Effect of hyperlipidemic serum and irradiation on wound healing in primary quiescent cultures of vascular cells.

After 8 weeks in culture, outgrowths from explants of aortic media of rhesus monkeys and New Zealand rabbits result in circular colonies of mature smooth muscle cells, quiescent in 10% serum. Such cultures were wounded by cutting out a 1.5-mm-wide strip. Migration of cells into the wound area was measured daily, and proliferation was assessed by [3H]thymidine incorporation. Migration began within 24 hr and at 7 days the defect was filled by proliferates of migrated cells. The cumulative labeling index was highest in the cells in the wound gap but was also increased in the remaining part of the culture. Wounding thus stimulated the uninjured portion of these primary cultures to proliferate, while in subcultures of these cells increase in [3H]thymidine incorporation was confined to the wound area. While hyperlipidemic serum has been shown to induce proliferation in unwounded cultures, it did not enhance cell replication elicited by wounding but reduced cell density and labeling index in the wound gap. Irradiation prior to wounding reduced cell proliferation to control values, while migration of cells was not significantly affected. In irradiated cultures, the inhibitory action of hyperlipidemic serum on cell migration became evident. Such quiescent cultures thus allow us to separate the effects of a specific injury on the proliferative and migratory responses of vascular smooth muscle.

Sequential change of DNA synthesis in cultured aortic smooth muscle cells stimulated by hyperlipidemic serum.

Smooth muscle cells from monkey aorta quiescent in 5% calf serum have been shown to be stimulated to renewed proliferation by hyperlipidemic serum or LDL from such serum. This proliferative response evidently is not dependent on platelet-derived growth factor present in our system in large quantities. The least exposure time required for reaction between the mitogen and the cells in order to initiate maximal DNA synthesis by this mechanism was studied using autoradiography. Stationary primary cultures and subcultures from monkey aortic media required at least 4 and 8 hr of contact with hyperlipidemic serum or LDL so that a significant number of cells reentered the mitotic cycle. Compared to the primary culture system, subcultures needed a slightly longer time of contact with serum to initiate DNA synthesis. Since there was no significant difference in labeling index between the primary cultures stimulated by serum for 8 and 48 hr and the subcultures exposed between 6 and 48 hr, it is concluded that a relatively brief stimulation commits the majority of responsive cells to reenter the cycle and initiate DNA synthesis.

Effects of normolipidemic high-density lipoproteins on proliferation of monkey aortic smooth muscle cells induced by hyperlipidemic low-density lipoproteins.

The primary outgrowth of medial explants of thoracic aorta from rhesus monkeys was used to study the influence of normolipidemic (N) high-density lipoproteins (HDL) on cell proliferation induced by hyperlipidemic (H) low-density lipoprotein (LDL). The experiments were initiated about 6 weeks after explantation when the cellular outgrowth had almost reached the stationary phase of growth. After being added to the culture media, 5% H-LDL induced another proliferative phase in the cultures, as measured by increase in culture area and [3H]thymidine incorporation. The cell proliferation stimulation by 5% H-LDL was prevented by adding 15% N-HDL along with the 5% H-LDL, so that the increment of colony size and incorporation rate of [3H]thymidine into nuclei were similar to those of a group maintained in a medium containing 5% N-HDL. In a second experiment the addition of 5% to 20% of N-HDL to the culture medium containing 5% H-LDL reduced the percentage of nuclei labeled by [3H]thymidine to control levels at all concentrations of N-HDL. In both experiments, the addition of N-HDL by itself at any concentration, from 5 to 20%, did not stimulate cell proliferation.

Radiosensitivity of vascular tissue. I. Differential radiosensitivity of capillaries: a quantitative in vivo study.

The effects of single doses of X radiation ranging from 200 to 2000 rad were studied by direct morphometry in vivo of the mature, stable microvasculature in rabbit ear chambers. Reproducible observations in vivo of the mature microvasculature were obtained by photomicrography of identical 0.033-mm2 sites in each ear chamber prior to and 1 and 5 days following single doses of X radiation. Measurements were made directly on color photomicrographs at a total magnification of 2000X. The microvessels were divided into two groups according to size: vessels greater than 10 microns in diameter (arterioles and venules), and vessels less than or equal to 10 microns in diameter (capillaries). Vascular length and outer and inner surface areas were measured directly on the projected photomicrographs, and vascular volumes and diameters were calculated from these measured parameters. Measurements of capillary length per unit surface area disclosed a decrease in capillary density with increasing dose, resulting in a calculated intercapillary distance in excess of 300 microns, conceivably associated with a decrease in oxygen delivery by the microvasculature. With this method, radiosensitivity of the capillaries was found to be significantly greater than that of larger vessels. Computerized histogram analysis of vascular length, surface area, and volume as a function of increasing diameter (1-micron bins) confirmed the significant difference in reduction of these measured parameters between the capillaries and the larger vessels. The total microvascular volume profile dominated by the volume of larger vessels did not change much 5 days after irradiation, although capillary volume was markedly reduced. Furthermore, the basic profile of the microvasculature showed a shift to larger diameters following irradiation, thus confirming the calculated dilatation of surviving vessels. Qualitative morphologic observations revealed considerable extravasation from the microvessels and formation of micropetechiae at the site of disrupted capillaries with subsequent inflammatory changes.

Radiosensitivity of vascular tissue. II. Differential radiosensitivity of aortic cells in vitro.

The cellular outgrowths from three layers of rabbit and monkey aorta were used as primary cultures. Irradiation of the tissue fragments at the time of explanation resulted in a reduction in outgrowth of 50% with a dose of 200 rad, and in a reduction of over 90% with doses of 300 rad and above. When comparable cultures were irradiated after 2 months in vitro as a mature actively metabolizing but slowly proliferating cell population, radioresistance was increased. Subcultures of medial smooth muscle cells irradiated during their logarithmic growth phase showed a linear dose response in the cell number parameter up to 150 rad. A dose of 250 rad resulted in complete flattening of the growth curve, with a reduction in labeling index, after a 3-hr terminal [3H]TdR pulse. On the other hand, the labeling index indicated some recovery 3 days after irradiation in cultures receiving less than 250 rad. Under the same experimental conditions, cells derived from the intima of the same aorta showed no recovery when increase in cell numbers over time, or the number of labeled cells per area, were used as parameters. Cells derived from adventitia showed a relative increase in the number of labeled cells per area 4 and 7 days after irradiation following an initial decrease on Day 1.

The effect of estradiol on the proliferation of rabbit aortic medial tissue culture cells induced by hyperlipemic serum.

The outgrowths of medial explants of thoracic aorta from New Zealand rabbits were used to study the influence of estrogen on cell proliferation. After 5-6 weeks of rapid growth in Basal Eagle Medium (BME) supplemented with 10% normal rabbit serum, such cultures reached a stationary phase during which they showed little mitotic activity and little further increase in surface area. Replacement of 5% of the normal serum with hyperlipemic rabbit serum resulted in a stimulation of these stationary cultures into a phase of renewed proliferation, which was measured directly as increase in cell culture size and by [3H]thymidine incorporation visualized by autoradiography. The addition of estrogen (estradiol, Progynon, Schering Corp.) in a concentration of 0.02 microgram/ml to the culture medium inhibited the proliferative effect induced by the hyperlipemic serum. On the other hand it had no effect on the growth rate of such explant cultures during their rapid growth phase if added at the time of explantation for 6 weeks. This would indicate that the inhibition of the hyperlipemic serum-induced proliferation by estrogen is not due to a toxic effect on mitosis in general. Cells exposed to estrogen tended to have larger amounts of intracellular lipid as visualized by oil red O staining. Moreover, prolonged exposure to estrogen resulted in a significant decrease in stainable collagen and elastin in these cultures.

The proliferative effect of platelets and hyperlipidemic serum on stationary primary cultures.

Outgrowths from explants of aortic media which have become stationary in the presence of a medium containing 10% normal serum have been studied. Homologous hyperlipidemic serum and especially its low density lipoprotein fraction has been shown to induce a second episode of proliferation in these cultures. Cell proliferation was evaluated by direct measurement of cell colony size and/or incorporation of [3H]thymidine visualized by autoradiography. The possibility has been investigated that the increase in arterial smooth muscle cell proliferation produced by hyperlipidemic serum might actually be due to a platelet factor present in that serum. Platelet-poor and platelet-rich sera were prepared from hyperlipidemic donors and added in a concentration of 5% to the culture medium. Both were equally effective in inducing proliferation; on the other hand, the addition of platelets from either hyperlipidemic or normolipidemic animals had no additive effect. The proliferation-stimulating effect of hyperlipidemic serum occurred even when the stationary cultures were maintained in a medium containing platelet-poor plasma serum for 2 weeks, prior to the addition of hyperlipidemic serum also derived from platelet-poor plasma. It is concluded that the proliferative effect of hyperlipidemic serum on stationary primary cultures does not depend on the presence of platelet-derived material. The implications of these observations on plaque formation are discussed.

Structural and functional changes of rhesus serum low density lipoproteins during cycles of diet-induced hypercholesterolemia.

Over the course of a 2-year study, two male rhesus monkeys underwent episodes of diet-induced hypercholesterolemia (from a diet supplemented with 25% coconut oil and 2% cholesterol) followed by regression phases in which the animals received a low fat Purina chow diet. During the induction of hypercholesterolemia, serum cholesterol, apo B, saturation of low density lipoprotein (LDL) cholesteryl ester fatty acyl chains, and the ability of the serum to stimulate cholesterol esterification by smooth muscle cells rose immediately and in parallel, whereas there was a lag period before the serum became mitogenic to smooth muscle cells. Concurrently, there were important changes in the density, size, chemistry, and concentration of the LDL species in the rhesus serum; induced LDL shifted from the LDL-II to the LDL-I density region with increasing cholesterol concentration. Both structural and functional changes were reversed upon return to a normal Purina chow diet, although at different rates. Serum cholesterol, apo B, and the rate of cholesterol esterification in smooth muscle cells promoted by the serum declined in parallel while the mitogenicity of the serum to smooth muscle cells and the degree of saturation of LDL cholesteryl ester fatty acids took longer to return to normal values. In fact, there was an immediate and dramatic rise in saturation upon reversal before the LDL cholesteryl ester fatty acyl chains returned to their normal composition. The Lp(a) particles did not increase in either concentration or size in response to the test diet, although the change in their lipid composition was similar to those of the other LDL species. The studies indicate that dietary manipulations affect the physicochemical properties of the LDL particles, and that the resultant structural alterations are accompanied by changed in vitro cellular response, suggestive of a greater atherogenicity.

Serum low density lipoproteins with mitogenic effect on cultured aortic smooth muscle cells.

Low density lipoprotein (LDL) subspecies of different size and lipid mass were isolated by density gradient ultracentrifugation from the serum of male rhesus monkeys (Macaca mulatta) fed both a low fat, low cholesterol commercial primate ration, and cholesterol-supplemented high-fat diets, as well as from the serum of human donors. The mitogenic effect of these lipoproteins was examined using primary cultures of rhesus aortic smooth muscle cells. It was observed that the smaller LDL (molecular weight 2.7 X 10(6) from normolipidemic monkeys and a small LDL (molecular weight 2.6 X 10(6) occurring in some normal human subjects exhibited no mitogenic action. In turn, the larger LDL subspecies (molecular weight greater than 3.0 X 10(6), and buoyant density less than 1.030 g/ml), whether from normolipidemic or hyperlipidemic monkeys, or from some normal human subjects, had a marked proliferative action. The results indicate that both hyperlipidemic and normal sera (both human and rhesus) contain mitogenic LDL species although in different amounts. LDL-III, the rhesus equivalent of human Lp(a) was not mitogenic despite its similarity on size and lipid composition to the stimulating particles. However, on the removal of most of its large sialic acid moiety, a clear mitogenic action was observed. The mechanisms responsible for the proliferative effect are unclear and may involve LDL mass, lipid composition, and surface charge although other speculations cannot at present be ruled out. Furthermore, since the small LDL subspecies of either rhesus or human origin were nonmitogenic and similar in mass to the LDL found in calf serum, the mitogenic response of the smooth muscle cells to large LDLs may depend on their early conditioning with the LDL of calf serum.

The mitogenic effects of methyl sterculate on aortic smooth muscle cells.

Cyclopropenoid compounds are derived from naturally occurring C19 long chain fatty acids which have been shown to increase mitosis in rat hepatocytes, to elevate serum cholesterol and to enhance atherosclerosis in hens. In order to study the direct effects of sterculi acid triglyceride on smooth muscle proliferation, sterculic acid triglyceride in Sterculia foetida seed oil extract was added to tissue culture media in concentrations of 2 x 10(-6) to 2 x 10(-3)mg/ml and tested on primary as well as subcultures of rabbit aorta smooth muscle cells. In stationary primary rabbit aortic smooth muscle cell cultures the outgrowth of explants treated with Sterculia foetida seed extract increased in diameter. This response was similar to the increase of cultures treated with hyperlipemic serum (which has been shown to stimulate smooth muscle cell proliferation in previous studies in this laboratory). Using autoradiography following a (3H) thymidine pulse, the percentage of labeled cells was increased in the sterculic acid triglyceride treated groups, as compared to controls. In trypsinized, subcultured rabbit aortic smooth muscle cells, Sterculia foetida seed oil extract resulted in an increased incorporation of (3H) thymidine per milligram of protein. These results, indicating increased cell proliferation, were significant at p less than 0.05.

Arterial foam cells with distinctive immunomorphologic and histochemical features of macrophages.

A variable population of fat-filled "foam" cells in diet-induced experimental arterial intimal plaques of rabbits and monkeys were analyzed for several features characteristic of macrophages. These included: 1) surface binding and phagocytosis of antibody-coated or complement-coated erythrocytes to detect specific surface receptors; 2) cytochemical tests and ultrastructural features to evaluate cell function and structure; and 3) rapid adherence to glass, a feature of macrophage activity, to isolate and identify a homogeneous population of fat-filled foam cells from excised and disrupted arterial lesions. Mixed populations of cells grown in culture from explants of lesions were also analyzed and lipid-filled cells were studied in histologic sections of adjacent lesions. Eighty to ninety percent of the easily dislodged glass-adherent cells from lesions had surface receptors for the Fc portion of immunoglobulin G and for the third component of complement. Coated red blood cells were readily phagocytized, but noncoated cells were not. Acid lipase activity was demonstrated in the Fc-receptor-positive cells. These cells were also devoid of ultrastructural features of smooth muscle. Among the cells growing or migrating out of explants, a population of large round foam cells possessed all of the macrophage features found in the glass-adherent cells from lesions and lacked ultrastructural characteristics of smooth muscle. Fusiform lipid vacuolated cells also grew out of the explants but did not exhibit surface receptors, failed to phagocytize coated or noncoated erythrocytes and did not stain for acid lipase activity; these cells showed distinctive morphologic features of smooth muscle. In histologic sections of nearby lesions foam cells that showed macrophage characteristics, ie, acid lipase activity and the presence of lysozymelike antigen, lacked ultrastructural smooth muscle features. Smooth muscle cells in lesion sections often contained lipid but demonstrated no lysozyme or acid lipase activity. The occurrence of a population of cells with several functional and structural features of macrophages among the lipid-laden cells of experimental diet-induced arterial lesions suggests that some foam cells may be derived from monocytes. An alternative explanation, that metabolically altered autochthonous arterial wall cells assume one or more characteristics of mononuclear phagocytes is less likely, since some of the markers used in these experiments are unrelated. Both explanations deserve further careful study.

Cellular proliferation, cellular death and atherosclerosis.

The multipotential smooth muscle cell (SMC) is the predominant cell in arterial media and intima, and the major cell type involved in proliferative lesions. Studies using a precursor of DNA, namely [3H] thymidine, and autoradiography have shown that proliferation of SMC is an important and early response to atherogenic stimuli, such as cholesterol feeding, surgically induced hypertension or endothelial injury. Cultures of aortic SMC offer excellent in vitro models to study the reactivity of these cells to recognized or suspected risk factors. Pure SMC cultures are obtained by placing small fragments of aortic media in a defined culture medium and their response evaluated by thymidine incorporation and/or increase in size of the outgrowing cell colony. Factors essential for adequate growth include normal serum constitutents, including the lipoproteins and a platelet derived factor. Replacing the normal serum with dietarily induced hyperlipemic serum results in greatly increased proliferation. The serum fraction largely responsible for this phenomenon is the low density lipoprotein. Simultaneous addition of high density lipoproteins partly suppress this reaction. Serum from diabetic or hypertensive--but normolipemic--animals similarly stimulates these cells to increased proliferation.

Stimulation of proliferation in stationary primary cultures of monkey aortic smooth muscle cells. Part 2. Effect of varying concentrations of hyperlipemic serum and low density lipoproteins of varying dietary fat origins.

The outgrowth of medial explants of thoracic aorta from Rhesus monkeys has used to study the influence of hyperlipemic serum on cell proliferation. After 5-6 weeks of rapid growth in BME plus 10% normal serum, the cultures reach a stationary phase during which they show little mitotic activity. When it replaces 5% of the normal serum in the media, hyperlipemic serum induces another proliferative phase in the cultures, as measured by [3H1thymidine incorporation and increase in culture area. Low density lipoprotein (LDL) has the greatest stimulatory effect, while high density lipoprotein (HDL) has no effect. Hyperlipemic serum or its LDL still stimulates the cells even when diluted to achieve cholesterol levels comparable to the values with normal serum or LDL. Normal LDL has no effect, even when concentrated to increase its cholesterol level in the media. Thus it appears that hyperlipemic LDL has a stimulatory effect on arterial smooth muscle cells which does not depend on its higher lipid or cholesterol level.