Junctional complexes in the early mammalian embryo.

Preimplantation embryos generate intercellular junctions during differentiation of the trophectoderm epithelium and the formation of the blastocyst. These membrane complexes comprise gap junctions, adherens junctions, tight junctions, and desmosomes, each performing fundamental roles in cellular communication, adhesion, and differentiation. The mouse embryo has been used as a model for the biogenesis of cell junctions. Their construction is achieved by temporally regulated gene expression programs. Mechanisms of junction membrane assembly include the timing of transcription, translation, and post-translational modifications of specific junctional proteins. Human embryos exhibit similar expression programs, and defects in these programs may contribute to reduced embryo viability.

Expression of alpha-smooth muscle actin, TGF-beta 1 and TGF-beta type II receptor during connective tissue contraction.

Closure of rat mesenteric perforation is considered to occur by connective tissue contraction, a process that has been shown to be stimulated by transforming growth factor-beta 1. In the present study, we assessed the expression of alpha-smooth muscle actin during closure by quantitative-reverse transcription-polymerase chain reaction and in situ hybridization. The expression of transforming growth factor-beta 1 and transforming growth factor-beta type II receptor was also estimated in mesenteric membranes and free peritoneal cells after wounding. A larger expression of alpha-smooth muscle actin was seen around the wound edges compared to unwounded tissue. Both alpha-smooth muscle actin and transforming growth factor-beta type II receptor were expressed during Days 0, 3, 5, 7, and 10. The expression of alpha-smooth muscle actin on Day 5 was > 100 times higher than on Day 0. Transforming growth factor-beta 1 was expressed in both membranes and free peritoneal cells of unoperated control animals but down-regulated after wounding, a finding that has not been reported previously. It reappeared on Days 7 and 10 in free peritoneal cells but not in perforated membranes. The enhanced expression of alpha-smooth muscle actin and down-regulation of transforming growth factor-beta 1 expression after wounding appears to be important phenomena in tissue contraction and repair.

Stimulation of protracted connective tissue repair in normal mice by transforming growth factor beta 1.

The repair and contraction during connective tissue repair of mesenteric perforations is prolonged in mice compared with rats. In the present study the stimulating effect of transforming growth factor beta 1 (TGF-beta 1) on different aspects of such repair of the mouse mesentery was assessed. The number of closed mesenteric perforations were counted on different days after operation and the free peritoneal cells were counted, the mitotic index was assessed, and actin distribution of fibroblasts around the perforation was studied with laser scanning confocal microscopy. TGF-beta 1 significantly increased the speed of closure and seemed to induce more actin in fibroblasts at the wound margin. It did not significantly influence the mitotic index, but fewer free peritoneal cells were obtained in mice treated with TGF-beta 1. We conclude that TGF-beta 1 is a potent stimulator of connective tissue repair and contraction in mice. The different methods of closure in rats and mice implicate different molecular responses in wounds and further studies on the stimulating effect of TGF-beta 1 may indicate basic fibroblastic cellular mechanisms that are active during contraction in connective tissue repair.

Contraction of collagen lattices by cells from Dupuytren's nodules.

The aim of this study was to see if nodular cells in Dupuytren's disease differed from dermal cells in their contractile capacity and motility. Ten surgical specimens from patients with Dupuytren's disease and contracture of the finger of more than 45 degrees were harvested and the nodular cells were explanted and cultured. Dermal fibroblasts from the forearm were used as control cells. Both types of cell had the same growth pattern. The morphology on confocal laser scanning microscopy was also similar in both types of cell. Dermal control cells caused significantly more contraction of collagen lattices compared with fibroblasts from nodules of Dupuytren's contracture. The F-actin content was equal in both groups. Platelet derived growth factor, PDGF-BB (but not PDGF-AA), increased the chemotactic activity of both cell types, but there were no differences between them. The results indicate that at a late state of the disease cells from Dupuytren's nodules lose their contractile capacity and regain a phenotype resembling that of dermal fibroblasts.

Competitive RNA templates for detection and quantitation of growth factors, cytokines, extracellular matrix components and matrix metalloproteinases by RT-PCR.

Detection of low-abundance mRNAs by reverse transcription-polymerase chain reaction (RT-PCR) has become a standard technique to determine gene expression by tissues and cells in culture. The ability to determine relative or absolute copy number of specific mRNAs has been difficult due to inadequate internal standards to control for sample-to-sample variation. The use of a synthetic RNA standard with identical sequences to the PCR primers allows reproducible quantitation between samples and assays. By designing multi-sequence templates, several specific mRNAs can be quantitated using a single template. Addition of multiple templates to a single RT reaction allows the quantitation of a large number of targets from as little as 4 micrograms of total RNA. In this report, we present a series of seven primer/template systems to detect and quantitate 52 specific messages, including 26 growth factors and receptors, 8 extracellular matrix components, 10 matrix-modifying enzymes and their inhibitors and 8 cytokines.

Impaired function of postoperative macrophages from zinc-deficient rats decreases collagen contraction. Brief report.

Zinc deficiency impairs connective tissue contraction in the perforated rat mesentery model. Since the rat mesentery is almost avascular, free peritoneal macrophages are important for mesenteric repair. Impairment of contraction may thus be caused either by a direct effect of zinc deficiency on tissue cells or by hampered macrophage function. To further elucidate the role of macrophages in tissue contraction, we studied their effect on lattice contraction. A number of typical functions of macrophages in zinc deficiency were also investigated. Lattice contraction was significantly impaired by conditioned medium from zinc-deficient macrophages. Zinc deficiency did not influence peripheral blood leukocyte number, but postoperatively the number of peritoneal macrophages increased on days 7 and 10. A significant release of lysosomal enzymes from macrophages was recorded during phagocytosis, whilst no difference was observed between controls and zinc-deficient macrophages. Superoxide anion generation during phagocytosis was not significantly increased in zinc deficiency. Conditioned medium from zinc-deficient macrophages was shown to impair lattice contraction in vitro and the results are compatible with impaired macrophage function as a cause of decreased connective tissue contraction in vivo.

Connective tissue repair in zinc deficiency. An ultrastructural morphometric study in perforated mesentery in rats.

OBJECTIVE: To quantify measures of healing in zinc-deficient and healthy rats. DESIGN: Randomized study. MATERIAL: 30 male Sprague-Dawley rats. INTERVENTIONS: Zinc deficiency was induced in half the rats. All rats underwent laparotomy and standard perforations were made in the small intestinal mesentery with a scalpel. At 1, 3, 5, 7 and 10 days after operation 6 rats were killed by overdose of anaesthetic agents and the specimens of the mesentery were fixed. MAIN OUTCOME MEASURES: Measurement of cellular volume density, surface density of the rough endoplasmic reticulum, and surface density of the plasma membrane. RESULTS: Perforations started to close on day 4, and most were closed by day 10. Cellular volume density reached its peak between days 3 and 5, as did surface density of rough endoplasmic reticulum. There were no significant differences between the two groups for either measurement. The surface density of the rough endoplasmic reticulum, however, was significantly higher in controls than in zinc deficient animals on days 3-10 (p less than 0.001). The surface density of the plasma membrane was significantly higher in zinc-deficient animals on days 1-3 (p less than 0.04), and in control animals on days 5-10 (p less than 0.01). CONCLUSIONS: Protein synthesis and formation of scar tissue were slightly lower in the zinc-deficient animals, and the higher plasma membrane surface density implies that contraction may be an important part of healing in the small intestinal mesentery in rats.