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Smooth muscle cells were grown from thoracic aortas of rats. The effect of insulin on the proliferation of these cells was studied by comparing the growth of cells in culture medium containing insulin and 1% fetal calf serum with growth of cells in culture medium containing only 1% serum and in culture medium containing 10% serum. Insulin in concentrations of 10, 50, 100, 1000 and 10 000 microunits/ml induced smooth muscle cells to stationary growth more rapidly than basal medium. This could be demonstrated as well in the logarithmic growth as in confluent cells grown in medium with 1% serum. However, the highest concentration of insulin did not stimulate growth to the same degree as medium containing 10% serum. Cells that were older in culture life (11th passage) did not show a growth response to insulin.

Serum contains various growth factors which regulate the proliferation of cells. We investigated the growth of cultured arterial smooth muscle cells under the influence of insulin-like growth factor I (IGF I), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF), and examined the effect of these growth factors on the binding of [125I] IGF I and on the binding of [125I]PDGF to these cells. IGF I, FGF, and PDGF stimulated [6-3H]thymidine incorporation into DNA of confluent cultures of cells which were incubated in modified Dulbecco's modified Eagle medium. However, the effect of these growth factors on DNA synthesis was much more potent in Dulbecco's modified Eagle medium with 1% fetal calf serum. FGF and PDGF potentiated the growth-promoting effect of IGF I. The binding of [125I]IGF I to the cells was increased after a preincubation with FGF and PDGF. The binding was potently increased by FGF (100 ng/ml) after a preincubation time of 30 min. There was an increase in binding during the first 3 h of preincubation followed by a decrease after 4-5 h. PDGF (10-1000 ng/ml) stimulated [125I]IGF I binding only after 2 h of preincubation. The stimulation was dose dependent. Maximal stimulation of the binding was observed after 3 h of preincubation followed by a decrease after 4-5 h of preincubation. Specific binding sites for PDGF on smooth muscle cells could be demonstrated too. A preincubation of confluent cells with IGF I caused a dose-dependent increase in [125I]PDGF binding. These results support the hypothesis that the regulation of the binding of a specific growth factor by a second growth factor is important for the control of cell growth.

Insulin-like growth factors (IGF) were isolated from human serum and compared with some biological actions of IGF supplied by Dr. J. Hapf, Zurich. Both factors were potent mitogens. They stimulated DNA-, RNA- and protein synthesis in cultivated human arterial smooth muscle cells. Furthermore, they enhanced the aminoacid transport. Our protein fraction (IGF Ulm) had a more potent biological activity than IGF (Zurich). Specific binding receptors for IGF (Zurich) on human arterial smooth muscle cells could be demonstrated. Specific binding of 125I-IGF (Zurich) was 10%. Half-maximal displacement was achieved by 250 ng/ml of unlabeled IGF (Zurich), by 1.2 micrograms/ml of IGF (Ulm), by 6.3 micrograms/ml of pro-insulin and by 17.8 micrograms/ml of insulin. In separate studies we could demonstrate that sera of normal adults, diabetic, acromegalic and hypophysectomized patients showed different growth-promoting activity in human arterial smooth muscle cells.

There are indications for the existence of an intrinsic renin angiotensin system in vascular walls, which is assumed to participate in blood pressure regulation and in pathogenesis of arterial hypertension. It was evaluated if and to what extent the decapeptide angiotensin (A) I, one of the natural substrates of A I converting enzyme (ACE), is degraded by other peptidases than ACE in rat vascular tissues. A I and A II degradation was studied in arterial and venous vascular wall extracts. The activities ranged between 0.068 ± 0.025 U and 0.044 ± 0.025 U. The enzymes involved were biochemically characterized by determination of isoelectric points (pI), pH optima, molecular weights and by investigation of their inhibition behavior in vitro. One potent A I degrading enzyme (AIDE) was identified with pI between 3.6 and 3.9, and pH optimum at 7.75. In vitro studies revealed that AIDE activity was not blocked by the specific ACE inhibitors MK 421 or MK 422 (both 11 nMol/ml). The molecular weight of AIDE ranged between 440,000 and 457,000. The results indicate that AIDE is not identical to ACE (pI 4.2–5.0; pH optimum 8.3). AIDE was also observed in aortic smooth muscle cells cultured in vitro. AIDE decreased following bilateral nephrectomy or administration of aldosterone combined with sodium chloride loading, whereas it was elevated in spontaneously hypertensive rats (Okamoto strain). Since AIDE metabolizes A I, one of the substrates of ACE, it may indirectly affect A II formation and bradykinin inactivation as well.