Senescence of aortic endothelial cells in culture: Effects of basic fibroblast growth factor expression on cell phenotype, migration, and proliferation

Abstract

Bovine aortic endothelial cells (BAEC) can be isolated in large numbers without major contamination by other cells and maintained in culture with a limited life span for about 100 population doublings. In order to study phenotypic changes of BAEC during long-term culture, stocks of different passages of BAEC were established and their morphological, migratory, and proliferative properties analyzed. Early-passage BAEC (passages 5-15) rapidly produce dense, cobblestone-like monolayers. Their growth beyond the monolayer configuration is characterized by the formation of an irregular network of spindle-shaped, crisscrossing BAEC growing either on top or beneath the monolayer, and by the assembly of elongated BAEC into well-differentiated capillary-like tubes. In contrast, senescent BAEC (passages 35-45) form perfect cobblestone monolayers that contain several, often multinucleated giant cells and a few capillary-like tubes but not the crisscrossing networks of their early-passage counterparts. The rates of BAEC migration and proliferation gradually decline during in vitro senescence. This decline is neutralized by exogenous basic fibroblast growth factor (bFGF) which elevates the migratory and proliferative capacities of early-passage and senescent BAEC to uniformly high levels. Northern blot analysis shows a gradual decline in bFGF message and an increase in laminin message during in vitro BAEC senescence. The present study supports the concept of autocrine growth regulation of BAEC and associates a decreased bFGF message with decreased rates of migration and proliferation as well as loss of the crisscrossing BAEC morphotype in senescent cultures.

The Effects of Hyperosmolarity on the Viability and Function of Endothelial Cells

“…Of the available asanguineous flu-up to 100 CPDLs [12,13]. ids, hypertonic solutions have been shown to signifiHypertonic media.…”

Xy99-5038 Promotes Long-Term Survival of Cultured Retinal Neurons

“…Numerous studies performed on cultured human fibroblasts (Wistrom & Villeponteau, 1990), lymphocytes (Pawelec et al, 1997), and several other types of mammalian cells (Augustin-Voss et al, 1993) demonstrated that normal dividing cells have a finite lifetime in vitro (Hayflick, 1998). During the last decade, significant progress was made in culturing of highly differentiated cell, such as neurons.…”

Transcriptional regulation of the Ras‐related protein TC21/R‐Ras2 in endothelial cells

“…Cells were routinely split at a 1:5 ratio and cultured up to 50 passages. Using these culture conditions, subconfluent BAE cells express high levels of endogenous bFGF up to passage 20 which will then gradually decline to undetectable levels during in vitro senescence [9]. Subconfluent migrating BAE cells were harvested at approximately 50% confluence 48 h after seeding.…”

“…For Northern blot analysis of TC21/RRas2 expression, 20 Ixg of total RNA was electrophoresed through a 1% agarose gel, capillary transferred onto nylon membranes (Hybond N, Amersbam, Germany), and used for hybridization with the random prime labeled TC21/R-Ras2 cDNA probe (DNA labeling kit, Pharmacia, Uppsala, Sweden). Hybridization with an 18S rRNA oligonucleotide was performed to confirm equal loading of the different lanes [9]. All Northern blot experiments have at least been performed twice with similar results.…”