Date of Award

1-1-1999

Embargo Period

1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Ophthalmology

College

College of Graduate Studies

First Advisor

Jian-Xing Ma

Second Advisor

Rosalie K. Crouch

Third Advisor

Maria G. Buse

Fourth Advisor

L. Lyndon Key, Jr.

Fifth Advisor

Julie Chao

Abstract

Abnormalities of the retinal vasculature contribute significantly to the development of diabetic retinopathy. In this study we have focused on two mechanisms which may be responsible-for the development and progression of this diabetic complication. Chronic hyperglycemia is associated with many of the vascular complications of diabetes mellitus; therefore, we examined mechanisms by which high-dose glucose damages retinal capillary endothelial cells. Our cell count results showed that high-dose glucose induces cell death in primary cultured human retinal capillary endothelial cells. Flow cytometry, in situ cell death detection, and transmission electron microscopy revealed that the cell death occurred via the apoptosis pathway rather than necrosis. Secondly, we focused on one of the two mechanisms that control vascular functions of vasodilation and vasoconstriction. Renin-angiotensin and tissue kallikrein-kinin are two counter-balancing systems which play important roles in local vascular regulation. Tissue kallikrein is a serine proteinase which cleaves kininogens to release vasoactive kinins. Kinins interact with the bradykinin receptors on the cell surface and mediated a variety of biological effects such as vasodilation, regulation of local blood flow and tissue metabolic rate, stimulation of cell proliferation, production of pain and inflammatory responses. The kallikrein-kinin system may play an important role in regulating vascular function in the eye. Kallikrein-binding protein (KBP) is a serine proteinase inhibitor (serpin) which forms a complex with tissue kallikrein and inhibits its proteolytic activity and thus reduces production of kinin. The results of RT-PCR followed by Southern blot analysis showed that components of the tissue kallikrein-kinin system are endogenously expressed in human ocular tissues. We showed that KBP expression was decreased in the retina and serum of the streptozotocin (STZ)-diabetic rat compared to the nondiabetic control rat as determined by enzyme-linked immunosorbent assay (ELISA) and Northern blot analysis. Moreover, KBP complex formation assay revealed that functionally active KBP was decreased in the serum of the STZ-diabetic animal. There are two hypotheses to explain decreased KBP in the development of diabetic retinopathy. First, KBP may be acting through the tissue kallikrein-kinin system. As a potential regulatory protein of tissue kallikrein, decreased KBP results in overproduction of bradykinin that consequently induces retinal vasodilation. Furthermore, it has been hypothesized that increased retinal blood flow, made worse by impaired autoregulation, is of pathogenic importance in the development of diabetic retinopathy. The second hypothesis is independent of interactions of KBP with kallikrein and kinin. Studies of growth hormone (GH) mechanism and animal growth regulation have identified KBP as one of the protein factors most strongly induced by GH in the liver. Several studies have shown that growth hormone is decreased in the STZ-diabetic rat. Thus, we examined the genetic dwarf rat to determine the in vivo effect of GH-deficiency of KBP expression. Serum ELISA and Northern blot analysis results showed a significant decrease in KBP expression in the dwarf rat compared to the normal rat; moreover, the GH-treated dwarf rat showed a significant increase in KBP levels compared to the untreated dwarf rat. Hepatic cells are the major suppliers to serum KBP and are directly exposed to serum insulin, glucose, and GH. To understand the decreased KBP expression in the dwarf rat and the STZ-diabetic rat, we have used cultured rat hepatocytes in vitro studies. We studied the effect of glucose and insulin, as well as GH, in cultured cells. RT-PCR followed by Southern blot analysis of the rat hepatocytes showed that GH increased KBP expression. Moreover, ELISA of the conditioned media from GH-treated rat hepatocytes supported these data showing a significant increase in KBP at the protein level. Understanding the pathogenesis of decreased KBP may contribute to our understanding of the physiological function of KBP as well as provide a potential target for drug or gene therapy in the treatment of diabetic retinopathy.

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