role of extracellular matrix in angiogenesis.
Contributors: University of Manchester. Faculty of Medicine.
Publisher: University of Manchester
Published: 1996
Place of publication: Manchester
Language: eng
Number of pages: 271
Description: Cultured endothelial cells may display two distinct phenotypes, characterised initially by their morphologies: (1)  cobblestone cells, which form a monolayer of closely apposed cells, similar to the endothelium lining mature vessels and (2) elongated or sprouting cells, which associate forming tubular structures or sprouts, reminiscent of the angiogenic vessels in vivo. The cobblestone phenotype is obtained when the cells are plated on a two-dimensional (2D) substratum, whereas the same cells display the sprouting phenotype when embedded within a three-dimensional (3D) matrix. 

Cobblestone and sprouting endothelial phenotypes are reversible. Several studies have reported that the transition from one to the other is accompanied by changes in gene expression; however, conflicting results have been published regarding the precise nature of these changes.  Discrepancies between different laboratories may be due to either heterogeneity amongst endothelial cells or the different culture conditions used. 

In this study, we have investigated the effects of various culture conditions on protein synthesis by bovine aortic endothelial cells (BAEC). We found that serum concentration, time in culture and cell proliferation did not affect protein synthesis by these cells. We demonstrated that cell density is the only culture condition that affects  protein synthesis. Total protein synthesis per cell was found to decrease with increasing cell density by both cobblestone cells (in 2D cultures) and  sprouting cells (in 3D cultures). 
The synthesis of certain matrix macromolecules was specifically affected by cell density. Namely, on 2D cultures (cobblestone) the relative levels of fibronectin, laminin and tenascin increased with cell density, whereas those of thrombospondin decreased with cell density. The regulation of expression for fibronectin and thrombospondin was found to be at the transcription level. 

In 3D cultures (sprouting cells), fibronectin levels increased and those of laminin decreased with increasing cell density. The direct relationship between fibronectin synthesis and cell density did not appear to be due to an increase in cell-cell contacts since the biosynthesis of this protein was inhibited when the sprouting cells were plated as multicellular aggregates. Our results suggest that at least some of the  changes attributed to the transition from the cobblestone to the sprouting phenotype may be due to cell density effects. 

Using immunocytochemistry, we demonstrated that the above matrix molecules were expressed in 2D cultures of sparse, confluent and post-confluent BAEC; the latter included cobblestone and sprouting cells. In these cultures, we found that the staining of fibronectin was mainly extracellular, whereas that of thrombospondin, tenascin and laminin was mainly cytoplasmic. Differences between cobblestone and sprouting cells were observed regarding the immunolocalisation of thrombospondin and tenascin.

Extracellular matrix molecules exert their biological role via integrins, which are heterodimeric glycoproteins capable of binding to matrix components and transmitting an intracellular signal upon activation. We have examined the localisation of certain integrins on post-confluent cultures of BAEC containing sprouting and cobblestone cells. The integrin subunits examined were: α1, α2, α3, α4, α5, αv, ß3 and the vitronectin receptor (VnR).  We found that both cobblestone and sprouting cells were equally stained by antibodies to integrin subunits α1, α2, α3, α4, α5, αv, ß3. In  contrast, sprouting cells stained preferentially with an antibody to the vitronectin receptor, which recognised the αvß3 and αvß5 integrins.  

The localisation of integrin subunits α1, α2, α3, α4, α5, αv, ß1, ß3 and αvß3 was next examined on histological sections of human tissues using immunohistochemistry.  The tissues were selected with the aim of comparing integrin expression in angiogenic and resting vessels. To that effect we examined tissues where angiogenesis is taking place, namely, granulation tissue, breast carcinoma and lung carcinoma, as well as their corresponding normal tissues.  We first had to choose a suitable pan-endothelial marker for localising the vessels and a method of quantitating the number of vessels stained.  We found that CD31 and von Willebrand factor (vWF) stained the same number of vessels. In quantitative analysis, we compared two methods of counting, namely average microvascular density (a-MVD) and highest microvascular density (h-MVD) and found a good correlation between the two. We decided to use vWF and a-MVD for practical considerations.  Adjacent sections were then stained with vWF and integrin antibodies.

We found that the integrin αvß3 complex was expressed by the majority of vessels in granulation tissue, breast carcinoma and lung carcinoma tissues and that its expression was markedly reduced or absent in the vessels of normal skin, normal breast and normal lung. These results indicate a clear difference between angiogenic and resting vessels. The expression of α4 and α5 integrin subunits was also enhanced in angiogenic vessels. In contrast,  α1, α2 and ß1 integrin subunits were equally expressed by all vessels in the resting and angiogenic tissues examined. Taken together our results suggest a combined role for matrix macromolecules and integrins during angiogenesis.
Notes: Thesis (Ph.D) - University of Manchester, Faculty of Medicine.
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