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Biomedical subjects

L A Ehrhart

Publications and source records attributed to L A Ehrhart.

At least 19 recordsLinked to original sources
PubMed1997

Homocysteine as a risk factor for vascular disease. Enhanced collagen production and accumulation by smooth muscle cells.

An increased plasma homocysteine level is an independent risk factor for vascular disease. However, the pathological mechanisms by which homocysteine promotes atherosclerosis are not yet clearly defined. Arterial smooth muscle cells cultured in the presence of homocysteine grew to a higher density and produced and accumulated collagen at levels significantly above control values. Homocysteine concentrations as low as 50 mumol/L significantly increased both cell density and collagen production. Cell density increased by as much as 43% in homocysteine-treated cultures. Homocysteine increased collagen production in a dose-dependent manner. Smooth muscle cells treated with homocysteine at concentrations observed in patients with hyperhomocysteinemia had collagen synthesis rates as high as 214% of control values. Likewise, collagen accumulation in the cell layer was nearly doubled in homocysteine-treated cultures. Addition of aquacobalamin to homocysteine-treated cultures controlled the increase in smooth muscle cell proliferation and collagen production. These results indicate a cellular mechanism for the atherogenicity of homocysteine and provide insight into a potential preventive treatment.

Animals↗
PubMed1995

Graft smooth muscle cells specifically synthesize increased collagen.

PURPOSE: Anastomotic intimal hyperplasia is characterized by smooth muscle cell (SMC) proliferation, but its final form is predominantly extracellular matrix. The purpose of this study was to compare collagen synthesis from graft SMC to that from adjacent native arterial SMC. METHODS: Thoracoabdominal bypass grafts were excised 20 weeks after implantation into canine models. SMC harvested from six anastomotic graft segments and adjacent native aorta were passaged twice, grown to near-confluence, and then assayed for collagen synthesis and total protein synthesis. In four of these sites type I alpha-1 procollagen mRNA levels were measured and normalized to glyceraldehyde-3-phosphate dehydrogenase. To control for increases in collagen synthesis associated with proliferation, SMC were plated at equal densities and tritium-thymidine incorporation and DNA concentration were determined. Data (mean +/- SE) were analyzed with two-factor ANOVA for repeated measures and paired Student t test and were considered significant if p < 0.05. RESULTS: There was no difference in thymidine incorporation and total protein synthesis between groups, but collagen synthesis (graft: 52.9 +/- 1.6 disintegrations per minute/ng DNA versus native: 42.6 +/- 1.9 dpm/ng DNA; p = 0.03) and collagen synthesis as a percentage of total protein synthesis (graft: 7.16% +/- 0.11% versus native: 5.8% +/- 0.14%; p = 0.001) increased significantly in graft SMC as compared to native SMC. Type I alpha-1 procollagen mRNA levels were higher in graft SMC, but this difference was not significant. CONCLUSIONS: Graft SMC specifically produce more collagen than SMC from adjacent native artery. This change does not simply reflect increases in either total protein synthesis or proliferation and may, in part, be due to increased collagen gene expression.

Analysis of Variance↗
PubMed1993

Basic fibroblast growth factor in the extracellular matrix suppresses collagen synthesis and type III procollagen mRNA levels in arterial smooth muscle cell cultures.

To determine the effects of an intact extracellular matrix on collagen synthesis, arterial smooth muscle cells (SMCs) were plated sparsely on a cell-free, SMC-derived matrix and examined the following day. Collagen synthesis during a 5-hour incubation by cells on the matrix was reduced to 67% of the control values obtained from cultures on plastic. Total protein synthesis was unaffected. Treatment of the matrix with heparitinase to remove basic fibroblast growth factor (bFGF) before seeding the SMCs abolished the inhibitory effect of the matrix on collagen synthesis. The inhibitory effect was also eliminated by treating the matrix with a neutralizing polyclonal antibody directed against bFGF. Collagen synthesis by SMC cultures grown in wells coated with purified bFGF was only 61% that of control cultures, whereas total protein synthesis remained unchanged. Slot-blot analysis revealed that the relative message level for alpha 1(III) procollagen was reduced in cultures grown on the preexisting matrix or on plastic precoated with bFGF, whereas the alpha 1(I) procollagen message was unaffected. These results demonstrate the ability of the extracellular matrix to modulate the synthesis of collagen by arterial SMCs and indicate that bFGF in the matrix is responsible for these effects.

Animals↗
PubMed1992

Cell density and proliferation modulate collagen synthesis and procollagen mRNA levels in arterial smooth muscle cells.

Collagen synthesis and procollagen mRNA levels were determined and compared in (1) sparse, rapidly proliferating smooth muscle cells (SMC); (2) postconfluent, density-arrested SMC; and (3) sparse, nonproliferating (mitogen-deprived) rabbit arterial SMC. Collagen synthesis per SMC was decreased by 70% in postconfluent versus proliferating cells. However, relative collagen synthesis, expressed as the percentage of total protein synthesis, increased from 3.7% in sparse cultures to approximately 7% in postconfluent cultures. Slot blot analyses demonstrated that the relative steady state alpha 1(I) and alpha 1(III) procollagen mRNA levels were also increased in postconfluent cultures when compared to sparse cultures. As with collagen synthesis per cell, the mRNA levels per cell for types I and III procollagen in postconfluent cells, determined by densitometry of blots, were likewise approximately half that found in sparse, proliferating cells. In a separate study to determine if cell-cell contact was necessary for eliciting these changes in collagen synthesis, we determined collagen synthesis in mitogen-deprived and proliferating SMC cultures at low density. Mitogen-deprived cultures synthesized only 10% the amount of collagen produced (per cell) by proliferating cultures in 10% fetal bovine serum. Relative collagen synthesis in proliferating and nonproliferating cultures was 5.0 and 8.3%, respectively. These results demonstrate elevated collagen synthesis, per cell, by proliferating cultures compared with nonproliferating cultures, regardless of whether cells were rendered quiescent by density arrest or by mitogen deprivation. Results also suggest a pretranslational mechanism for the regulation of collagen synthesis in rabbit aortic smooth muscle cells.

Animals↗
PubMed1987

Compartmentalization of proline pools and apparent rates of collagen and non-collagen protein synthesis in arterial smooth muscle cells in culture.

Rates of collagen and non-collagen protein synthesis in rabbit arterial smooth muscle cells (SMC) were determined by using the specific (radio)activity of [3H]proline in the extracellular, intracellular, and prolyl-tRNA pools. The intracellular free proline specific activity was only 25% of the extracellular value in cultures incubated for 12 h in 0.25 mM-proline. The specific activity of prolyl-tRNA was less than 10% of the extracellular specific activity. Increasing the extracellular proline concentration 10-fold (to 2.5 mM), while keeping the extracellular specific activity of proline constant, resulted in equilibration of the specific activities of intracellular and extracellular free proline, but the specific activity of prolyl-tRNA remained at less than 10% of the extracellular specific activity. Therefore, calculated rates of collagen and non-collagen protein synthesis were greatly underestimated using the intracellular or extracellular specific activity of proline. SMC were also incubated with 0.1 mM-[14C]ornithine in 0.25 nM or 2.5 mM non-labelled proline to examine synthesis de novo of proline and prolyl-tRNA from ornithine. In SMC cultures containing 0.25 mM unlabelled proline, the specific activity of intracellular ornithine was approx. 45% of the extracellular specific activity, due to the production of unlabelled ornithine. The specific activity of ornithine-derived intracellular free proline in SMC incubated with 2.5 mM-proline was significantly lower than in SMC incubated in 0.25 mM-proline, due to the influx of unlabelled proline. However, a corresponding difference in the specific activity of [14C]prolyl-tRNA between SMC in 0.25 mM- or 2.5 mM-proline was not observed. Ornithine-derived [14C]proline was incorporated into proteins in a manner different from that of exogenously added radiolabelled proline. A much higher proportion of the proline synthesized de novo was channelled into collagen synthesis relative to total protein synthesis. Together, these results show that intracellular proline pools are highly compartmentalized in arterial SMC. They also suggest that proline synthesized from ornithine may enter a prolyl-tRNA pool separate from that of proline entering from the extracellular medium.

Animals↗
PubMed1987

Collagen binding in clinical isolates of Staphylococcus aureus.

Collagen binding was examined in 90 strains of Staphylococcus aureus derived from patient samples. Slightly under one-half (39 of 90) of the S. aureus strains bound collagen. Collagen binding in S. aureus did not correlate with either immunoglobulin G or fibronectin binding by these organisms. Chi-square analysis of isolates obtained from patients with complicated bacteremia (bacteremia associated with deep tissue infection) compared with isolates from patients with uncomplicated bacteremia (bacteremia without other infection) showed that the former strains were significantly more likely to have collagen-binding ability. Subcloning of primary isolates from patients with bacteremia showed that all clones from individual patients were either all positive for collagen binding or all negative, suggesting a common clonal origin for this characteristic. The ability to bind collagen could not be induced in strains lacking collagen affinity by repeated subculture in media supplemented with collagen.

Collagen↗
PubMed1986

Substratum influence on collagen and fibronectin biosynthesis by arterial smooth muscle cells in vitro.

Collagen, fibronectin, and nonfibrous protein biosynthesis were examined in cultures of rabbit arterial smooth muscle cells grown on tissue culture plastic precoated either with rabbit plasma fibronectin or bovine serum albumin. Cells seeded into fibronectin-coated wells appeared to reach confluence more quickly than counterparts grown on albumin-coated surfaces. Measurement 3H-thymidine incorporation into DNA by these cultures suggested that this was probably a consequence of more rapid and efficient cell attachment rather than an increased rate of proliferation of smooth muscle cells grown on fibronectin. In preconfluent cultures, the rates of collagen and fibronectin biosynthesis were reduced to 34 and 57%, respectively, on a per-cell basis in cultures grown on fibronectin-coated surfaces compared with cells grown on albumin-coated plasticware. In preconfluent cultures grown on fibronectin-coated surfaces, a greater percentage of the total fibronectin synthesized was incorporated into the cell layer. The distribution of newly synthesized collagen between culture medium and cell layer, however, was not affected by alteration of substratum composition. There was no difference in the rate of synthesis of noncollagen proteins between the two groups of preconfluent cells. In postconfluent cultures the rates of collagen and fibronectin biosynthesis were equivalent in both albumin- and fibronectin-treated cultureware. In preconfluent cultures, analyses of procollagens showed that the overall amounts of both types I and III procollagens were reduced in fibronectin-treated wells, indicating the reduction in collagen synthesis to be general and not type-specific. Although type V procollagen biosynthesis was not detected in either preconfluent group, it was found in postconfluent cultures. The reduction of fibronectin synthesis in cells grown in fibronectin-coated wells was significant as early as 4 hours after plating. Together, these findings suggest that cultured arterial smooth muscle cells are capable of deriving information from their substratum and regulating the biosynthetic rates of extracellular matrix components in response to the immediate needs of the cell.

Animals↗
PubMed1986

Collagen binding to Staphylococcus aureus.

Staphylococcus aureus can bind soluble collagen in a specific, saturable manner. We have previously shown that some variability exists in the degree of collagen binding between different strains of heat-killed, formaldehyde-fixed S. aureus which are commercially available as immunologic reagents. The present study demonstrates that live S. aureus of the Cowan 1 strain binds amounts of collagen per organism equivalent to those demonstrated previously in heat-killed, formaldehyde-fixed bacteria but has an affinity over 100 times greater, with Kd values of 9.7 X 10(-11) M and 4.3 X 10(-8) M for live and heat-killed organisms, respectively. Studies were also carried out with S. aureus killed by ionizing radiation, since this method of killing the organism seemed less likely to alter the binding moieties on the surface than did heat killing. Bacteria killed by exposure to gamma radiation bound collagen in a manner essentially indistinguishable from that of live organisms. Binding of collagen to irradiated cells of the Cowan 1 strain was rapid, with equilibrium reached by 30 min at 22 degrees C, and was fully reversible. The binding was not inhibited by fibronectin, fibrinogen, C1q, or immunoglobulin G, suggesting a binding site for collagen distinct from those for these proteins. Collagen binding was virtually eliminated in trypsin-treated organisms, indicating that the binding site has a protein component. Of four strains examined, Cowan 1 and S. aureus ATCC 25923 showed saturable, specific binding, while strains Woods and S4 showed a complete lack of binding. These results suggest that some strains of S. aureus contain high-affinity binding sites for collagen. While the number of binding sites per bacterium varied sixfold in the two collagen-binding strains, the apparent affinity was similar. The ability of S. aureus to bind collagen with high affinity may provide a mechanism for bacterial adhesion to host tissue and thereby play a role in the invasive characteristics of this organism.

Binding, Competitive↗
PubMed1985

Specific binding of collagen to Staphylococcus aureus.

In this study the specific binding of soluble type I collagen to several strains of Staphylococcus aureus was investigated. Both type I procollagen and soluble, pepsin-treated, lathyritic rat skin type I collagen bound to these bacteria in a manner which could be blocked by the addition of gelatin to the binding assay. Saturation binding studies showed more than one class of binding sites for [125 I]-lathyritic rat skin collagen to be present with each bacterium of the Cowan I strain containing approximately 135 high affinity sites with an apparent KA of 2.3 X 10(7)M-1. Like Cowan I strain, American Type Culture Collection (ATCC) strain 25923 also bound type I collagen. IgG inhibited collagen binding in a dose dependent manner. This observation together with the finding that the protein A-deficient Wood strain did not bind collagen suggested that protein A might be the collagen binding site. However, failure of protein A-Sepharose to bind soluble collagen or protein A in solution to inhibit binding of collagen to Cowan I cells suggests that bacterial protein A does not mediate the binding. Addition of fibronectin to the binding assay did not affect the level of collagen binding, suggesting a) that the collagen binding site is different from the fibronectin binding site and b) that fibronectin does not mediate the binding of collagen to these cells. These results demonstrate a new example of bacterial binding to an extracellular matrix protein and suggest a possible mechanism whereby Staphylococcus aureus may adhere to mammalian tissue.

Animals↗
PubMed1984

A single-step gel-filtration method for the isolation of procollagens from cell culture conditioned medium.

Procollagens are the major proteins secreted into the conditioned medium of cultured arterial smooth muscle cells. Methods for the isolation and quantification of these macromolecules have traditionally required preliminary salt precipitation of procollagens from the conditioned medium followed by cellulose ion-exchange chromatography. The method described here exploits the elongated conformation of soluble procollagens and allows the direct recovery of procollagens from culture medium by a single gel-filtration chromatographic step under nondissociating conditions. Procollagens are isolated in high yield and show minimal processing by procollagen N- or C-terminal peptidase activity. This method results in rapid recovery of highly purified procollagens, free of most proteoglycans or other products of smooth muscle cell metabolism.

Animals↗
PubMed1984

Modulation of types I and III procollagen synthesis at various stages of arterial smooth muscle cell growth in vitro.

Collagen synthesis was monitored in cultures of rabbit arterial smooth muscle cells (SMC). Both the rate of collagen synthesis per cell and collagen synthesis as a percent of total protein synthesis were measured at specific intervals from 1 to 14 days after inoculation of smooth muscle cells. The proportions of types I and III collagen present in the conditioned incubation medium and in the cell layer were also examined. After inoculation the cells displayed population expansion typical of SMC in which growth slowed but did not cease after the cells attained confluence. Collagen synthesis rates, expressed as [14C]hydroxyproline per cell, were eight-fold higher in preconfluent cells. In these cultures collagen accounted for more than 20% of the newly synthesized, 14C-labeled protein present as trichloroacetic acid (TCA)-insoluble material in 24 h culture media. In post-confluent cultures, this percentage was reduced to about 7% of the total protein synthesized. Synthesis rates of both collagen and non-collagen protein decreased with increasing time after inoculation. However, the rate of decline of collagen synthesis was three times greater than that seen for non-collagen protein. Early cultures synthesized relatively more type I than type III procollagen. The type I to type III ratio was highest at day 3 and declined after that time to day 14. While the synthesis of both types decreased with increasing age, type I declined at a greater rate resulting in a predominance of type III procollagen secretion by older cultures. We conclude that protein synthesis in general and collagen synthesis in particular are quantitatively and qualitatively dependent upon the growth stage of SMC in vitro.

Animals↗
PubMed1983

Arterial response to laser operation for removal of atherosclerotic plaques.

The cellular response of normal and atherosclerotic aortic intima after exposure in vivo to a 0.9 mm diameter carbon dioxide laser was examined in hypercholesterolemic swine with light and electron microscopy to evaluate tissue damage, thrombosis, and healing. At energy levels of greater than 5 joules, laser burns appeared as craters less than 1 mm in depth and 2 mm in diameter. Two days after the operation, craters were filled with platelet-fibrin thrombi that did not protrude above the level of adjacent endothelium. The internal elastic lamina was exposed 1 to 2 mm around the crater. This area was surrounded by a ring of densely packed leukocytes at the edge of the normal endothelium. Two weeks after the operation, the depressed crater surface was mostly reendothelialized with small, closely packed endothelial cells. The subjacent thrombus contained numerous phagocytic cells with inclusion of fibrin, erythrocytes, and membranous debris. Proliferative invaginations containing medial smooth muscle cells, mitotic figures, and collagen extended into the pit from the lateral aspects. Eight weeks after the operation, the burned area was still depressed and therefore less occlusive than adjacent lesion areas, and a fibrous cap had formed over the remaining necrotic area. The results suggest that a focused, low-energy carbon dioxide laser can be used to remove focal atherosclerotic plaques from arteries without inducing excessive thrombogenicity. Rapid healing, including reendothelialization and intimal fibrous scarring, with minimal damage to surrounding tissue, was observed.

Animals↗
PubMed1980

Aortic collagen, elastin and non-fibrous protein synthesis in rabbits red cholesterol and peanut oil.

Alteration of the fatty acid composition of atherogenic test diets has been a widely recognized method for influencing the character and severity of atherosclerotic lesions. The addition of peanut oil or coconut oil to cholesterol-supplemented diets has been shown to produce lesions of a fibrous nature in several species. In the present study, addition of 8% peanut oil to a 2% cholesterol diet accelerated the formation of atherosclerotic lesions which were more fibrous after only 90 days than those previously seen in rabbits even after 6 months on a diet supplemented with cholesterol alone. Collagen, elastin and non-fibrous protein synthesis were all increased over control values, as previously seen in aortas from rabbits given cholesterol supplementation alone. However, the addition of peanut oil to the 2% cholesterol diet produced a preferential increase in the rate of aortic collagen synthesis per unit dry, defatted weight compared with the increases seen in elastin, non-fibrous protein or total protein synthesis. Collagen deposition in proliferative intimal plaques was evident by histological examination. These focal accumulations, however, did not result in significant increases in either total collagen content of the whole descending thoracic aorta or in collagen concentration expressed per unit of dry, defatted weight. These data suggest that, while a portion of the increased synthetic rates may be a direct result of aortic hyperplasia, the proportionally greater increase in collagen synthesis in these lesions is attributable to the addition of peanut oil to the atherogenic diet. Although the lesions produced in this experiment lacked the overt fibrosis seen in man and in some forms of experimentally induced atherosclerosis, the relative synthetic rates of collagen, elastin and nonfibrous protein described here suggest that even a small preferential increase in collagen synthesis compared with non-collagen protein synthesis may gradually lead to a more fibrous lesion.

Animals↗
PubMed1980

Inhibition of collagen and non-collagen protein synthesis in cultured aortic smooth muscle cells by hyperlipoproteinemic serum.

The response of rabbit arterial tissue to prolonged extreme hyperlipoproteinemia is the development of a state of enhanced in vitro protein synthesis where severe atherosclerosis is present. In order to examine this stimulation, arterial cells were grown in cell culture and exposed to hyperlipemic serum as a part of the growth medium. Rather than observing an overall increase in protein synthesis as seen in atherosclerotic tissue, the cells exposed to hyperlipemic serum produced less collagen and non-collagen protein per cell than counterparts exposed to normolipemic homologous serum. We conclude that exposure to hyperlipemic serum for six days in vitro causes enhanced cellular proliferation, as seen by others, and a general perturbation of protein synthesis which may be related to cell population density.

Animals↗
PubMed1977

Studies on a beta-migrating high density lipoprotein.

An unusual serum lipoprotein (Lp) profile was detected in a Japanese family. A double beta-Lp was observed when serum was subjected to polyacrylamide gel electrophoresis. The slower migrating beta-Lp was identified as a subfraction of high density lipoprotein (HDL). It was present in the d 1.063--1.21 fraction, migrated to the position designated as the midband L.1 (sinking pre-beta-lipoprotein) by Mead, M.G. and Dangerfield, W.G. (1974) (Clin. Chim. Acta 51, 173--182) [1], and reacted against human anti-beta-Lp antiserum. This lipoprotein contained greater amounts of triglyceride than the usual beta-lipoprotein and could not be clearly detected by paper electrophoresis. Individuals exhibiting this high density midband lipoprotein appeared to be heterozygous for an autosomal dominant gene. Although other reports have indicated the possibility of a positive association between the occurrence of serum lipoproteins with unusual eletrophoretic mobility and premature ischemic heart disease, no such correlation was demonstrable in these subjects.

Adolescent↗