Date of Award

12-1-1990

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Anatomy and Cell Biology

Abstract

The purpose of this study was to determine the effects of adenosine and hypoxia on the proliferation of microvascular pericytes from bovine retina. This was accomplished through: (1) development of in vitro methods which allowed consistent isolation and propagation of homogeneous retinal pericyte cultures; (2) manipulation of oxygen partial pressure in the culture chamber; (3) culture of retinal pericytes in physiologically relevant concentrations of various vasoactive substances.Retinal pericytes were obtained, by homogenization and enzyme digestion, from aseptically isolated bovine retinal vessels. Efficacy of the culture methods in generating a homogeneous population of pericytes was determined using immunocytochemical markers of microvascular cells, and fluorescence microscopy. Pericytes in culture were identified by their characteristic morphology, lack of reactivity to factor VIII-related antigen antibodies, and lack of uptake of acetylated low-density lipoprotein. Pericytes exhibited reactivity to monoclonal antibodies to both smooth muscle-specific actin and a pancreatic $\beta$-cell surface antigen. Effects of exposure to adenosine or hypoxia on pericyte proliferation were evaluated through quantitation of cell number using an electronic cell counter. Metabolites of adenosine and cyclic AMP modifiers were also tested for any effects on retinal pericyte proliferation.Exposure of pericytes to adenosine resulted in a decreased proliferation rate when compared to untreated controls. Differences in cell number were greatest (p $<$ 0.01) following 8 to 12 days in vitro. Polyadenylic acid mimicked adenosine-mediated pericyte inhibition. An adenosine antagonist, 8-phenyltheophylline, abrogated the effects of both adenosine and polyadenylic acid, suggesting the adenosine-mediated growth inhibition involved receptors. Adenosine appeared to act through the A$\sb2$ receptor, as treatment with the A$\sb2$ receptor specific analog 5$\sp\prime$-$N$-ethylcarboxamidoadenosine gave results similar to adenosine at concentrations near the $K\sb{\rm m}$ for the A$\sb2$ receptor. Since A$\sb2$ receptors are linked via G proteins to adenylate cyclase, the effect of adenosine on pericyte proliferation may involve a transient elevation of cellular cyclic AMP levels. Results from pertussis toxin experiments also suggest a role for the inhibition of adenylate cyclase in this process. The results of the present study suggest that transient elevation of cellular cyclic AMP levels inhibits proliferation of retinal microvascular pericytes following treatment with adenosine in vitro.

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