Extracellular matrix permits the expression of von willebrand’s factor, uptake of Di-I-acetylated low density lipoprotein and secretion of prostacyclin in cultures of endothelial cells from rat brain microvessels

Abstract

Microvascular endothelial cells from the adult rat brain were cultured on Matrigel and found to express many differentiated properties including secretion of prostacyclin (PGI2) and von Willebrand's factor (vWF). Brain microvascular endothelial cells (BMECs) were purified by dextran and percoll gradients after enzymatic treatment and cultured under various conditions. BMECs that were plated on Matrigel stained positively for factor VIII-related antigen and incorporated Di-I-acetylated low density lipoprotein, whereas BMEC plated on fibronectin, gelatin, or uncoated dishes did not express any of the above properties which are characteristic of endothelial cells. vWF was measured by a sensitive ELISA in the culture media of BMECs plated on different types of matrices. Specificity of the anti-human vWF antibodies for the rat vWF was verified by immunoabsorption on a solid phase, sodium dodecyl sulfate, and Western blot analysis. BMECs also secreted vWF into the culture media only when the cells were plated on Matrigel, and this secretion was augmented after a 6 h incubation with an interleukin-1 tumor necrosis factor-alpha mixture, but not by lipopolysaccharide. From different matrices tested, only Matrigel permitted the secretion of PGI2 by BMECs. Cells also proved to be sensitive to mechanical stimulation and became refractory to secretagogue if the mechanical stimulation was serially repeated. Under the best conditions, stimulation of the cells with bradykinin (1 microM) substantially increased PGI2 secretion. These data indicate that growth of BMECs on Matrigel in vitro permits the expression of classical endothelial cell markers in a manner similar to the behavior of these cells in situ.

Reorganization of endothelial cord‐like structures on basement membrane complex (Matrigel): Involvement of transforming growth factor β1

“…The relationship between endothelial cell differentiation and proliferation is well known (Ingber and Folkman, 1989). In addition to the expression of endothelial differentiation markers (Doron et al 1991), a variety of cells when cultured on Matrigel lead to the expression of differentiation markers as well as inhibition of proliferation (Kubota et al, 1988;Vukicevic et al, 1990;Kibbey et al, 1992). We found that soluble factor(s1 released from Matrigel inhibited DNA synthesis of endothelial cells on plastic plates.…”

“…The reconstituted basement membrane protein mixture derived from the Engelbreth-Holm-Swarm (EHS) murine sarcoma induced differentiation in a variety of cell types: mammary epithelial cells (Streuli et al, 19911, hepatocytes (Bissell et al, 19871, embryonic lung cells (Schuger et al, 19901, bone cells (Vukicevic et al, 1990), Sertoli cells (Hadley et al, 1990), and pheochromocytoma cells (Schwarz et al, 1990). The culturing of microvascular endothelial cells on Matrigel resulted in the increased expression of factor VIII and uptake of acetylated low density lipoprotein, both classical markers for endothelial cell differentiation (Doron et al, 1991). Although the exact mechanism of the Matrigel-induced differentiation remains unknown, biologically active peptide sequences within laminin may play an important role in the process.…”

Prostanoids regulate angiogenesis acting primarily on IP and EP4 receptors

“…Major constituents of matrigel are structural proteins such as laminin, entactin, collagen and heparan sulfate proteoglycans. It has emerged that complex substrates such as matrigel are essential for the expression of classical endothelial genes and their response to prostacyclin (Doron et al, 1991). The importance of endothelial adhesion to the extracellular matrix through integrins and other factors in response to prostacyclin has been demonstrated (Aburakawa et al, 2013); this suggests that the effects of prostacyclin in HUVECs might be influenced by interactions with the extracellular matrix (Orpana et al, 1997).…”

Adhesion Molecules on Normotensive and Hypertensive Rat Brain Endothelial Cells

“…The isolation and cultivation of cerebromicrovascular EC from SHR, WKY and SD rats was performed with some modification as previously described (23). Propagated EC (1 x 10 4 /100 J,Lllwell) were passaged and grown to confluency on 1% gelatin-coated coverslips or 96-well flat bottom microtiter plates at 37°C in humidified atmosphere of 5% COiair in M-199 medium (containing 25 mM Hepes buffer, Earle's salts and L-glutamine) with 20% heat-inactivated FCS, 90 J,Lg/ml heparin, 100 J.Lg/ml Streptomycin, 100 U/ml penicillin G, and 0.25 J.Lg/ml amphotericin B as fungizone (all from GIBCO, Long Island, NY) and 20 J.Lg/ml endothelial cell growth supplement (Collaborative Research, Bedford, MA).…”