The effect of insulin (10--10 000 mU/1) on the proliferation of cultured human arterial smooth muscle cells was studied. Smooth muscle cells were cultivated by explanation. Cells from the third to the fifth subculture were used. Proliferation was studied by growth curve experiments. Insulin stimulated cell proliferation in all concentrations (p less than 0.001). Growth was however stimulated more by a medium containing 10% fetal calf serum. The highest concentration of insulin produced only 35% of the effect of 10% fetal calf serum. Our results support the hypothesis that insulin may play a role in atherosclerosis.

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.

Insulin-like growth factors (IGF) were isolated from human serum and compared with some biological actions of IGF supplied by Dr. J. Hapf, Zürich. 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 (Zürich). Specific binding receptors for IGF (Zürich) on human arterial smooth muscle cells could be demonstrated. Specific binding of 125I-IGF (Zürich) was 10%. Half-maximal displacement was achieved by 250 ng/ml of unlabeled IGF (Zürich), 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.

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.

SUMMARY Investigations were performed on components of the renin-angiotensin system (RAS) in homogenate extracts of vascular tissue and aortic smooth muscle cells cultivated in vitro. Determinations of isoelectric points and pH optima indicated the existence in aortic homogenate extracts of two local angiotensin I (AI)-forming enzymes (AIFE) that were different from those of plasma, renal cortex, veins, and aortic smooth muscle cells. The pH optima for Al-converting enzyme (ACE) from vascular tissues, aortic smooth muscle cells, and plasma were in the same range (pH 8.0-8.5), and in agreement with those measured previously in other tissues. In contrast, in vitro studies with the ACE inhibitors MK-421 and MK-422 and measurement of isoelectric points suggested that aortic ACE was different from the plasma enzyme. AIFE and ACE activities were found to be elevated in spontaneously hypertensive rats (SHR). The biochemical characteristics of the enzymes investigated in the vascular tissue of SHR were not different from those of the normotensive controls. AI-and All-degrading enzymes were found both in aortic tissue and in aortic smooth muscle cells. One potent Al-degrading enzyme different from ACE was observed in aortic tissue. A high ratio of AI/AII immunoreactivities in arterial walls suggests the availability of renin substrate, and that Al-degrading enzymes are the rate-limiting enzymes for AH formation. were the first to demonstrate the existence of arterial and also venous wall renin by using systematically performed biochemical methods. In the last few years, there has been renewed interest in the vascular reninangiotensin system (RAS). Now, as before, it is thought to play a role in the regulation of blood pressure and in the development and maintenance of hypertension. The present investigation continues our previous studies in this field 4 ' 3 and was undertaken to recognize, quantify, and partly characterize those components of