Heterogeneity in collagen biosynthesis by sprouting retinal endothelial cells

Abstract

Bovine retinal microvascular endothelial cells can display two distinct and reversible morphologies in culture: 'cobblestone' and 'sprouting'. The cobblestone morphology resembles the resting cells lining the lumen of mature vessels while the sprouting morphology resembles the angiogenic cells involved in the formation of new vessels. Retinal cells displayed some heterogeneity in the shape of the cells making up the cobblestone monolayer. In contrast, all cell lines displayed an identical sprouting morphology. We have investigated the synthesis of matrix macromolecules by retinal endothelial cells displaying either the cobblestone or the sprouting morphology. Type IV was the only collagen synthesised by eight different lines of early-passage (between one and six) cobblestone endothelial cells. Collagen types I and III were not detected in these cultures. In contrast, heterogeneity was observed in the types of collagen synthesized by four lines of early-passage cells displaying the sprouting morphology. That is, two lines synthesised collagen types, II, III and IV, whereas two other lines continued to synthesise only type IV collagen. Both cobblestone and sprouting cells synthesised fibronectin and thrombospondin, although the relative amounts of these macromolecules varied with culture conditions. The pattern of collagen synthesis by cobblestone cells was also affected by in vitro "ageing": 4/5 lines examined above passage eight synthesised collagen types I, III and IV. Our results indicate that there is heterogeneity in the sprouting phenotype displayed by retinal endothelial cells, and that this phenotype is not necessarily associated with the synthesis of type I collagen. We suggest that differences in the spectrum of matrix macromolecules synthesised by sprouting endothelial cells may play a role in the control of angiogenesis.

Cell sheet detachment affects the extracellular matrix: A surface science study comparing thermal liftoff, enzymatic, and mechanical methods

“…Type IV collagen (Coll IV) is believed to function as a scaffold by which other ECM proteins and constituents bind and align 50, 51. Although Coll IV is known to be the primary type of collagen in basement membranes secreted from endothelial cells, it has previously been found that endothelial cells may respond to minute differences in their surroundings (e.g., in the media or the type of substrate used) by extruding differing amounts or types of collagen 52, 53. For this reason, we have chosen to perform immunoassay on both Coll IV and collagen type I (Coll I).…”

Synergistic stimulation of gamma-glutamyl transpeptidase and alkaline phosphatase activities by retinoic acid and astroglial factors in immortalized rat brain microvessel endothelial cells

“…The transition from the cobblestone to the sprouting morphology is associated with changes in matrix biosynthesis and in matrix degrading proteolytic enzymes (Iruela-Arispe et al, 1991;Canfield and Schor, 1994). The induction of such changes by factors added t o the culture medium may modulate the formation or stability of three-dimensional structures.…”

Basic Cell Culture Protocols

“…The lateral matrix chemistry, found between homotypic cells, consists of CAMs and proteoglycans, but no collagens, fibronectins, or laminins (55)(56)(57)(58)(59)(60)(61)(62)(63)(64)(65)(66)(67)(68)(69)(70).…”

“…MEC from human, bovine, sheep, rabbit, rat, and mouse have been cultivated from brain (56)(57)(58)(59)(60), retina (61)(62)(63)(64), skin (65), lung (66)(67)(68), intestinum (69), heart (70--72), liver (73), corpus luteum (74,75), meniscus (76), and synovium (77). Since investigations are performed in the field of rheumatology in our laboratory, techniques appropriate for isolating, cultivating, and immunostaining synovial microvascular EC (SMEC) are described in this chapter.…”