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CARP, a cardiac ankyrin repeat protein, is up-regulated during wound healing and induces angiogenesis in experimental granulation tissue.

Cardiac ankyrin repeat protein (CARP) was identified by subtractive hybridization as one of a group of genes that are rapidly modulated by acute wounding of mouse skin. Quantitative RT-PCR showed that CARP was strongly induced during the first day after wounding (157.1-fold), and the high level persisted for up to 14 days. Immunohistochemistry and in situ hybridization revealed that CARP was expressed in skeletal muscle, vessel wall, hair follicle, inflammatory cells, and epidermis in the wound area. To examine the effects of CARP on wound healing, we developed an adenoviral CARP vector to treat subcutaneously implanted sponges in either rats or Flk-1(LacZ) knock-in mice. Four days after infection, CARP-infected sponges in rats showed a remarkable increase in the vascular component in granulation tissue as compared to Ad-LacZ controls. This result was confirmed by CD34 immunostaining. By 7 days post-infection of sponge implants in Flk-1(LacZ) knock-in mice, granulation tissue showed many more LacZ-positive cells in Ad-CARP-infected sponges than in virus controls. Ad-CARP treatment also induced neovascularization and increased blood perfusion in rabbit excisional wounds in and ischemic rat wounds. These findings indicate that CARP could play a unique role in therapeutic angiogenesis during wound healing.

Angiogenic Proteins↗

[Reactions of granulation tissue fibroblasts in regenerating skin to chronic hypoxia].

An investigation of the effect of chronic hypoxia on fibroblasts of the granulation tissue of a regenerating wound showed that an appreciable portion of fibroblasts degenerated and died. Some of the cells retained their capability to synthesize collagen but its excretion outside the cell was disordered. It also demonstrated an inhibition of fibrillogenesis and thus delayed regeneration of an experimental lesion.

Animals↗

[Effect of contrical on the activity of dehydrogenases and their isoenzymes in the muscles and organs of animals with developing granulation tissue].

Studies of the influence of contrycal on condition of the enzyme systems of the muscles, liver, kidneys and heart of rats with developing granulation tissue showed contrycal, a protease inhibitor, to have a stimulating effect on the LDH and MDH activity and on their isozyme spectrum. The effect of the inhibitor was expressed in the changes occurring in the state of the enzyme system, both at the site of damage (granulations and the underlying tissue) and in a number of internal organs -- the liver and kidneys.

Animals↗

Localization of the fibronexus at the surface of granulation tissue myofibroblasts using double-label immunogold electron microscopy on ultrathin frozen sections.

An intimate transmembrane complex of fibronectin-containing extracellular fibers and actin microfilaments termed the fibronexus has been observed at the adhesive surface of fibroblasts in vitro [19] and along the plasmalemma of myofibroblasts in vivo [22]. Although the observation of coincident actin and fibronectin immunofluorescence patterns in the latter work strongly suggested that the fibronexus is localized at the myofibroblast surface, we only obtained morphological evidence for its existence with electron microscopy. Therefore, in the present study, we have utilized a double-label immunoelectron microscopic technique to localize fibronectin and actin simultaneously in the putative fibronexuses of myofibroblasts within guinea pig granulation tissue, formed 7 to 9 days after skin wounding. This method employed rabbit antifibronectin and mouse anti-actin antibodies, followed by species-specific secondary antibodies conjugated to colloidal gold particles of different sizes. These probes were applied to the surfaces of ultrathin frozen sections mounted on grids. We found that fibronectin and actin were specifically localized on the respective external and internal components of myofibroblast fibronexuses. Our results suggest that specific transmembranous fibronectin-cytoskeletal complexes play an important role in the cohesion of granulation tissue.

Actins↗

[Relationship between wound healing and TNF, MDA and SOD contents in granulation tissues of rats in the first week].

An experimental model was used to determine the changes in TNF, MDA and SOD in granulation tissues during natural wound healing after skin excision on rats. Our results indicated that the changes in TNF and SOD exhibited a curve of V. The levels of TNF and SOD were lower on day 7 than day 3. The levels of MDA rose gradually, especially on day 7. A positive correlation was shown between TNF and MDA (at days 3, 5, 7), also between TNF and SOD (at day 3, 7). When the concentration of TNF was lower than 90 Pg/mg protein, the process of wound healing was best, while wound healing was hindered when the levels of SOD were low. The results suggest that in the process of wound repair there are influential changes in the contents of TNF, MDA and SOD. Lower levels of TNF and higher levels of SOD are apparently beneficial to wound healing after trauma.

Animals↗

Three-dimensional ultrastructural distribution of cytoplasmic interdigitation between endothelium and pericyte of capillary in human granulation tissue by serial section reconstruction method.

Two- and three-dimensional electron microscopic observations in the capillaries with angiogenetic sprout of human granulation tissue revealed the cytoplasmic interdigitations (CID) between the endothelium and the pericyte, which were composed of unit of cytoplasmic projection and an indentation. Two types of interdigitation were observed: the interdigitation composed of cytoplasmic projection from the pericyte plugging into the endothelial indentation, CID(P-E); the interdigitation which had a reverse mode of composition of the cytoplasmic projection and indentation to the former one, CID(E-P). We investigated three-dimensional distribution of the two kinds of interdigitation in two cases of the capillaries with angiogenetic sprout. The two kinds of interdigitation exhibited a remarkable difference in the three-dimensional distribution. The CID(P-E) was rather uniformly distributed over the parent capillary endothelial area (6.4 per endothelium in average) and was not observed at all in the angiogenetic area; the other type of cytoplasmic interdigitation, CID(E-P), was located in only the angiogenetic sprouting endothelial area (3.5 per endothelium in average). The cytoplasmic interdigitation between the endothelium and the pericyte in vivo was the only site for cell contact and seemed to be involved in a conducting function which is biochemically proved to exist in vitro between the two cells.

Capillaries↗

Identification of cytokine-induced neutrophil chemoattractants (CINC), rat GRO/CINC-2 alpha and CINC-2 beta, produced by granulation tissue in culture: purification, complete amino acid sequences and characterization.

Four basic neutrophil chemotactic factors (chemokines) have been purified from conditioned medium of granulation tissue obtained from carrageenin-induced inflammation in the rat. On the basis of their N-terminal amino acid sequences, one of the chemokines was identical with rat GRO/cytokine-induced neutrophil chemoattractant (CINC) which we reported previously, and another was identical with rat macrophage inflammatory protein-2 (MIP-2). Two other chemokines were novel chemoattractants related to MIP-2. The novel chemokines are referred to as rat GRO/CINC-2 alpha and CINC-2 beta, and consequently CINC and rat MIP-2 are renamed rat GRO/CINC-1 and CINC-3 respectively. The complete amino acid sequences of purified CINC-2 alpha and CINC-3 were determined by analysis of the fragments isolated from proteinase V8-treated CINCs. The cDNA for CINC-2 beta was cloned by reverse transcription/PCR amplification using specific primers starting with total RNA extracted from lipopolysaccharide-stimulated rat macrophages. A comparison of the amino acid sequence encoded by the cDNA with the N-terminal amino acid sequence of purified CINC-2 beta revealed that mature CINC-2 beta is a 68-residue chemoattractant produced by cleavage of a 32-residue signal peptide. The difference in amino acid sequences between CINC-2 alpha and CINC-2 beta consisted of only three C-terminal residues. Rat GRO/CINC-2 alpha is a major chemokine, and the four purified chemokines have similar chemotactic activity, suggesting that they contribute to neutrophil infiltration into inflammatory sites in rats.

Amino Acid Sequence↗

The influence of locally deposited fibrin on the biomechanical properties of developing granulation tissue in rats.

A recently developed method for the occlusion of fistulas with a fibrin clot has stimulated studies concerning the effects of deposited fibrin on the formation of granulation tissue (g.t.). In this study the influence of artificially implanted fibrin on the biomechanical properties of developing g.t. was investigated in rats. Perforated Teflon cylinders either empty or fibrin filled, were implanted subcutaneously for two weeks. The g.t. from fibrin filled cylinders was found to adhere significantly better to the cylinder walls which is an important property in the case of fistula closure with fibrin. Mechanical testing, however, showed that the maximum load and the maximum strain were significantly higher in cylinders implanted empty. However, the collagen concentration was no higher in cylinders implanted empty suggesting that in the newly formed g.t. studied, factors other than the collagen concentration determined the strength of the tissue.

Animals↗