Total Collagen Deposition Research Articles

Early captopril treatment inhibits DNA synthesis in endothelial cells and normalization of maximal coronary flow in infarcted rat hearts.

Cardiac remodeling due to myocardial infarction (MI) includes myocyte hypertrophy, collagen deposition, a rise in DNA synthesis, and normalization of initially diminished maximal coronary bloodflow. Previously, we demonstrated that early captopril treatment can prevent the rise in total DNA synthesis, collagen deposition and hypertrophy. In the present experiments, we investigated the effects of captopril or perindoprilat treatment on cardiac endothelial cell proliferation and maximal coronary flow. MI was induced by ligation of the left coronary artery in Wistar rats. Sham-operated and infarcted rats were treated with captopril (12 mg/kg.d s.c.) from either day 0-21 (early) or day 21-35 (late) after surgery. In isolated retrogradely perfused rat hearts, maximal coronary flow was determined following maximal dilatation with nitroprusside and adenosine (1 mM each). In separate groups, sections of hearts of sham-operated and MI rats treated with BrdU (day 7-14) and either captopril or perindoprilat (1 mg/kg.d s.c.; day 0-14) were double stained with a monoclonal anti-BrdU antibody and the lectin GSI. The total fraction of DNA synthesizing cells and its proportion of endothelial cells was determined. Maximal coronary flow was completely normalized in MI hearts within three weeks after surgery. Early captopril, but not late captopril, inhibited the normalization of maximal coronary flow in MI hearts (Early: sham, 27.4 +/- 1.0; MI, 21.2 +/- 1.4 ml/min; P < 0.05; mean +/- SEM) without affecting the hypertrophic response. The total fraction of DNA synthesizing cells was significantly increased in MI hearts (sham: 7.6 +/- 1.9; MI: 14.9 +/- 2.2%). The proportion of endothelial cells, however, was comparable in sham-operated and infarcted hearts (sham: 30 +/- 3; MI: 33 +/- 3%). Both early captopril and perindoprilat treatment inhibited total DNA synthesis in MI hearts. Only in captopril pre-treated hearts, this inhibition was associated with a disproportionate inhibition of the endothelial cell proliferation (10.3 +/- 2.0%). Early captopril treatment inhibits endothelial cell proliferation and coronary vessel growth following MI, which seems to be partly due to inhibition of the renin angiotensin system.

Inhibition of the matrix metalloproteinase system in a rat model of chronic cyclosporine nephropathy

Chronic cyclosporine A (CsA)-induced nephropathy is histologically characterized by tubular lesions, the interstitial recruitment of inflammatory cells, arteriolopathy and focal interstitial fibrosis. Recent studies show that the intrarenal inhibition of matrix degradation and recruitment of monocytes/ macrophages into the kidney plays a critical role in the development of renal interstitial fibrosis. We examined the expression of components of the matrix metalloproteinase (MMP) system and plasminogen activator inhibitor type-1 (PAI-1) in kidneys from rats injected daily s.c. during three weeks with CsA (10, 15 or 20 mg CsA/kg body wt) or vehicle solution. In all CsA-treated rats, serum creatinine levels were significantly elevated compared to control levels. The extent of CsA-induced atrophy was not influenced by the dosage during a three-week CsA treatment. The administration of CsA did not significantly increase total cortical interstitial collagen deposition, whereas alpha-smooth muscle actin expression was significantly increased in all CsA-treated rats. Analysis of the different subpopulations of inflammatory cells recruited into the chronically injured kidney revealed a marked influx of macrophages into fibrotic cortical foci of CsA-treated rats. The number of cortical macrophages was highest in the group receiving the highest CsA dose. PAI-1 antigen, present in proximal tubular lysosomes in kidneys from all experimental groups, stained very intensely in atrophic tubules in CsA-treated rats. Both stromelysin and interstitial collagenase mRNA were expressed in the kidneys of control rats, but their message transcription remained unaltered after CsA treatment. In contrast, the expression of tissue inhibitor of matrix metalloproteinase type 1 (TIMP-1) was significantly increased after CsA treatment. TIMP-1 mRNA was undetectable in renal sections from sodium-depleted vehicle-treated animals using the in situ hybridization (ISH) technique. ISH of selected renal sections of CsA-treated rats identified the cells responsible for the increased TIMP-1 message transcription after CsA administration, mainly as interstitial cells and also as visceral and parietal epithelial cells. These results suggest that the locally increased expression of TIMP-1 rather than a decrease of matrix metalloprotease expression, contributes to the development of CsA-induced focal interstitial fibrosis in the rat.

Altered collagen metabolism and delayed healing in a novel model of ischemic wounds

Cellular mechanisms occurring in the healing wound have been well described in various animal models. However, the events associated with wound healing seen in ischemic skin have not been as thoroughly defined. In this series of experiments, we created a novel model of excisional skin wounds under gradient ischemia to study the cellular and extracellular events leading to delayed healing. We hypothesized that altered collagen metabolism accounts for delayed wound healing in ischemic skin. Three pairs of 4 mm punch wounds were made 4 days after bipedicle skin flaps were created on the dorsum of rats. Sham-operated control animals had the same punch wounds without flap creation. The kinetics of excisional wound healing were measured by means of computerized planimetry. In addition, wounds were excised with a 6 mm trephine, radiolabelled with ((3)H)-proline and in vitro collagen synthesis determined as collagenase digestible protein along with quantitation of DNA content. Total collagen deposition was determined as 4-hydroxy-L-proline by high-performance liquid chromatography, and wounds were histologically evaluated. Data was analyzed by means of two-way analysis of variance. Although control wounds healed by day 10, flap wounds consistently had greater surface area on days 2, 4, 6, 8, and 12 (p < 0.001). Relative collagen synthesis (% collagen/noncollagen protein), as measured by an in vitro synthesis method, showed no statistically significant differences between flap and controls wounds. However, the total collagen content (deposition) as measured by 4-hydroxy-l-proline was significantly lower in flap wounds compared with controls on days 7 (p < 0.05) and 9 (p < 0.001). In addition, a significant increase occurred in DNA content in the flap wounds on days 7 (p < 0.05) and 9 (p < 0.001) versus control wounds. These data indicate that, in ischemic wounds, significantly less collagen is deposited despite the inherent ability of the tissue to synthesize appropriate levels of collagen. Because the in vitro collagen synthesis technique only assesses the ability of the tissue to synthesize collagen in a well oxygenated environment, one cannot be assured that the tissue expresses this potential in vivo. However, these data are consistent with the hypothesis that the delay in wound closure is due to an alteration in collagen metabolism which results in a net decrease in collagen accumulation. Because of the observed increase in DNA within the ischemic wounds, we suggest that there is prolonged inflammation in these wounds which may enhance collagen degradation through the release of proteases. In addition, there may be an inability of the tissue to maintain appropriate levels of collagen in this inflammatory wound environment.

Deposition and selective degradation of structually-abnormal type I collagen in a collagen matrix produced by osteogenesis imperfecta fibroblasts in vitro

Collagen matrix deposition and turnover were studied in skin fibroblasts from a control and from a patient with lethal perinatal osteogenesis imperfecta (OI) identified as a Gly667 to Arg substitution in the alpha 1(I) chain. A culture system where ascorbic acid was included to stimulate collagen matrix formation over extended culture periods was used. Serial extraction of the control cell collagen matrix confirmed that a substantial mature crosslinked collagen matrix was formed in the control fibroblast cell layer. In contrast, total collagen deposition by the OI fibroblasts was poor, with the quantity of collagen deposited only about a quarter of that of the control cells. Detailed analysis of the OI fibroblast matrix revealed that the mutant collagen chains were incorporated into the collagenous matrix. These data indicate that, when grown with ascorbate in long-term culture, OI fibroblasts reproduced the abnormal matrix deposition pattern of OI tissues in vivo. The overall dramatic reduction in collagen matrix formation was not accounted for by reduced collagen production, since during the period of matrix deposition (days 8-12) the rate of production by the OI cells was only slightly less than that of the control cells. The incorporation of the newly-synthesized OI collagen into the matrix was less efficient than in control cells, reflecting the cooperative nature of matrix deposition. The fate of this mutant collagen containing the Gly to Arg charge-change was followed in the matrix by a pulse-chase experiment and two-dimensional electrophoresis. These data demonstrated that the mutant incorporated into the matrix was unstable, with the proportion of mutant declining during the chase. The deposition of the mutant monomers into a pool more accessible to proteolytic degradation indicated that the mutant and normal collagens did not copolymerize to form collagen fibers of even collagen distribution, but rather the mutant collagen was either enriched on the exposed surfaces of mixed-composition fibers, or was unable to form copolymers efficiently and polymerized into mutant-only fibrillar assemblies more prone to proteolytic attack.

Colchicine and wound healing

Rats with incised and sutured wounds and having subcutaneously implanted polyvinyl alcohol sponges were treated with various doses of colchicine. Breaking strength of wound tissue and biochemical analysis of sponge-induced granuloma tissue were compared with similar measurements in pair-fed controls. Colchicine reduced breaking strength of wound scar tissue significantly while lysyl oxidase activity in sponge tissue was not affected. Synthesis of collagenous and noncollagenous protein was stimulated by colchicine but accelerated collagen synthesis did not result in increased deposition of collagen in sponge tissue. The increased urinary excretion of hydroxyproline in colchicine-treated rats was paralleled by loss of body weight. Colchicine-induced cytotoxicity was also identified in fibroblasts from sponge granuloma tissue examined by transmission electron microscopy. We conclude that some reduction of total collagen deposition and breaking strength of wound tissue in colchicine-treated animals may have been the result of general toxicity of the drug. A specific effect of colchicine on collagen metabolism cannot be ruled out.