Hypoxia-induced proliferative response of vascular adventitial fibroblasts is dependent on g protein-mediated activation of mitogen-activated protein kinases.

Hypoxia has been shown to act as a proliferative stimulus for adventitial fibroblasts of the pulmonary artery. The signaling pathways involved in this growth response, however, remain unclear. We tested the hypothesis that hypoxia-induced proliferation of fibroblasts would be dependent on distinct (compared with serum) activation and utilization patterns of mitogen-activated protein (MAP) kinases initiated by Galpha(i/o) proteins. We found that hypoxia stimulated increases in DNA synthesis and growth of quiescent fibroblasts in the absence of exogenous mitogens and also markedly augmented serum-stimulated growth responses. Hypoxia caused a transient activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), the time course and pattern of which was somewhat similar to that induced by serum but which was of lesser magnitude. On the other hand, hypoxia-induced activation of p38 MAP kinase was biphasic, whereas serum-stimulated activation of p38 MAP kinase was transient, and the magnitude of activation was greater for hypoxia compared with that of serum stimulation. ERK1/2, JNK1, and p38 MAP kinase but not JNK2 were necessary for hypoxia-induced proliferation because PD98059, SB202190, and JNK1 antisense oligonucleotides nearly ablated the growth response. JNK2 appeared to act as a negative modulator of hypoxia-induced growth because JNK2 antisense oligonucleotides led to an increase in DNA synthesis. In serum-stimulated cells, antisense JNK1 oligonucleotides and PD98059 had inhibitory effects on proliferation, whereas SB202190 led to an increase in DNA synthesis. Pertussis toxin, which blocks Galpha(i/o)-mediated signaling, markedly attenuated hypoxia-induced DNA synthesis and activation of ERK and JNK but not p38 MAP kinase. We conclude that hypoxia itself can act as a growth promoting stimulus for subsets of bovine neonatal adventitial fibroblasts largely through Galpha(i/o)-mediated activation of a complex network of MAP kinases whose specific contributions to hypoxia-induced proliferation differ from traditional serum-induced growth signals.

Hypoxia-induced pulmonary vascular remodeling: contribution of the adventitial fibroblasts.

Vascular repair in response to injury or stress (often referred to as remodeling) is a common complication of many cardiovascular abnormalities including pulmonary hypertension, systemic hypertension, atherosclerosis, vein graft remodeling and restenosis following balloon dilatation of the coronary artery. It is not surprising that repair and remodeling occurs frequently in the vasculature in that exposure of blood, vessels to either excessive hemodynamic stress (e.g. hypertension), noxious blood borne agents (e.g. atherogenic lipids), locally released cytokines, or unusual environmental conditions (e.g. hypoxia), requires readily available mechanisms to counteract these adverse stimuli and to preserve structure and function of the vessel wall. The responses, which were presumably evolutionarily developed to repair an injured tissue, often escape self-limiting control and can result, in the case of blood vessels, in lumen narrowing and obstruction to blood flow. Each cell type (i. e. endothelial cells, smooth muscle cells, and fibroblasts) in the vascular wall plays a specific role in the response to injury. However, while the roles of the endothelial cells and smooth muscle cells (SMC) in vascular remodeling have been extensively studied, relatively little attention has been given to the adventitial fibroblasts. Perhaps this is because the fibroblast is a relatively ill-defined cell which, at least compared to the SMC, exhibits few specific cellular markers. Importantly though, it has been well demonstrated that fibroblasts possess the capacity to express several functions such as migration, rapid proliferation, synthesis of connective tissue components, contraction and cytokine production in response to activation or stimulation. The myriad of responses exhibited by the fibroblasts, especially in response to stimulation, suggest that these cells could play a pivotal role in the repair of injury. This fact has been well documented in the setting of wound healing where a hypoxic environment has been demonstrated to be critical in the cellular responses. As such it is not surprising that fibroblasts may play an important role in the vascular response to hypoxia and/or injury. This paper is intended to provide a brief review of the changes that occur in the adventitial fibroblasts in response to vascular stress (especially hypoxia) and the role the activated fibroblasts might play in hypoxia-mediated pulmonary vascular disease.

Chronic hypoxia induces exaggerated growth responses in pulmonary artery adventitial fibroblasts: potential contribution of specific protein kinase c isozymes.

Enhanced proliferation of adventitial fibroblasts is a major contributor to the structural remodeling of the pulmonary artery (PA) that occurs during hypoxia-induced pulmonary hypertension. The mechanisms responsible for the exuberant growth of fibroblasts are unknown; however, protein kinase C (PKC) isozymes have previously been shown to be important in the enhanced growth properties of immature PA fibroblasts. We tested the hypotheses that PA adventitial fibroblasts from neonatal calves exposed chronically to hypoxia after birth would express augmented growth responses compared with fibroblasts from the control adventitia and that these properties would be associated with selective changes in expression of PKC isozymes. We studied the effects of serum, purified mitogens, and hypoxia on the growth of aggregate populations of fibroblasts isolated from the PA of neonatal control calves (Neo-C) and calves chronically exposed to hypoxia for 2 wk beginning on Day 1 of life (Neo-Hyp). Neo-Hyp fibroblasts demonstrated higher proliferative capabilities than did Neo-C cells in response to all the stimuli tested. Importantly, hypoxia was found to act synergistically with peptide mitogens (platelet-derived growth factor, basic fibroblast growth factor, insulin-like growth factor-I) to stimulate growth in Neo-Hyp but not in Neo-C cells. Using PKC-isozyme nonselective and selective inhibitors and immunoblot analysis, we found differences in utilization of PKC isozymes in Neo-Hyp and Neo-C fibroblasts and have identified PKC-betaI and -zeta as key contributors to the augmented growth of Neo-Hyp fibroblasts. Although the activity of PKC-betaI and -zeta isozymes was increased by hypoxia in serum-deprived Neo-C and Neo-Hyp fibroblasts, under normoxia, quiescent Neo-Hyp fibroblasts had higher PKC-zeta-specific activity than did Neo-C cells. These results suggest that neonatal PA adventitial fibroblasts acquire new growth properties in the setting of hypoxia- induced pulmonary hypertension and that the augmented proliferative characteristics of the Neo-Hyp fibroblasts might be associated with changes in specifc PKC isozyme expression and activation patterns.

Fibulin-1, vitronectin, and fibronectin expression during avian cardiac valve and septa development.

BACKGROUND: Extracellular matrix (ECM) proteins have been implicated as mediators of events important to valvuloseptal development (reviewed by Little and Rongish, Experentia, 51:873-882, 1995). The aim of this study was to identify connective tissue ECM proteins present at sites of valvuloseptal morphogenesis, and to determine how their patterns of expression change during the developmental process. METHODS: Immunofluorescence microscopy was used to examine the distribution of fibulin-1, vitronectin, and fibronectin in the embryonic chicken heart over a broad developmental time frame (Hamburger and Hamilton stages 14 to 44), emphasizing stages that illustrate endocardial cushion formation, growth, fusion, and development into valvuloseptal components. RESULTS AND CONCLUSIONS: Fibulin-1 immunolabeling was concentrated in endocardial cushions, notably at boundaries with the myocardium, during stages when the cushions are differentiating into valvular and septal components. Fibulin-1 was detected in the endocardial cushions prior to their seeding with cushion cells, but became undetectable by early midgestation. Vitronectin expression was similar to fibulin-1, but less restricted in its distribution. Vitronectin was observed before endocardial cushion cell migration commenced and persisted until the formation of prevalvular structures (early midgestation) in the atrioventricular cushions. Vitronectin remained detectable in the semilunar valves until late midgestation. Fibronectin was present in the endocardial cushion region and in portions of the endocardium and myocardium throughout the stages presented. Our data suggests that the ECM of the endocardial cushions undergoes remodelling in a regionally and temporally specific manner which corresponds with morphogenetic changes during valvuloseptal development.

Distribution of connective tissue proteins during development and neovascularization of the epicardium.

OBJECTIVE: The epicardium is the site of initial cardiac neovascularization and formation of the coronary circulatory system. Recent evidence indicates that vascular progenitor cells are influenced by the connective tissue proteins of their extracellular environment, yet little is known about the composition or function of the embryonic epicardial extracellular matrix (ECM). This study examines the distribution of ECM proteins during the migration, growth and maturation of epicardial cells and also during the development of the coronary vascular network. METHODS: Immunofluorescence microscopy was used to determine the distributions of vitronectin, fibronectin and a newly described fibrillin-like protein, the JB3 antigen, in the embryonic chicken heart. Immunoblot analysis was performed to compare the relative electrophoretic mobilities of the JB3 antigen and fibrillin-1. RESULTS: The data show that vitronectin and fibronectin are present at sites of initial migration of the epicardial cells. The expression of vitronectin (and also fibronectin) becomes more pronounced as the epicardium thickens, undergoes remodeling and differentiates. The JB3 antigen is prominently expressed in the coronary arteries, allowing visualization of their connection to the systemic circulation and to the heart muscle, as well as vessel wall formation and organization. Immunoblot analysis suggests that the JB3 antibody recognizes a fibrillin-like polypeptide that is distinct from fibrillin-1. CONCLUSIONS: The observed distributions of vitronectin and fibronectin are consistent with roles in migration of epicardial cells, in remodeling of the epicardium and as substratum components during blood vessel formation. The observed distribution of the JB3 antigen indicates a structural/organizational role in coronary arterial wall assembly and suggests that the JB3 antibody be considered an early marker for maturing coronary arteries.

Temperature sensitivity of equine herpesvirus isolates: a brief review.

This article reviews the findings on temperature sensitivity of equine herpesvirus isolates with an emphasis on equine herpesvirus 3, etiological agent of equine coital exanthema. The hypothesis is presented that the relative apathogenic nature of this herpesvirus may be an indirect result of its inability to synthesize and/or process glycoproteins needed by the virus to produce infectious virions at the normal body temperature of its natural host. It is suggested that equine herpesvirus 3 is the more evolved and naturally attenuated member of the equine herpesviruses.

Molecular pathogenesis of equine coital exanthema (ECE): temperature sensitivity (TS) and restriction endonuclease (RE) fragment profiles of several field isolates.

Examination of six field isolates of equine herpesvirus 3, the causative agent of equine coital exanthema, indicates that all were temperature sensitive (ts) at the body temperature, 39 degrees C, of their host (Equine asinus and callabus) when grown in cell culture. The isolates were characterized by fingerprint analysis with the restriction endonucleases XbaI, EcoRI, BamHI and Hind III to establish possible epidemiologic relatedness. Three of the six isolates may be considered related. Variation in the mobility of the BamHI-A and Hind III-K fragments indicates that a small plaque isolate may contain a 5.7 kb insert of DNA in the unique short region of the genome.