Objective: Human vascular smooth muscle cells (VSMC) prepared by enzyme dispersal have been reported to proliferate more slowly and remain more differentiated than VSMC derived by explant culture. The aim of this study was to investigate whether these differences are attributable to the production of transforming growth factor beta (TGF beta), a major modulator of VSMC properties in culture, by enzyme dispersal, but not explant, cultures.
Methods: Human aortic VSMC cultures were prepared by enzymatic dissociation of aortic media or by outgrowth of cells from explants derived from the vessel media. Properties, including rate of proliferation and differentiation, of the two different types of culture obtained from 10 donors (age 3-54 years, of either sex) were compared. The role of TGF beta as mediator of these differences was investigated.
Results: VSMC from enzyme dispersal cultures proliferated with a long doubling time in 20% fetal calf serum of 68(SEM 2) h, reaching a low saturation density with no "hills and valleys", and the cells were predominantly of stellate morphology. VSMC from explant cultures proliferated with a shorter doubling time of 35(2) h, reached a higher saturation density with "hills and valleys", and showed a spindle shaped morphology. The enzyme dispersed cells, but not the explant cells, released TGF beta into the medium. Addition of exogenous TGF beta to the explant cells extended the doubling time, while addition of a neutralising antibody to the enzyme dispersed cells decreased the doubling time. Tamoxifen, an anti-oestrogenic drug, decreased the rate of proliferation of the explant cells (ED50 = 50 nM; n = 3) but not enzyme dispersed cells by stimulating production of TGF beta. The explant and enzyme dispersal cultures also differed in response to growth factors. The explant cultures stimulated DNA synthesis in response to platelet derived growth factor (PDGF) AA and BB. Enzyme dispersal cultures stimulated DNA synthesis in response to PDGF-BB but response to AA was weaker and also variable.
Conclusions: Human explant derived VSMC systematically differ from enzyme dispersed VSMC obtained from the same donor tissue. Wherever the potential role of TGF beta was investigated, the differences between enzyme dispersal and explant cultures were due to autocrine production of TGF beta by the enzyme dispersal, but not explant cultures.