Clonal growth of bovine vascular endothelial cells: fibroblast growth factor as a survival agent.

Abstract

Bovine vascular endothelial cells do not grow when cultured at low density unless fibroblast growth factor is included in the culture medium. When endothelial cells obtained from the intimal surface of fetal and adult aortas were seeded at low density (8 cells per cm2), they formed small colonies of large, irregular, vacuolated cells. At very low density (0.3 cells per cm2) they did not survive. The addition of fibroblast growth factor to endothelial cells maintained at such low densities resulted in the formation of vigorously growing colonies of small, uniform cells. Electron microscopy showed that the cultured endothelial cells had the fine structure characteristics of endothelial cells. Immunofluorescence microscopy revealed antihemophilic factor (Factor VIII) antigen in the cells. Our results demonstrated that fibroblast growth factor permits the survival of endothelial cells plated at extremely low cell density. With the use of fibroblast growth factor, endothelial cell clones are easily produced. Endothelial cells constitute the inner lining of the blood vascular system. Because of their location at the interface between blood and tissue, they are the chief elements involved in the permeability of blood vessels (1, 2). Abnormalities of endothelial cell structure and function are prominent in the pathology of a number of diseases of blood vessel walls such as thromboangiitis (3) and microangiopathy (4).Since the continuity of the vascular endothelium is essential for the survival of the organism, the elucidation of the factors involved in endothelial cell survival and proliferation is important. Their survival and proliferation can be examined most easily in tissue culture.Recently, several factors that promote the growth of cells in culture have been identified. One of the most potent is the fibroblast growth factor (FGF) which stimulates the growth of a variety of mesoderm-derived cells (5).Three observations have led us to examine the effect of FGF on vascular endothelial cells: (i) FGF is a potent mitogen for Balb/c 3T3 cells (6). Although these cells are commonly referred to as fibroblasts, their morphology and the fact that they can produce vasoformative sarcomas in vivo (7) Dulbecco's modified Eagle's medium (DME) supplemented with 10% calf serum (Gibco) and 100 ng/ml of FGF in an atmosphere of 12% CO2 in air to maintain the pH of the medium between pH 7.3 and 7.5. Bovine fetuses of 4 months' gestation (45 cm, crown to rump), bovine adult aortic arches, and bovine umbilical cords were obtained from a local slaughterhouse.Endothelial Cell Culture. The aortic arches were opened lengthwise with a scalpel and the intimal surface was washed with Ca++-free phosphate-buffered saline to remove blood. The endothelial cell layer was removed by gently scraping the intimal surface with a grooved director. The grooved director was dipped in 10 ml of DME with 10% calf serum. This technique gave cell populations composed of 99% endothelial cells.For purer populations, a cotton swab was used instead of a ...

Migration of Mononuclear Cells Expressing β-Actin Through the Adventitia into Media and Intima in Coronary Arteriogenesis and Venogenesis in Ischemic Myocardium

“…β-MNCs express CD 34 , a nonspecific marker for bone marrow origin, 17) but not CD 31 , a marker for ECs, 17,29) vimentin, a marker for SMCs and myofibloblasts, 30) α-SMA, a marker for contractile phenotype of SMCs and fibroblasts/myofibroblasts, [19][20][21] SM-1, a marker for matured SMCs, 20) or N-cadherin, a marker for fibroblasts and myofibrolasts. 21) β-MNCs are different from SMC progenitor cells or fibroblasts which do not express β-actin and CD 31 .…”

Aortic endothelial and smooth muscle histamine metabolism in experimental diabetes.

“…Animals were held for 2 or 4 weeks following overt manifestation of diabetes, e.g., hyperglycemia, glycosuria; in the 4-week group, additional rats were given 10 units of insulin (lletin NPH, Eli Lilly and Company, Indianapolis, Indiana) subcutaneously in the late afternoon daily during the fourth week. Animals were killed by decapitation, the thoracic aorta excised, and endothelial cells removed as described by Gospodarowicz et al 16 Verification of isolated cell types (endothelial and smooth muscle) involved both transmission electron microscopy of fresh isolates and phase contrast microscopy of primary cell cultures established from these cell isolates (see below).…”

The fate of the primary diploid population during spontaneous transformation of growth factor‐supplemented murine cell cultures

“…In a t least one cell culture system (bovine vascular endothelial cells (8,9)), growth factor supplementation has been successful in eliminating both the typical growth crisis and the associated loss of diploid cells. This result has encouraged us to attempt to extend the very short precrisis stage of primary rodent cells, hoping to make available more long-term cell cultures of murine origin.…”