This resubmitted renewal application proposes to extend the current work on the remodelling of the pulmonary microvascular bed in hypoxic hypertension. Our principal goal is to identify mechanisms by which the process is triggered. As before, the main focus is on the precursor smooth muscle cell or pericyte (PC). Hypoxic exposure of rats is used as a reproducible animal model that exhibits similar changes to those seen in human disease. Previous studies in the model have established that in hypoxia, PC in distal arteries undergo a series of changes: hypertrophy, DNA replication (possibly linked to proliferation), and increased matrix production, all within the first week. Each of these changes contributes to an increase in pulmonary vascular resistance, but deposition of matrix is likely to have the most permanent restrictive effect. The trigger(s) for these changes is currently unknown: there are many putative candidates, but in this application we propose to test the contribution of low oxygen itself. Our main hypothesis is that the external trigger acts on the PC to change autocrine production. We focus on two likely autocrines, TGFbeta and PDGF, because they are powerful stimulators of matrix production and their known growth effects are relevant to those seen in remodelling. Since TGFbeta modulates the expression of PDGF, we postulate that it is the autocrine principally affected by initial trigger events. Recent pilot studies have demonstrated that steady-state mRNA levels of TGFbeta1 increase in whole lung homogenates of rats exposed to hypoxia. Immunohistochemistry has localized TGFbeta1 in the distal arteries. Furthermore, PC in culture express TGFbeta at message and protein levels. Although TGFbeta expression is clearly affected in vivo, its direct effect on cells in vitro does not readily explain some of the changes observed in remodelling; for example, it inhibits DNA synthesis and proliferation. Since PDGF stimulates these, we postulate that the changes observed in the lung depend on a dynamic balance between TGFbeta and the specific PDGF sub-types it induces. We will test this in vitro, employing PC obtained from the distal lung. For comparison, we will use smooth muscle cells from the main pulmonary artery where the timing and type of remodelling is different than in the periphery and less likely to reflect the direct influence of hypoxia. We will determine the expression of the two autocrine factors and their effects on specific cellular functions relevant to remodelling - growth and matrix production - and specify which receptors mediate these responses. We will test the effect of in vitro hypoxia on TGFbeta expression, using newly available methods that will allow us to probe hypoxia-sensitive mechanisms of transcriptional regulation. In vivo, we will use autoradiography and Feulgen microspectrophotometry to identify changes in DNA replication and ploidy levels and relate them temporally to change in the expression of genes for the two autocrine factors and for matrix proteins by in situ hybridization and immunohistochemistry. By focusing on stimuli and response mechanisms intrinsic to the cell principally involved, the project has a relevance to types of hypertension of different etiology or of idiopathic origin and will thus point to therapies that have a general application.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL041811-06
Application #
2220161
Study Section
Lung Biology and Pathology Study Section (LBPA)
Project Start
1988-04-01
Project End
1994-11-30
Budget Start
1993-12-01
Budget End
1994-11-30
Support Year
6
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Pharmacology
Type
Schools of Dentistry
DUNS #
053785812
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Fukunaga, T; Davies, P; Zhang, L et al. (1998) Prolonged high intermittent positive-pressure ventilation induces airway remodeling and reactivity in young rats. Am J Physiol 275:L567-73
Wong, H R; Finder, J D; Wasserloos, K et al. (1996) Transcriptional regulation of iNOS by IL-1 beta in cultured rat pulmonary artery smooth muscle cells. Am J Physiol 271:L166-71
Boota, A; Zar, H; Kim, Y M et al. (1996) IL-1 beta stimulates superoxide and delayed peroxynitrite production by pulmonary vascular smooth muscle cells. Am J Physiol 271:L932-8
Pitt, B R; Weng, W; Steve, A R et al. (1994) Serotonin increases DNA synthesis in rat proximal and distal pulmonary vascular smooth muscle cells in culture. Am J Physiol 266:L178-86
Davies, P; Patton, W (1994) Peripheral and central vascular smooth muscle cells from rat lung exhibit different cytoskeletal protein profiles but similar growth factor requirements. J Cell Physiol 159:399-406
Johnson, B A; Lowenstein, C J; Schwarz, M A et al. (1994) Culture of pulmonary microvascular smooth muscle cells from intraacinar arteries of the rat: characterization and inducible production of nitric oxide. Am J Respir Cell Mol Biol 10:604-12
Weng, W; Reynolds, I J; Jani, J P et al. (1994) Desensitization of 5HT2 receptors by protein kinase C activation in distal pulmonary vascular smooth muscle cells in culture. Microcirculation 1:129-35
Kubiak, J; Mitra, M M; Steve, A R et al. (1992) Transforming growth factor-alpha gene expression in late-gestation fetal rat lung. Pediatr Res 31:286-90
Pitt, B R; Brookens, M A; Steve, A R et al. (1992) Expression of pulmonary metallothionein genes in late gestational lambs. Pediatr Res 32:424-30
Davies, P; Reid, L (1991) Hypoxic remodeling of the rat pulmonary arterial microcirculation assessed by microdissection. J Appl Physiol 71:1886-91

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