Studies on isolated pericytes have been hindered by the lack of specific biochemical markers with which to facilitate identification and purification of this cell type. The pericyte is defined by a strictly anatomical criterion in vivo, namely its anatomical location within the microvascular basement membrane and surrounding the endothelia (1). In dissociated cultures, pericytes have been identified by morphological and growth characteristics (2-5). A recent study (6) has shown that pericytes can be differentiated from endothelial cells and smooth muscle cells by immunofluorescent staining with antibodies directed against actin isoforms. Endothelial cells were shown to express only nonmuscle actin in stress fibers, whereas smooth muscle cells expressed only muscle actin in stress fibers . In contrast, pericytes were shown to express both muscle and nonmuscle actins in stress fibers . The literature contains additional reports of differential distribution of cytoplasmic and cytoskeletal/contractile proteins in vascular cell types (7-9). However, while the differential distribution of cytoplasmic proteins allows identification of vascular cells in vitro, antibodies to these proteins cannot be used as selective agents for purifying cell types as the cells must be fixed and/or permeabilized to make these antigens accessible to antibodies .In this study we report the characterization of an mAb (3G5) that appears to be a specific marker for pericytes within the microvasculature . The 3G5 mAb has previously been shown to react with brain cells, pancreatic islet cells, adrenal medullary cells, thyroid follicular cells, renal glomerular cells, and peripheral blood T lymphocytes (10, 11). Nonetheless, within the microvasculature, the 3G5 mAb is a unique and useful pericyte marker and a selective agent for enriching these cells.
Although one of the earliest findings of diabetic retinopathy is altered capillary permeability, metabolic factors in diabetes that may increase the permeability of capillaries to fluorescein are unknown. We have studied the effect of a variety of vascular and retinal cells and hyperglycemia on the diffusion rate of fluorescein. These studies were performed with a cell culture system that mimics the cross-section of a capillary by having two chambers separated with a porous membrane that can support the growth of cells on either side of the membrane. The addition of a confluent layer of endothelial cells or retinal pigmented epithelial (RPE) cells inhibited fluorescein diffusion between the two chambers 20- and 300-fold, respectively, after cells were cultured for greater than 5 days. Exposure of endothelial cells to 400 mg/dl glucose for either 3 or 100 days did not affect the barrier function of these cells. The barrier function of capillary endothelial cells isolated from BB rats with chronic diabetes and from nondiabetic animals did not differ. In contrast to endothelial cells and RPE cells, arterial smooth muscle and pericytes, which are not known to form tight junctions, did not inhibit the diffusion of fluorescein more than 2-fold. Surprisingly, the dual culture of endothelial cells with either retinal pericytes or smooth muscle cells resulted in a 50-fold increase in the rate of fluorescein diffusion, showing a disruption of the endothelial barrier. In summary, the intercellular connections between endothelial and epithelial cells that are responsible for the barrier to fluorescein diffusion are not functionally affected by chronic exposure to hyperglycemia or diabetic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)
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