Characterization of Clonal Vascular Smooth Muscle Cell Lines Derived from Young and Old Fischer 344 Rats

Abstract

A significant finding with aging humans (and aging animal models) is that blood vessels lose their ability to respond to beta-adrenergic receptor stimuli. Therefore, they produce less cyclic adenosine monophosphate (cAMP) and have decreased vasorelaxation with advancing age. This change likely contributes to hypertension, insufficient blood flow, and atherosclerosis. Our goal was to develop a vascular smooth muscle cell culture model that replicates the molecular and biochemical changes observed in blood vessels with advancing age. A clonal selection strategy was used to produce cell lines from 2-, 6-, 12-, and 24-month-old male Fischer 344 rat aortae. Cultures were validated as smooth muscle cells with immunocytochemistry positive for α-actin and negative for von Willebrand factor VIII. Positive staining for G protein-coupled receptor kinase 2 indicated presence of this adrenergic receptor regulator. A total of n = 5 clones from n = 7 animals for each age group were initially analyzed for cAMP accumulation under three conditions: basal, isoproterenol stimulated, and forskolin stimulated. Results found that at passage3, there was a significant reduction in cAMP accumulation to isoproterenol. However, this reduction disappeared by passage6. Secondary analysis segregated clones into phenotypic age groups independent of donor animal age. Segregation identified n = 3 clones per group. At passage3, the age-related change in the beta-adrenergic change was magnified. However, even with segregation, the adrenergic response was lost by passage6. Our results show that early passaged clonal vascular smooth muscle cell cultures maintain their aging, adrenergic phenotype. Two separate strategies to identify age-representative phenotypes into later passage were unsuccessful.