Partial purification of smooth muscle cell derived growth factor

Abstract

The serum free medium conditioned by cultured rabbit aortic smooth muscle cells was partially purified using ultrafiltration and heparin affinity chromatography. Incorporation of [³H]-thymidine (³H-TdR) into cell DNA was used to measure the mitogenic activity of the fractions from chromatography for NIH 3T3 fibroblasts. The molecular weight and the iso-electric point of these fractions were determined by NaDodSO₄-polyacrylamide gel electrophoresis (SDS-PAGE) and iso-electric focusing, respectively. The results showed that the protein eluted in 1. 0–1. 6 mol/L NaCl from the heparin-Sepharose was mitogenic for 3T3 cells, and this protein had a molecular weight of 22. 8–26. 7 ku and an iso-electric point of about 4. 6. The fact that the above-mentioned biochemical properties differed from that of PDGF, IGF and FGF suggests that this mitogenic protein may be a separate growth factor.

Keywords

growth factor / smooth muscle cell / purification / atherosclerosis

References

[1] SchwarteC J, WalenteA J, SpragueE A, et al.. The pathogenesis of atherosclerosis: An overview. Clin Cardiol, 1991, 14(suppl 1): 1-1

[2] RossR. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature, 1993, 326(6 423): 801-801

[3] 1991, 6(3): 216

[4] MoscatelliD, PrestaM, RifkinD B. Purification of a factor from human placenta that stimulates capillary endothelial cell protease production, DNA synthesis and migration. Proc Natl Acad Sci USA, 1986, 83(7): 2-091

[5] DempseyE C, StenmarkK r, McMurtryI F, et al.. Insulin-like growth factor I and protein C activaiton stimulate pulmonary artery smooth muscle cell prolieration through separate but synergistic pathways. J Cell Physiol, 1990, 144(1): 159-159

[6] DaviesG E, StarkG R. Use of dimethyl suberimidate, A cross-linking reagent, in studying the subunit structure of oligomeric proteins. Proc Natl Acad Sci USA, 1970, 66(3): 651-651

[7] OakleyB R, KirschD R, MorrisN R. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem, 1980, 105(2): 361-361

[8] SarzaniR, BrecherP, ChobanianA V. Growth factor expression in aorta of normotensive and hypertensive rats. J clin Invest, 1989, 83(4): 1-404

[9] FolkmanJ, KlagsbrunM. angiogenic factors. Science, 1987, 235(4 787): 442-442

[10] SjölundM, HedinU, SejersenT, et al.. Arterial smooth muscle cells express platelet derived growth factor (PDGF) A chain mRNA, secete a PDGF-like mitogen and bind-exogenous PDGF in a phenotype and growth state-dependent manner. J Cell Biol, 1988, 106(2): 403-403

[11] ShingY, FolkmanJ, SullivanmR, et al.. Heparin affinity: purification of a tumor-derived capillary endothelial cell growth factor. Science, 1984, 223(4 642): 1-296

[12] MorisakiN, KoyamaN, MorisS, et al.. Effects of smooth muscle cell derived growth factor (SDGF) in combination with other growth factors on smooth muscle cells. Atherosclerosis, 1989, 78(1): 61-61