Raymond P. Boot-Handford. Department of Biochemistry & Molecular Biology .... V.L., Cannon, F.B., Laurie, G.W. & Martin, G.R. (1986). Biochemistry 25,312-318.
368s Biochemical SocietyTransactions ( 1 991 ) 19
Maintenance of collagen IV expression by bovine retinal p e r i c y t e s isolated directly o n t o M a t r i g e l film. Alan D. Murdoch, Ann E. Canfield*, Michael E. Grant and Raymond P. Boot-Handford. Department of Biochemistry & Molecular Biology and Diabetes Research Centre, University of Manchester, Oxford Road, Manchester M13 9FT, and *C.R.C. Department of Medical Oncology, Christie Hospital & Holt Radium Institute, Manchester M20 9BX, U.K. The biosynthetic phenotype and cell biology o f retinal microvascular pericytes in normal and pathological states is of great interest due to the involvement of this cell type in diseases such as diabetic retinopathy and in angiogenesis [ 11. The anatomical location of pericytes within the retinal capillary basement membrane suggests that a major secreted biosynthetic product in vivo would be collagen IV. However, when placed in culture, the predominant collagenous species secreted by pericytes is collagen I with only trace amounts of collagen IV being apparent [ 11. We decided to investigate whether the low level of collagen IV synthesis exhibited by cultured retinal pericytes was a consequence of dedifferentiation due to the culture conditions employed. In particular, we were interested in determining whether growing these cells on a basement membrane substratum (Matrigel) rather that the routinely employed gelatin films would enhance collagen IV synthesis since basement membrane matrices have been shown to be effective in maintaining the in vivo pattern of gene expression in other cell types that normally exist upon a basement membrane matrix [e.g. 21. Bovine retinal pericytes were isolated and characterised as described [see 11 and cultured either on plastic tissue culture dishes coated with a film of gelatin or Matrigel or on a 2mm thick gel of Matrigel in Eagles MEM + 20% donor calf serum. Films of gelatin (O.lmg/ml solution) and Matrigel (prepared as described in [31 and used at lmg proteidml) were generated by coating the surface of culture dishes with the designated sterile solution, leaving for 20 min, decanting the excess liquid and then washing with serum-free medium. Matrigel gels were prepared by pipetting an appropriate volume of sterile, icecold Matrigel (10mg proteidml) to give a depth of 2mm, allowing the Matrigel to set by warming to room temperature over 30 min, and finally equilibrating the gel with culture medium. Cultures were labelled for 24 h with L-[ 5-3HIproline and proteins secreted into the medium analysed by SDS/PAGE fluorography [ 11. Northern analysis was performed on total cellular RNA as described [41. In the first series of experiments, primary or early passage cultures of pericytes isolated directly onto gelatin films were subcultured and plated onto films of gelatin, Matrigel or onto Matrigel gels. Cells plated onto films of either gelatin or Matrigel exhibited the typical pericyte morphology [ 1I. However, cells plated on top of a Matrigel gel attached to the surface but remained rounded a n d gradually extended into the gel, thin dendrite-like processes that branched and made contacts with similar processes from other cells. The major collagen type synthesized by cells plated under these different culture conditions was type I collagen. Surprisingly, culturing the cells on either a film
or a gel of Matrigel did not increase the synthesis of collagen IV in comparison with the cells grown on films of gelatin. In a second series of experiments, bovine retinal pericytes were isolated directly and subsequently passaged once on films of Matrigel. Pericytes isolated in this way appeared identical morphologically to those isolated on gelatin films. These cultures were then passaged onto films of either Matrigel or gelatin and the synthesis of collagen IV examined at confluence. Collagen IV, assessed by SDWAGE fluorography of the labelled culture media, was found to be a major biosynthetic product of pericytes isolated directly onto Matrigel films whether subsequently passaged onto films of either Matrigel or gelatin. The authenticity of the collagen IV produced by pericytes isolated directly onto films of Matrigel was confirmed by its susceptibility to highly purified bacterial collagenase and by its immunoprecipitation with an antiserum to collagen IV. Total cellular RNA was isolated from the pericyte cultures exhibiting high levels of collagen IV synthesis and analysed for various matrix mRNAs by northern blotting. mRNAs encoding a 1(I) collagen and laminin B l were detected readily by hydridization with human- and mouse-derived cDNAs. However, both human- and mouse-derived cDNAs encoding either al(1V) or a2(IV) collagen failed to produce detectable signals upon hybridization to the same blots. This study demonstrates clearly that collagen IV is a major biosynthetic product of retinal pericytes isolated directly onto a basement membrane-like substratum but not if isolated onto gelatin. Two obvious explanations for this finding are either that different subsets of this cell type are obtained dependant upon the nature of the matrix used in the initial isolation or that the nature of the matrix to which they first attach has a profound effect upon the biosynthetic phenotype exhibited subsequently by pericytes in culture. Although biochemical analysis has confirmed the authenticity of the collagen IV synthesized by bovine retinal pericytes isolated on Matrigel films, the chain composition of this macromolecule remains in doubt. Human and mouse cDNAs for the a 1 and a 2 chains of collagen (IV) did not cross-hybridize with bovine pericyte mRNA. This could be due to significant differences in the nucleotide sequence of bovine compared to humadmouse collagen (IV) a -chains although this appears unlikely [5]. Alternatively, these cells may be synthesizing a collagen IV with a chain composition other than the usual a 1(IV)2a2(IV). This study was supported by the R.N.I.B., t h e University of Manchester (R.A. Needham Fund) and the C.R.C. [ l ] Canfield, A.E., Allen, T.D., Grant, M.E., Schor, S.L. & Schor, A.M. (1990) J. Cell Sci. 96, 159-169 [2] Bissell, M.J. & Ram, T.G. (1989) Environmental Health
Perspectives 80,61-70 [3] Kleinman, H.K., McGarvey, M.L., Hassell, J.R.,Star, V.L., Cannon, F.B., Laurie, G.W. & Martin, G.R. (1986) Biochemistry 25,312-318 [4] Boot-Handford, R.P., Kurkinen, M. & Prockop, D.J. (1987) J. Biol. Chem. 262, 12475-12478 [5] Butterworth, H., Murdoch, A.D. & Boot-Handford, R.P. (1991) Biochem. SOC.Trans. This Vol.
