Katti Fischer-Dzoga

Stimulation of proliferation in stationary primary cultures of monkey and rabbit aortic smooth muscle cells: I. Effects of Lipoprotein Fractions of Hyperlipemic Serum and Lymph

Abstract Medial explants of thoracic aorta from rhesus monkey or rabbit show outgrowth within 5–10 days when cultured in 90% basal Eagles medium and 10% normal serum. After 5–6 weeks of rapid growth, cell colonies around the explant reach a stationary phase with low mitotic activity. Homologous hypercholesterolemic serum and lymph stimulate these stationary cultures into another proliferative phase, when 5% of the normal serum is replaced in the culture medium. Measurement of increase in colony size and evaluation of [ 3 H]thymidine incorporation by autoradiography serve to indicate increased proliferation. Low density lipoproteins (LDL) from hyperlipemic serum and lymph have the greatest stimulatory effect, while high density lipoproteins (HDL) and the lipid-free bottom fraction, as well as LDL from normal serum and lymph, have no effect. These stationary cultures are valuable for the study of agents suspected of having a stimulatory effect on cell proliferation of aortic lesions in vivo .

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.

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.

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.

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.

Effect of hyperlipemic serum lipoproteins on the lipid accumulation and cholesterol flux of rabbit aortic medial cells

Abstract The effects of hyperlipemic rabbit serum and its lipoproteins on the lipid accumulation in cultured rabbit aortic medial cells were evaluated in this study. Hyperlipemic serum stimulated the accumulation of cholesterol esters in these cells. Cells exposed to 5–40% normal serum had equivalent amounts of cholesterol ester, while cells exposed to only 2% hyperlipemic serum showed a 2–5-fold increase in cholesterol ester. This happened in spite of the fact that 2% hyperlipemic serum contained much less cholesterol than did 40% normal serum. Most of the cholesterol ester accumulation occurred in the initial 12 h and it could be reproduced by incubating these cells in a medium containing hyperlipemic low density lipoproteins. Cells exposed to low concentrations of hyperlipemic serum, as well as those in high concentrations of normal serum, also showed up to a 2-fold increase of free cholesterol and a smaller increase of triglycerides. This appeared to be a function of increased lipid level in the culture medium, not a specific effect of hyperlipemic serum. Furthermore, culture media containing a high lipid level, either from hyperlipemic serum or a high concentration of normal serum, showed a progressive increase in free cholesterol and a concomitant decline of cholesterol esters. These results indicated that the cells hydrolyzed a large amount of cholesterol ester. The present data do not permit a distinction to be drawn between hydrolysis in the cell and on the cell membrane. It is clear, however, that cells were required for hydrolysis and it is not due to enzymes excreted into the culture medium.

The role of hyperlipidemic serum on the proliferation and necrosis of aortic medial cells in vitro.

Abstract The effects of hyperlipidemic rabbit serum on the proliferation and death of subcultured rabbit aortic medial cells were evaluated. The cultured cells were grown from the aortic media of normal adult male rabbits, prelabeled with [ 3 H]thymidine, and then trypsinized, pooled, and subcultured. Either normal or hyperlipidemic rabbit serum was added to the culture media. Proliferative activity was measured by cell count and by the isotopic dilution of the prelabeled DNA, with corrections made for cell loss. Cell loss, presumed to represent mainly cell death, was assessed from the total loss of [ 3 H]thymidine from the cultured cell population. The cells grown in basal medium of Eagle containing 10% normal serum showed a 25% loss in the first 3 days after trypsinization. The loss rate stabilized at 1.5% per day 5 days after the subculture. When these cells were exposed to hyperlipidemic serum, the rate of cell loss showed a 5.5-fold increase in the initial 3 days, and thereafter returned to the levels seen in the presence of normal serum. Cells exposed to hyperlipidemic serum also showed a 25–50% increase in proliferative activity. These alterations have been demonstrated in both the early proliferative phase and later stationary phase. Cells grown in hyperlipidemic serum were larger on the average and showed a 50% increase in protein content per cell.

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.

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.

Arterial Smooth Muscle Cells in Tissue Culture

Smooth muscle cells are among the most remarkable of the mesenchymal cells. They have long been of interest to biologists and physiologists. It is not surprising that they were grown in vitro by Carrell, Maximow, and other pioneers who first cultured diploid cells, since it is relatively easy to grow the cells in pure culture from explants (Carrell and Burrows, 1910; Maximow, 1925). The outgrowths from these explants in turn provide cells which are readily subcultured.