809 (0.3 or 1.0 mg X kg-1 X d-1) reduced the neointimal cross-sectional area by up to 37% measured 14 days after balloon dilatation. In the porcine studies, animals were treated with vehicle or test compound beginning 2 days before and extending 28 days after experimental angioplasty. Left anterior descending, left circumflex, and/or right coronary arteries were injured by inflation of commercially available angioplasty balloons with placement of coiled metallic stents. Infusion of L-158,809 (1 mg X kg-1 X d-1), L-163,082 (1 mg X kg-1 X d-1), or L-164,282 (1.5 mg X kg-1 X d-1) in the study animals yielded plasma drug levels sufficient either to chronically block or, for L-164,282, to spare pressor responses to exogenous Ang II. Neither L-158,809, L-163,082, nor L-164,282 had statistically significant effects (P=.12, P=.75, and P=.48, respectively, compared with vehicle-treated controls) on neointimal thickness (normalized for degree of injury) measured by morphometric analysis at day 28 after angioplasty. CONCLUSIONS: These findings indicate that chronic blockade of Ang II receptors by either site-selective or balanced AT1/AT2 antagonists is insufficient to inhibit intimal hyperplasia after experimental coronary vascular injury in the pig. The results further suggest that, unlike in the rat carotid artery, Ang II is not a major mediator of intimal thickening in the pig coronary artery.

Platelet-derived growth factor and transforming growth factor-beta regulate plasminogen activator inhibitor-1 synthesis in vascular smooth muscle cells.

Bovine vascular smooth muscle cells (SMC) were examined for production of plasminogen activator inhibitor-1 (PAI-1) which may play a key role in regulating the fibrinolytic system. Growth-arrested SMC released active PAI (101 arbitrary units (AU)/10(6) cells/24 h) and a latent form of PAI (880 AU/10(6) cells/24 h) into the conditioned medium (CM). The levels of PAI were significant since 880 AU of PAI could inhibit approximately 1 microgram of tissue plasminogen activator. The extracellular matrix of SMC also contained PAI activity; however, the level was 17-fold less than that observed in the CM. SMC-PAI was a rapid inhibitor of tissue plasminogen activator (kass greater than 10(7) M-1 S-1) and was identified as a 45-kDa protein immunologically related to endothelial cell PAI-1. PAI-1 comprised 20 and 30%, respectively, of the newly synthesized protein detected in the CM and extracellular matrix of SMC. The SMC growth modulators, platelet-derived growth factor and transforming growth factor-beta, induced PAI-1 activity and protein synthesis by 2- and 3-fold, respectively, in a dose- and time-dependent manner. The increases in PAI-1 activity and protein synthesis were ascribed to elevated levels of PAI-1 mRNA as judged by Northern blot analysis of total RNA prepared from control and platelet-derived growth factor- and transforming growth factor-beta-treated cells. Increases in PAI-1 mRNA levels were evident 1 h after growth factor treatment and were maximal after 4 h. PAI-1 mRNA levels were unaffected by cycloheximide treatment. The results indicate that SMC synthesize and release PAI-1 which could regulate the normal fibrinolytic environment of the arterial wall. During atherosclerosis or after vascular injury increases in platelet-derived or locally produced mitogens may stimulate further PAI-1 synthesis and generate a prothrombotic state.

Metastatic choroidal melanoma. Hepatic binding protein reactivity toward a liver-metastasizing clone.

A clone of B16 malignant melanoma cells with a preference for metastasis to the liver was isolated and characterized. The parent tumor cells (F1) were injected in the tail vein of C57BL/6 mice, and the resultant liver colonizing cells were isolated and then subcultured for two to three weeks. The cells were then reinjected into the next series of mice. After five such passages, a clone (L4) of melanoma cells was obtained that metastasized almost exclusively to the liver. A hepatic binding protein (HBP) was isolated from rabbit liver that agglutinated neuraminidase-treated F1 and L4 malignant melanoma cells. Different agglutination titers found between the parent and liver-metastasizing clone demonstrated differences in cell-surface properties between the parent tumor and the liver-metastasizing clone. These results demonstrate that malignant melanoma cells can be selected for preferential liver metastasis and can be recognized and agglutinated by specific HBPs. Metastasis from human uveal malignant melanoma may occur by similar mechanisms.

Cell-mediated immunity in herpes corneal stromal disease.

Regional draining lymph nodes (RDLN) from rabbits with herpes virus disciform keratitis (defined as corneal edema or corneal edema with concomitant epithelial lesions) were tested for general as well as specific immune cap-city. Although general RDLN immune capacity did not seem to be impaired, specific reactivity to herpes antigens was found only in those rabbits which presented with disciform keratitis and concomitant epithelial lesions; specific reactivity to herpes antigens was not found in those animals which presented with disciform edema alone. Additionally, the unilateral nature of the RDLN immune response during ocular infections of unequal severity was demonstrated.

Scanning electron microscope study of herpes simplex virus experimental disciform keratitis.

Scanning electron microscopy of the endothelium of experimental disciform keratitis revealed corneal endothelial changes which distinguished disciform oedema from the more progressive stages of disciform keratitis. The endothelium of corneas with disciform oedema were wholly intact and characterised by subtle morphological alterations. In contrast, the more progressive stages of disciform keratitis were characterised by massive destruction and denudation of the endothelium. The significance of these observations is discussed.

Scanning electron microscopic observation of two retinoblastoma cell lines.

Two continuous retinoblastoma cell lines were observed by scanning electron microscopy. Both cell lines spontaneously grow as a suspension of round cells in clusters, chains, and unique ring (rosette) formations. Scanning electron microscopy of suspension cells reveals some variation in the number and frequency of surface adornments such as blebs, lamellipodia, and microvilli. Although the cell lines are nonadherent to substratum and therefore assume a spherical form, highly villous cells are not characteristic of the entire cell populations. When WERI-Rb1 and Y79 are seeded onto a polyornithine-treated substrate, attachment and growth as adherent cultures are evident. With selective attachment on a positively charged substrate, we observe alteration of membrane architecture with the extension of cytoplasm and filopidia. In addition, WERI-Rb1 cell-to-substratum adhesion induces morphological changes suggestive of neuronal cell differentiation.

Characterization of a new continuous cell line derived from a human retinoblastoma.

A new continuous cell line derived from a human retinoblastoma has been established. This cell line, WERI-RB1, has been maintained in vitro since December 1974. The purpose of this investigation was to characterize WERI-Rb1 on the basis of morphology, growth, tumorigenicity, cytogenetics, and to compare this cell line with Y79, a human retinoblastoma cell line established at another institution. Morphologically, both cell lines were similar; each spontaneously grew as a suspension of small round cells in grapelike clusters. Each exhibited growth of cells in rosettes, as well as unusual chain formations. Growth rates differed: the population-doubling times for WERI-Rb1 and Y79 were 96 and 33 hr, respectively. When the negative surface charge on a plastic tissue culture flask was changed, each cell line grew as a monolayer. Y79 could be cloned in soft agar; WERI-Rb1 could not. An inoculm of 10(7) WERI-Rb1 or Y79 cells produced a retinoblastoma in test rabbits. Karyological examination showed each cell line to have a stable, near diploid chromosome number. Although large markers were observed in each line, they shared no common marker.