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Biomedical subjects

M B Stemerman

Publications and source records attributed to M B Stemerman.

At least 55 records · Page 3Linked to original sources
PubMed1987

Absolute quantitative autoradiography of low concentrations of [125I]-labeled proteins in arterial tissue.

We developed a method for absolute quantitative autoradiographic measurement of very low concentrations of [125I]-labeled proteins in arterial tissue using Kodak NTB-2 nuclear emulsion. A precise linear relationship between measured silver grain density and isotope concentration was obtained with uniformly labeled standard sources composed of epoxy-embedded gelatin containing glutaraldehyde-fixed [125I]-albumin. For up to 308-day exposures of 1 micron-thick tissue sections, background grain densities ranged from about two to eight grains/1000 micron 2, and the technique was sensitive to as little as about one grain/1000 micron 2 above background, which correspond to a radioactivity concentration of about 2 x 10(4) cpm/ml. A detailed statistical analysis of variability was performed and the sum of all sources of variation quantified. The half distance for spatial resolution was 1.7 micron. Both visual and automated techniques were employed for quantitative grain density analysis. The method was illustrated by measurement of in vivo transmural [125I]-low-density lipoprotein [( 125I]-LDL) concentration profiles in de-endothelialized rabbit thoracic aortic wall.

Animals↗
PubMed1985

Coagulation in the elderly.

The elderly have few bleeding disorders related exclusively to the aging process. However, abnormalities of coagulation are seen frequently in this group as complications of other illnesses. Thus a good understanding of basic coagulation physiology and testing will provide the necessary tools for therapeutic guidance.

Aged↗
PubMed1985

Proliferation of smooth muscle cells in the thoracic aorta after injury to the abdominal aorta: evidence for a humoral mediator in experimental arteriosclerosis.

We tested the hypothesis that circulating humoral material(s) can induce vascular smooth muscle cells to synthesize DNA and to proliferate. Either the entire aorta or its abdominal segment was balloon de-endothelialized in four groups of rabbits. In the first group (control), the entire aorta was injured, and no further procedures were carried out. In a second group (reinjury), the abdominal aortic segment was reinjured 4 days after the initial de-endothelialization procedure. A third group (sham) had a second sham operation 4 days after initial injury. In a fourth group (abdominal only), the abdominal aortic segment was injured on two occasions 4 days apart. There was a rise in the specific activity of 3H-thymidine incorporation into smooth muscle cell DNA (DNA-SA) of the thoracic segments, which began 12 hours after reinjury, peaked within 24 hours at 335 +/- 63 dpm/micrograms DNA (+/- SEM), and returned to control level within 72 hours. The DNA-SA of the thoracic aorta of control rabbits and sham-operated animals 4.5 days after the initial injury was 86 +/- 19 and 48 +/- 8 dpm/micrograms DNA, respectively. There was no rise in DNA-SA in the thoracic aorta of animals in which the abdominal aorta was injured twice. Intimal cell nuclei per 0.1 mm internal elastic lamina were counted 3 days after the second injury and showed similar differences between doubly injured and control animals. Platelet accumulation, as measured by chromium 51 platelet attachment to the aortic surface, was increased in the abdominal segment 12 hours after reinjury.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Rabbit aortic medial thickness under relaxed and specified simulated in vivo conditions.

A method is presented for measuring the thickness of the intima-media layer of the normal rabbit descending thoracic aortic wall under both relaxed (excised) and specified simulated in vivo conditions. The in vivo conditions were simulated by maintaining the aorta in situ at its normal longitudinal extension while perfusing its lumen at the normal mean arterial pressure with a mixture of liquid silicone polymer and a catalyst, thus providing physiological radial distension. After the rubber cured, both relaxed and extended-distended tissue segments were obtained from adjacent sites on the same aorta. These tissue segments were fixed and further processed for measurement of their medial thicknesses by light microscopy. This data was used to estimate the ratio of the medial thickness of the relaxed, excised aorta to that under in vivo conditions, 1.72 +/- 0.15. This information is required for quantitative analysis of data obtained from previous studies of in vivo macromolecular transport across the rabbit thoracic aortic wall.

Animals↗
PubMed1982

Growth of human foreskin fibroblasts in a serum-free, defined medium without platelet-derived growth factor.

The hormones which support growth, in vitro, of normal, neonatal human foreskin fibroblasts were determined. Whereas thrombin and hydrocortisone were major growth stimulants, platelet-derived growth factor was not. Human foreskin fibroblasts grew in a serum-free, biochemically defined medium consisting of epidermal growth factor (100 ng/ml), insulin (100 ng/ml), transferrin 10 micrograms/ml), thrombin (1 microgram/ml), ascorbic acid (10 micrograms/ml), and hydrocortisone (5 x 10(-5) M) in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12, supplemented with ovalbumin (1 mg/ml) and trace elements. The growth achieved was comparable to that achieved with 5% fetal bovine serum. Neither platelet-derived growth factor, fibroblast growth factor, nor somatomedin activity increased proliferation. This serum-free medium, designated Defined Medium F, provides a biochemically defined system for growth and limited subcultivation of human foreskin fibroblasts in vitro.

Blood Platelets↗
PubMed1982

Vascular smooth muscle cell growth kinetics in vivo in aged rats.

Age is a risk factor in the development of atherosclerosis. In this study we investigated the hypothesis that proliferation of vascular smooth muscle cells (SMCs), an integral part of atherosclerotic plaque formation, changes with age. SMC growth kinetics of old rats (21-24 months) were compared to those of young adult rats (3-4 months). Rat aortas were denuded of their endothelium and the animals were killed after [3H]thymidine and Evans blue injections at 0-28 days after denudation. Incorporation of [3H]thymidine into SMC peaked in the young animals by day 2, whereas the older animals responded to endothelial removal with greater incorporation at day 2 and a more sustained rate of incorporation peaking at day 4. The [3H]thymidine incorporation curves decreased sharply from their peaks at 2 and 4 days, respectively, and paralleled each other after day 7. [3H]Thymidine uptake reflected the subsequent SMC intimal growth as measured morphometrically, with old animals showing greater numbers of intimal SMC than did the younger animals. The difference in response of SMC to injury with age suggests that aging produces a change in the vascular SMC that enhances proliferation. This change in response implies that the more pronounced atherosclerotic plaque growth seen with aging may be a result of an age-related increase in response to injury rather than merely the accumulation of time-related intimal change.

Aging↗
PubMed1982

Organizational behavior of human umbilical vein endothelial cells.

Culture conditions that favor rapid multiplication of human umbilical vein endothelial cells (HUV-EC) also support long-term serial propagation of the cells. This is routinely achieved when HUV-EC are grown in Medium 199 (M-199) supplemented with fetal bovine serum (FBS) and endothelial cell growth factor (ECGF), on a human fibronectin (HFN) matrix. The HUV-EC can shift from a proliferative to an organized state when the in vitro conditions are changed from those favoring low density proliferation to those supporting high density survival. When ECGF and HFN are omitted, cultures fail to achieve confluence beyond the first or second passage: the preconfluent cultures organize into tubular structures after 4-6 wk. Some tubes become grossly visible and float in the culture medium, remaining tethered to the plastic dish at either end of the tube. On an ultrastructural level, the tubes consist of cells, held together by junctional complexes, arranged so as to form a lumen. The smallest lumens are formed by one cell folding over to form a junction with itself. The cells contain Weibel-Palade bodies and factor VIII-related antigen. The lumens contain granular, fibrillar and amorphous debris. Predigesting the HFN matrix with trypsin (10 min, 37 degrees C) or plasmin significantly accelerates tube formation. Thrombin and plasminogen activator had no apparent effect. Disruption of the largest tubes with trypsin/EDTA permits the cells to revert to a proliferative state if plated on HFN, in M-199, FBS, and ECGF. These observations indicate that culture conditions that do not favor proliferation permit attainment of a state of nonterminal differentiation (organization) by the endothelial cell. Furthermore, proteolytic modification of the HFN matrix may play an important role in endothelial organization.

Cell Differentiation↗
PubMed1981

Vascular injury: platelets and smooth muscle cell response.

The blood platelet appears to play an important role in the pathogenesis of the atherosclerotic plaque. Using a platelet specific antigen, platelet factor 4 (PF4), we have demonstrated that PF4 released from platelets enters the vessel wall. Smooth muscle cell (s.m.c.) proliferation in vivo was examined by using a new technique for measuring [3H]thymidine incorporation. With this technique, we have shown that a remote vascular injury can cause s.m.c. proliferation, presumably mediated by a humoral agent. Endothelial dysfunction, in turn, may be caused by a sustained, mild hypercholesterolaemia. This permeability dysfunction may provide circulating s.m.c. mitogens with access to the vessel, wall, thus allowing s.m.c. proliferation in areas of non-desquamated endothelium. These experiments from the basis for a modification of the hypothesis implicating platelets in atherogenesis.

Animals↗
PubMed1981

Hormonal requirements for growth of arterial smooth muscle cells in vitro: and endocrine approach to atherosclerosis.

In this study the hormonal requirements for the growth of arterial smooth muscle cells in vitro were determined. A serum-free, biochemically defined medium, supplemented with the relevant hormones, permitted proliferation and propagation of normal diploid mammalian arterial smooth muscle cells. Serum-free, hormone-supplemented cultures spontaneously formed atherosclerotic plaque-like nodules. Thus atherosclerosis may be mediated by a complex endocrine system.

Animals↗
PubMed1981

Serial propagation of human endothelial cells in vitro.

Human umbilical vein (HUV) endothelial cells were grown for 15 to 21 passages at a split ratio of 1:5 (at least 27 population doublings) on a human fibronectin (HFN) matrix in Medium 199 supplemented with fetal bovine serum (FBS) and endothelial-cell growth factor (ECGF). This system also permitted the growth of HUV endothelial cells at cell densities as low as 1.25 cells/cm2. In addition to delaying the premature senescence of HUV endothelial cells, ECGF also reduced the serum requirement for low-density HUV endothelial-cell growth; 2.5% serum and ECGF yields half-maximum growth as compared to high serum controls. Significant HUV endothelial-cell growth was also observed in medium supplemented with either ovine hypophysectomized (HYPOX) serum, plasma-derived serum (PDS), or HYPOX-PDS in the presence of ECGF, suggesting that neither the pituitary nor the platelet contributes to HUV endothelial-cell growth.

Animals↗
PubMed1981

The effect of hypophysectomy on experimental endothelial cell regrowth and intimal thickening in the rat.

We studied the influence of hypophysectomy on endothelial cell regrowth and intimal thickening following aortic endothelial removal. Modification of this influence by replacement doses of glucocorticoids, mineralocorticoids, thyroxin, and growth hormone also was investigated. Young adult male rats were used, and endothelial removal was achieved by the balloon catheter method. Percent endothelial regrowth and percent intimal thickening were quantified 1 or 2 weeks after injury (3 or 5 weeks after hypophysectomy). One week after injury, endothelial regrowth was 64% in hypophysectomized rats and only 54% in controls. At 2 weeks, values were 80% and 79% respectively. Intimal thickening (percent of wall thickness due to intima) at 1 week was 11% in hypophysectomized and 14% in control rats. At 2 weeks, values were 13% and 22%, respectively. All differences between hypophysectomized and control rats at 2 weeks were statistically significant. Neither endothelial regrowth nor intimal thickening in hypophysectomized rats was altered by hormone replacement. Comparison of areas in which endothelium had not regrown suggested that hypophysectomy had a direct effect on intimal smooth muscle cell proliferation. Thus, hypophysectomy suppresses intimal thickening and accelerates endothelial regrowth after wall injury. Neither effect depends on certain known hormones, and these effects are to some extent independent of one another. These findings are relevant to recent work on growth factors and atherogenesis.

Animals↗
PubMed1981

The morphological and biochemical characterization of a line of rat promegakaryoblasts.

Biochemical and morphological evidence is presented to support the characterization of a rat bone-marrow-derived cell line (RPM) as an analog of the promegakaryoblast. The conditions for in vitro growth and maturation of the RPM line are described. Rapid proliferation of the RPM line is readily achieved when cultures are supplemented with moderate levels of fetal bovine serum (FBS). The proliferative compartment is a small blast-like cell. Immunofluorescent staining demonstrates that the RPM cells contain factor VIII:antigen and fibrinogen in their cytoplasm. The cells secrete, into their conditioned medium, a potent mitogenic activity for rat aortic smooth muscle cells. When incubated under conditions of relative serum deprivation, the cells stop proliferating and undergo a process of maturation. The sequence of maturation is described as stage I (promegakaryoblast--the proliferative compartment); stage II (immature megakaryocyte or promegakaryocyte); stage III (mature megakaryocyte). The stage III cells release, from their cytoplasm, small membrane-bound vesicular bodies containing lavender granules and cytoplasmic organelles. These have been designated stage IV. The RPM line may provide a useful model for the in vitro study of megakaryocyte maturation and the synthesis of megakaryocyte-specific proteins.

Animals↗
PubMed1981

The interaction of platelets with aortic subendothelium: inhibition of adhesion and secretion by prostaglandin I2.

We have studied the effect of prostaglandin I2 on platelet turnover, attachment to the subendothelium, and secretion following balloon deendothelialization of the rabbit aorta. Survival of 51Cr-labeled platelets in the balloon-injured animals remained normal. Thirty minutes after injury, there were 4.52 X 10(6) platelets/sq cm attached to the aortic surface, which was 87% covered by platelets. Although plasma platelet factor 4, as measured by radioimmunoassay, did not rise above the normal level of 6.8 +/- 2.6 ng/ml (mean +/- SEM) during the first hour after balloon injury, platelet factor 4 antigen was detected within the vessel wall by direct immunofluorescence within 30 min of injury. An infusion of 650-850 ng/kg/min prostaglandin I2 completely inhibited platelet aggregation and reduced surface coverage by 84% and platelet attachment by 63%. Animals given 50-100 ng/kg/min prostaglandin I2, which only partially inhibited platelet aggregation, had 70% of the aortic surface covered by platelets. Platelet factor 4 antigen was also detected within the aortic wall. Platelet attachment was normal in animals that had been given 850 ng/kg/min prostaglandin I2 prior to balloon injury but sacrificed after the infusion was stopped and ex vivo platelet aggregation had returned to normal.

Animals↗
PubMed1980

Vascular permeation of platelet factor 4 after endothelial injury.

Antibody to platelet factor 4 was used to demonstrate permeation of this factor into the blood vessel wall after endothelial injury in rabbits. The presence of platelet factor 4 antigen in the vessel wall after removal of the endothelium was shown by immunofluorescence 10 and 30 minutes after injury but not 240 minutes afterward. This study demonstrates that factors carried by platelets can enter the vessel wall and that the movement of these platelet products into the vasculature is a short-lived, self-limiting process.

Animals↗