Open AccessIdentification of multiple genes in bovine retinal pericytes altered by exposure to elevated levels of glucose by using mRNA differential display.
Author(s) -
Lloyd Paul Aiello,
G. A. Robinson,
You-Wei Lin,
Yoshihiko Nishio,
George L. King
Publication year - 1994
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.91.13.6231
Subject(s) - pericyte , biology , differential display , gene expression , messenger rna , microbiology and biotechnology , gene , retinal , northern blot , western blot , blot , endocrinology , genetics , biochemistry , endothelial stem cell , in vitro
Loss of capillary pericytes, a characteristic finding in diabetic retinopathy, is strongly associated with hyperglycemia. The pathologic aberrations associated with diabetic retinopathy are localized primarily in the retinal capillaries and are only poorly reversed by subsequent euglycemic control. Since hyperglycemia significantly inhibits pericyte growth in culture, we investigated the regulation of gene expression in retinal pericytes exposed to physiologic (5.5mM) and pathologic (20 mM) glucose concentrations. By utilizing modifications of the mRNA differential display technique, over 14,000 mRNA species were screened, and 35 candidate clones were obtained. Partial DNA sequence demonstrated that 25 of these were distinct genes, including 7 known, 16 previously unreported, and 2 sequences with known homologues. Northern blot analysis demonstrated altered gene expression in 10 (40%), undetectable signals in 12 (48%), and nonregulation in 3 (12%). Genes with glucose-regulated expression included those encoding fibronectin (51% +/- 15%, P = 0.003; mean percentage of control +/- SD), caldesmon (68% +/- 18%; P = 0.026), two ribosomal proteins (201% +/- 72%, P = 0.011; 136% +/- 16%, P = 0.036), Rieske FeS reductase (66% +/- 17%; P = 0.029), three previously unreported sequences (57%, 167%, 271%), and molecules homologous to autoantigens (213%) and tyrosine kinases (down 16- to 33-fold). Caldesmon protein concentrations in pericytes and smooth muscle cells demonstrated decreases by Western blot analysis concordant with mRNA levels. These studies identify genes whose expression is significantly altered after 7 days of exposure to elevated glucose levels and provide new targets for understanding the adverse effects of hyperglycemia on vascular cells. In addition, this study provides strong support for the use of differential mRNA display as a method to rapidly isolate differentially expressed genes in metabolic systems.