L. Allen Ehrhart

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.

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.

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.

Stimulation of aortic protein synthesis in experimental rabbit atherosclerosis

Collagen, elastin and non-fibrous protein synthesis were measured in the aortas of male New Zealand white rabbits fed a diet containing 2% cholesterol for 140 or 180 days. At these time periods increases in aortic cholesterol and cholesteryl esters were evident. The atherosclerotic lesions induced were predominantly of the foam cell type although some areas of early fibrous lesion formation were noted. These changes in lipid concentration and arterial morphology were accompanied by a significant increase in collagen synthesis as determined by the formation of [14C]hydroxyproline. This increase, however, was not confined specifically to collagen since both elastin and non-collagenous proteins were also being synthesized at a higher rate. The two-fold increase in the rates of both fibrous and non-fibrous protein synthesis may in part be a consequence of marked intimal hyperplasia necessitating a general increase in protein synthesis.

Aortic collagen, elastin and non-fibrous protein synthesis in rabbits fed 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.

Effects of diets rich in saturated fatty acids with or without added cholesterol on plasma lipids and lipoproteins.

Semi-synthetic diet I which contained 16% hydrogenated coconut oil and 5% cholesterol, and diet II, identical to I but without cholesterol supplement, were fed to dogs for four months to determine the effects of added cholesterol on lipemia produced by diets high in saturated fatty acids (FA) and lacking essential FA. In addition, diet I was fed to another group of dogs for 12 to 16 months. The initiation of lipemia was very similar in all experimental animals. Plasma from dogs on diets I and II showed significant increases in lipid concentration and changes in FA per cent composition within the first week, as compared to controls, while during the first month there was no difference in lipid concentration or FA distribution in all lipid fractions between I and II. At the 10th and 16th weeks plasma total and free cholesterol and phospholipid were significantly higher in the group on diet I, with the cholesterol supplement, than on diet II with no added cholesterol, but there was no difference in triglyceride concentration between these groups. Dogs on diet I for 12 to 16 months showed a further and substantial increase in plasma FA concentration; these changes were most marked in cholesteryl esters. Little or no lipoprotein with electrophoretic and ultracentrifugal properties of alpha-lipoprotein was present in the plasma. Immunotechniques showed that it was present. The composition of dietary FA had great influence in producing this hyperlipemia. Lipemia produced was not a simple reflection of the FA in these diets as evidenced by the increase in some FA, e.g., C16∶1, which was absent in the experimental diets and C18∶1, which contributed only 3.4% of the FA. Large increases in palmitoleate and oleate indicate synthesis or mobilization or both from other tissues. Diets composed predominantly of saturated medium chain length fatty acids, with or without added cholesterol were equally effective in the initiation of hyperlipemia. Data also suggest that added cholesterol is necessary for sustaining hyperlipemia.

Enhanced synthesis and accumulation of collagen in cholesterol-aggravated pigeon atherosclerosis☆

Atherosclerotic segments of pigeon aorta synthesized collagen at four times the rate found in normal aorta (Athero = 2071 +/- 1339 ng/g/h; Control = 497 +/- 192 ng/g/h; P less than 0.025). Similar results were obtained when synthesis was expressed per mg DNA. Elevation in collagen synthesis was relatively specific, collagen accounting for 4% of total protein synthesis in lesion-free aorta and 11.5% in atherosclerotic aorta. Substantial increases in total collagen were observed in atherosclerotic aortas (Athero = 9.9 +/- 3.1 mg/aorta; Control = 6.0 +/- 1.3 mg/aorta; P less than 0.05). Ultrastructural studies revealed the accumulation of large amounts of dense fibrillar collagen in the sub-endothelial region of the plaque. Plaque cells contained multiple vacuoles, an extensive rought endoplasmic reticulum and many mitochondria, suggesting active protein synthesis. It is concluded that increased collagen biosynthesis and deposition is an important metabolic derangement in lipid-rich atherosclerotic lesions whihc promotes their gradual conversion to fibrous plaques.

Familial hyperlipoproteinemia in beagles.

Abstract Canine serum lipoproteins (Lp) were studied by electrophoretic and immunologic techniques. Two of five normal beagles on a regular diet were spontaneously “hyperlipidemic”. Using the double-layer separating gel, polyacrylamide gel (PAG) block electrophoresis method, α 1 -, α 2 -, and β-Lps were consistently detected. While α 1 -Lp was the major serum Lp in normolipidemic dogs, α 2 - and β-Lps were elevated in the “hyperlipidemic” animals. The α 1 -Lp and α 2 -Lp exhibited A 1 and A 2 antigenic components and β-Lp had B and C components. We propose that in all probability α 1 - and α 2 -Lp have similar A 1 , A 2 components, but that β-Lp contains both B and C components. This suggests that α 1 - and α 2 -Lps, particularly in beagles with familial hyperlipoproteinemia are distinct Lp entities with similar lipid composition and with two common major antigenic components. The albumin fraction of PAG block electrophoretograms contained lipid materials other than free fatty acids. This albumin-lipid complex may be similar to that observed in guinea pig serum. On the basis of this study, the α 2 -Lp of dogs is a distinct and important Lp moiety which is elevated in the early stages of both familial and experimental canine hyperlipoproteinemia.

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.

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.