Glycoproteins from experimental granulation tissue and their effects on collagen synthesis in embryonic chick tendon cells.

Buffer-soluble and pronase-liberated glycoproteins from experimental granulation tissue were fractionated by gel filtration and DEAE-cellulose chromatography. The age of the granuloma was reflected in the gel filtration pattern. Two glycoproteins were isolated, purified to homogeneity and analyzed for their carbohydrate and amino acid compositions. The collagen synthesis in embryonic chick tendon cells was measured in the presence of these fractions, which were found to be inhibiting even at 10(-6) M. These glycoproteins may be significant in the feedback regulation of the development of granulation tissue and fibrosis.

Superoxide dismutase does not cause scar thinning after myocardial infarction.

Previous studies demonstrated that treatment with superoxide dismutase, a scavenger of superoxide anions, limits the extent of myocardial injury in a canine preparation of regional myocardial ischemia and reperfusion. Little is known, however, about the effects of superoxide dismutase on the healing of a myocardial infarct. Therefore, this study was performed to determine whether treatment with superoxide dismutase during myocardial ischemia impairs formation of scar tissue after infarction. Dogs received 2 hour infusions of superoxide dismutase or albumin (controls) by way of the left atrium beginning 15 minutes before and ending 15 minutes after a 90 minute occlusion of the left circumflex coronary artery. Six weeks later the animals were killed. Two-dimensional echocardiography was performed before surgery and before induced death. Wall thickening in the central ischemic zone was decreased at 6 weeks compared with baseline studies (p less than 0.05), but the decrease was similar for both groups. The hydroxyproline concentrations (microgram/mg dry weight) of the scar tissue in the superoxide dismutase and control groups, respectively, were 35.3 +/- 3.8 and 28.7 +/- 5.0 (p less than 0.05). The ratios of the scar thickness to normal wall thickness were superoxide dismutase 0.91 +/- 0.03 and control 0.89 +/- 0.03 (p greater than 0.05). Thus, superoxide dismutase had no adverse effect on wall thickening or scar formation assessed 6 weeks after myocardial infarction, and may be useful to limit oxygen radical-mediated damage during reperfusion of the ischemic myocardium.

Thrombospondin in early human wound tissue.

We examined partial thickness incised human wounds of 2, 3, 5, 7, and 14 days of age for the presence of thrombospondin by immunostaining and light microscopy. At 2, 3, 5, and 7 days after wounding, thrombospondin is present primarily at the cut edges of the lateral and deep margins of the wound. It appears to be cleared from these extracellular matrix sites, and is no longer detectable in those sites in most 14-day-old wounds. Thrombospondin staining is present, however, in increased amounts around the vascular channels within and adjacent to the 7- and 14-day wounds in increased amounts relative to vascular channels distant from the wound. Our observations are consistent with known in vitro data regarding the binding of thrombospondin to fibrin and components of the extracellular matrix, as well as with data showing that proliferating endothelial cells secrete more thrombospondin than quiescent endothelial cells. These data support the hypothesis that thrombospondin plays a role in the early organization of the extracellular matrix of wounds.

Distinction between two molecular species of type V collagen from human post-burn granulation tissues.

Human type V collagen was purified from post-burn granulation tissues, and was demonstrated to exist in two different molecular assemblies consisting of [alpha 1(V)]2 alpha 2(V) and alpha 1(V)alpha 2(V)alpha 3(V) heterotrimers which are designated as type V(112) and V(123) collagens, respectively, in this paper. The two molecular species were separated by salt fractionation at neutral pH under non-denaturing conditions. When crude type V collagen was dialyzed against phosphate-buffered saline at 4 degrees C, mainly collagen V(112) precipitated, leaving collagen V(123) in the solution. Type V(112) collagen, but not type V(123), precipitated at 0.15 M NaCl in 50 mM Tris-HCl buffer (pH 7.5), whereas the type V(123) molecule precipitated at 4.5 M NaCl in the same buffer. When the crude type V collagen was electrophoresed under non-denaturing conditions, two bands were observed; and it was confirmed that the fast-migrating band was composed of [alpha 1(V)]2 alpha 2(V) and the slow-migrating band was alpha 1(V)alpha 2(V)alpha 3(V). Both alpha 1 and alpha 2 chains of V(112) showed biochemical properties that were very similar, if not identical, to those of the corresponding alpha chains of V(123) judging from amino acid compositions, peptide mapping patterns obtained following treatment with cyanogen bromide and lysyl endopeptidase, and periodic acid Schiff and concanavalin A stainings. Alpha 3 chain, in contrast, was distinct from both alpha 1 and alpha 2 chains. The amino acid composition and peptide maps of alpha 3 chain were similar to some extent, but not identical, to those of the alpha 1 chain. The intensity of carbohydrate stainings of the alpha 3 chain was clearly different from that of the alpha 1 chain. The negatively stained segment-long-spacing crystallites of the two molecular species exhibited an identical banding pattern. The crystallite derived from collagen V(112) was usually in a dimeric form exhibiting the C-C terminal junction, but that of collagen V(123) was mostly in a monomeric form. Differences between the two molecular species is ascribed to the presence of the alpha 3 chain in collagen V(123).

Alpha-smooth muscle actin, a differentiation marker of smooth muscle cells, is present in microfilamentous bundles of pericytes.

alpha-Smooth muscle (alpha-sm) actin, an isoform typical of smooth muscle cells (SMC) and present in high amounts in vascular SMC, was demonstrated in the cytoplasm of pericytes of various rat and human organs by means of immunocytochemistry at the electron microscopic level. In SMC and pericytes, alpha-sm actin was localized in microfilament bundles, strengthening the assumption that it is the functional isoform in these cell types and supporting the assumption that pericytes exert contractile functions.

Diabetes-induced increases in vascular permeability and changes in granulation tissue levels of sorbitol, myo-inositol, chiro-inositol, and scyllo-inositol are prevented by sorbinil.

In a recently developed animal model, we investigated the pathogenesis of diabetic vascular disease and demonstrated that 125I-albumin permeation is markedly increased in new "granulation tissue" vessels formed in subcutaneous tissue after the onset of diabetes. The studies described in this report were undertaken to examine the effects of an aldose reductase inhibitor on diabetes-induced increases in vascular permeability in this animal model. 125I-albumin permeation was assessed 3 weeks after the subcutaneous implantation of sterile preweighed polyester fabric (to stimulate angiogenesis) in diabetic male Sprague-Dawley rats, in controls, and in diabetic rats given sorbinil approximately 12 or approximately 25 mg/kg/d mixed in ground rat chow. Sorbinil administration prevented the diabetes-induced increase in vascular permeability by approximately 60% at the lower dose and by approximately 80% at the higher dose without affecting body weight or plasma glucose levels. Diabetes-induced changes in tissue levels of sorbitol, myo-inositol, scyllo-inositol, and chiro-inositol were also prevented by the high dose of sorbinil (data were not obtained for the lower dose). These observations are consistent with evidence linking diabetic cataracts and neuropathy to imbalances in sorbitol/inositol metabolism and support the hypothesis that diabetic vascular disease as well as neuropathy and cataracts are mediated by excess metabolism of glucose through the polyol pathway. Furthermore, these observations suggest that increased vascular permeability associated with diabetic microangiopathy in humans may be prevented by inhibitors of aldose reductase without the need to normalize blood glucose levels.

Time-dependent changes of collagen crosslinks in the socket after tooth extraction in rabbits.

Time-dependent changes of the reducible collagen crosslinks in the healing tissue of rabbit tooth extraction wounds were analyzed chromatographically. The ratio of dihydroxylysinonorleucine to hydroxylysinonorleucine in the collagen from normal alveolar bone was 4.4. This value increased about four times, on the 10th day after tooth extraction, coinciding with the phase of active woven bone formation, and then decreased rapidly toward a normal value on the 14th day after tooth extraction. The data suggest that active biosynthesis and fibrillogenesis of bone collagen precede the morphological completion of lamellar bone formation.

Hyaluronidase activity of rabbit skin wound granulation tissue fibroblasts.

The objective of this work was to identify and compare hyaluronidase activities of normal dermal and dermal wound granulation tissue fibroblasts. Direct evidence of the fibroblast as a source of tissue hyaluronidase was obtained. Fourth passage rabbit dermal fibroblasts were harvested on culture days 4, 8, 14, 18, and 22. Hyaluronidase activity and [35S]-sulfate- or [3H]-glucosamine-labeled glycosaminoglycans (GAGs) were monitored. Hyaluronidase assays were performed on medium and cellular fractions at the designated intervals. Enzyme activity of cellular fractions for both normal dermal and 14-day post-wound granulation tissue fibroblasts increased progressively through culture day 8. Thereafter (days 14-22), an eight-fold drop in cellular activity was coupled with cell death and emergence of hyaluronidase activity in medium fractions. Marked increases in degradation of secreted matrix components were concurrent with lysis-induced release of hyaluronidase. In this culture system, hyaluronidase activity was confined exclusively to cellular fractions and was released into the medium only under non-physiological conditions conducive to cellular death and lysis. Accordingly, this work suggests that previously reported skin wound hyaluronidases may be of fibroblastic origin and that susceptible GAGs are not degraded extracellularly, but, rather, must be internalized as a prerequisite to depolymerization.

cAMP levels in reactive tissues around dimethylpolysiloxane solid implants.

By the aid of radioimmunologic assay, the authors measured the cAMP content of the reactive capsules around solid dimethylpolysiloxane implants in mice at different times; they also measured the same substance in some human connective tissues (granulation tissue and normal dermis) and compared together all the values they obtained. Different concentrations of cAMP in different tissues seem to reflect correspondent histologic findings, since they vary according to them. These values also seem to indicate a close correspondence between the development of the process of wound healing and of the foreign-body reaction following the implantation of alloplastic materials. On the basis of these data, the authors suggest an experimental therapeutic trial to enhance peripheral cAMP synthesis in order to control the process of reactive capsule constitution.