Increased expression of connective tissue growth factor in the infarct zone of experimentally induced myocardial infarction in rats

Connective tissue growth factor (CTGF), a 36- to 38-kDa peptide, is selectively induced by transforming growth factor-beta and has been suggested to contribute to tissue repair. To test the hypothesis that CTGF is expressed in myocardial infarct tissue following acute myocardial infarction (AMI), we examined CTGF expression after AMI was experimentally induced in rats. Myocardial infarction was induced by left coronary artery ligation in male Sprague-Dawley rats. Northern blotting demonstrated that the CTGF mRNA expression on days 2, 7 and 14 was increased by 6-, 23- and 8-fold, respectively, compared to that in the pre-ligation hearts. In situ hybridization revealed CTGF mRNA signals on day 2 in myocytes in the infarct marginal zone and spindle-shaped mesenchymal cells (presumably myofibroblasts and fibroblasts) located between surviving myocytes in the infarct peripheral zone. On day 7, the signals were observed in the inner lesion of the infarct around infarct granulation tissue. Western blotting demonstrated that the CTGF protein expression on days 2, 7 and 14 was increased compared to the pre-ligation hearts. Immunopositive staining for CTGF was observed in the inner lesion of the infarct tissue on day 7. In conclusion, the findings demonstrated the increased expression of CTGF in the infarct tissue. Myocytes in the infarct marginal zone and spindle-shaped mesenchymal cells (presumably myofibroblasts and fibroblasts) were the cells responsible for CTGF production.     

Remodeling of alveolar walls after elastase treatment of hamsters. Results of elastin and collagen mRNA in situ hybridization

Treatment of hamster lungs with porcine pancreatic elastase (PPE) causes emphysema and a decrease in lung elastin content, which returns to control level by Day 30. To explore the mechanism of alveolar wall remodeling after elastolytic injury, we examined the expression of elastin and alpha1(I) collagen mRNAs by in situ hybridization at 1, 2, 3, 5, 7, and 30 d after intratracheal PPE. The lungs of control animals displayed weak signals for elastin and alpha1(I) collagen mRNA in pleura, large arteries, veins, and airways. There was little or no signal in respiratory air space walls. Increased expression of elastin and alpha1(I) collagen mRNA began by Day 1 after PPE and reached an asymptote by Day 3 that was maintained by elastin until Day 7; expression of alpha1(I) collagen mRNA waned earlier. Elastin and, to a lesser extent, alpha1(I) collagen mRNA were heavily expressed in pleura, blood vessels, and airways. Analysis of serial sections showed elastin message was minimal in the walls of respiratory air spaces and when present, at 3, 5, and 7 d, was primarily found at the free margins of alveolar septa. Collagen message was very sparse in respiratory air space walls. By 30 d, elastin mRNA expression was reduced but still above control levels and emphysema was widespread and severe. Rank score of elastin mRNA expression in individual subpleural air spaces showed a positive correlation with air space size. In conclusion, most expression of elastin and alpha1(I) collagen mRNA occurs in the pleura, airway, and vascular walls. In respiratory air space walls, expression of elastin mRNAs occurs in damaged tissue at free septal margins.     

Spatial distribution of collagen type I mRNA in Paracentrotus lividus eggs and embryos

We have identified the presence of type I collagen (COLL1alpha) mRNA in Paracentrotus lividus unfertilized egg, indicating a maternal origin of this mRNA. By in situ whole mount hybridization the spatial distribution of COLL1alpha mRNA in egg and embryo at different developmental stages was established. Moreover, the presence of COLL1alpha gene in Paracentrotus lividus genome was analyzed by Southern blot experiments. The localization pattern indicates that the maternal mRNA is placed in the fertilized egg in a fixed position, relative to the embryonic axes. Furthermore, the embryonic expression is spatially restricted during development, suggesting involvement in sea urchin embryo cell specification events. The presence of two bands in Southern blot hybridization may indicate that two genes specific for COLL1alpha are present in the sea urchin genome.     

Expression of rat homeobox gene, rHOX, in developing and adult tissues in mice and regulation of its mRNA expression in osteoblasts by bone morphogenetic protein 2 and parathyroid hormone-related protein

The rat homeobox gene, rHox, was cloned from a rat osteosarcoma cDNA library. Southwestern and gel mobility shift analyses showed that rHox binds to the promoter regions of collagen (alpha1)I and osteocalcin genes while transient transfection with rHox resulted in repression of their respective promoter activities. In situ hybridization studies showed that rHox mRNA was widely expressed in osteoblasts, chondrocytes, skeletal muscle, skin epidermis, and bronchial and intestinal epithelial cells, as well as cardiac muscle in embryonic and newborn mice. However in 3-month-old mice, rHox mRNA expression was restricted to osteoblasts, megakaryocytes, and myocardium. Bone morphogenetic protein 2, a growth factor that commits mesenchymal progenitor cells to differentiate into osteoblasts, down-regulated rHox mRNA expression by 40-50% in UMR 201, a rat preosteoblast cell line, in a time- and dose-dependent manner. In contrast, PTH-related protein (PTHrP), recently shown to be a negative regulator of chondrocyte differentiation, significantly enhanced rHox mRNA expression in UMR 106-06 osteoblastic cells by 3-fold at 24 h while at the same time down-regulating expression of pro-alpha1(I) collagen mRNA by 60%. Expression of rHox mRNA in calvarial osteoblasts derived from PTHrP -/- mice was approximately 15% of that observed in similar cells obtained from normal mice. In conclusion, current evidence suggests that rHox acts as a negative regulator of osteoblast differentiation. Furthermore, down-regulation of rHox mRNA by bone morphogenetic protein 2 and its up-regulation by PTHrP support a role of the homeodomain protein, rHox, in osteoblast differentiation.     

The regulation of collagen in fetal skin wounds: mRNA localization and analysis

The deposition of collagen in fetal skin wounds has been shown in several animal models. The authors used a radiolabeled RNA antisense probe, complementary to the mRNA for the alpha-1 chain of human procollagen type I, to assess regulation of this collagen species in fetal and adult rabbit wounds. Dorsal skin wounds were placed on fetal and maternal animals at the beginning of the third trimester, and were harvested 3, 5, and 7 days later. In situ RNA/RNA hybridization was performed on suitable specimens, and morphometric analysis was carried out with a computerized LECO image analyzer. Fetal wounds exhibited an inflow of mesenchymal cells that produced collagen type I at levels higher than the surrounding tissue; this activity was highest on days 3 and 5 after wounding. Adult wounds had increased fibroblast presence by day 7, producing collagen type I at levels higher than those of adjacent unwounded tissue. Morphometric analysis of the signal produced by in situ hybridization and of the number of cells producing the signal in a given field showed that fetal wounds appear to produce collagen type I by an increase in the number of cells in the area of the wound--not by induction of the gene for procollagen type I. In contrast, adult wounds had both fibroblast migration and induction of procollagen type I mRNA synthesis. These findings imply multilevel regulation of collagen production in the adult and posttranslational regulation in the fetus.     

Vascular adventitial cell expression of collagen I messenger ribonucleic acid in anti-glomerular basement membrane antibody-induced crescentic nephritis in the rabbit. A cellular source for interstitial collagen synthesis in inflammatory renal disease

BACKGROUND: Scarring in the interstitial compartment of the renal cortex heralds a poor prognosis in many forms of renal injury, however, the mechanism through which glomerular inflammation leads to interstitial scarring is not understood. In a model of anti-GBM disease in the rabbit, development of crescentic glomerulonephritis is associated with marked interstitial fibrosis and decreased renal function. We previously demonstrated that collagen accumulation in the model was preceded by increases in collagen I and IV mRNA and that these changes were primarily extraglomerular at early time points when inflammation was predominantly intraglomerular. In order to identify the cellular origins of extraglomerular collagen synthesis in this model, in situ hybridization using an alpha 2(I) procollagen probe was performed. EXPERIMENTAL DESIGN: A 602 bp rabbit alpha 2(I) procollagen cDNA was cloned using a PCR strategy and sequenced. The nucleotide sequence of the coding region was 94% identical with the human alpha 2(I) procollagen sequence. Northern blots were performed to define conditions of specific hybridization of the anti-sense riboprobe. Tissue sections from normal rabbit kidneys and from kidneys 4, 5, 7, 10 and 14 days after injection of anti-GBM antibody were hybridized with 35S-labeled sense and anti-sense riboprobes. Cells containing alpha 2(I) mRNA were identified by autoradiography and mRNA abundance was quantitated by grain density. RESULTS: No specific hybridization was detected with the sense probe at any time. alpha 2(I) mRNA was undetectable with the anti-sense probe in normal kidney sections. In contrast, the anti-sense probe hybridized specifically at all time points after induction of anti-GBM disease. In agreement with previous filter hybridization studies, on day 4, when inflammation was predominantly intraglomerular, cells in the periarterial adventitial compartment of renal cortex hybridized strongly. At later time points, labeling was also present in the interstitial spaces, the periglomerular region, in Bowman's space and in the glomerular tuft itself. CONCLUSIONS: We conclude that perivascular adventitial cells are among the first to respond to glomerular inflammation and represent a pool of cells that subsequently contribute to interstitial and glomerular scarring.     

Eukaryotic expression of recombinant biglycan. Post-translational processing and the importance of secondary structure for biological activity

Biglycan is a small chondroitin sulfate proteoglycan found in many tissues and is structurally related to decorin, fibromodulin, and lumican. The biological function of biglycan is poorly understood, although several studies have indicated interaction with other extracellular matrix components. We have initiated studies of structural and functional domains of biglycan by transient eukaryotic expression using the vaccinia virus/T7 bacteriophage expression system. A recombinant vaccinia virus, vBGN4 encoding the mature biglycan core protein as a polyhistidine fusion protein under control of the T7 phage promoter was expressed in HT-1080 cells and UMR106 cells. The structure of the recombinant biglycan secreted by these cells was defined by analyzing molecules labeled in the presence of [35S]sulfate, [3H]glucosamine, and [35S]methionine. Glycoforms of biglycan were separated by imidazole gradient elution, under non-denaturing conditions, and comprised: a large proteoglycan form substituted with two chondroitin sulfate chains of molecular mass approximately 34 kDa (HT-1080 cells) or approximately 40 kDa (UMR106 cells); a small proteoglycan form substituted with two chondroitin sulfate chains with a median molecular mass approximately 28 kDa; and a core protein form secreted devoid of glycosaminoglycan chains. All the glycoforms were substituted with two N-linked oligosaccharides, and the disaccharide composition of the two glycosaminoglycan populations were identical. Approximately 70% of the recombinant biglycan secreted by HT-1080 cells was substituted with chondroitin sulfate chains, whereas about 50% of the biglycan expressed by UMR106 cells was substituted with chondroitin sulfate chains. Infection with vBGN4 in both HT-1080 and UMR106 cells resulted in the production of approximately 10 mg of biglycan/10(9) cells per 24 h. The native recombinant biglycan was shown to bind to collagen type V and the complement protein, C1q. However, when the secondary structure of recombinant biglycan was disrupted by exposure to 4 M guanidine hydrochloride, the affinity for collagen type V was dramatically reduced. These data demonstrate the importance of secondary structure to the function of this small proteoglycan.     

Stage-specific expression of pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA in the rat seminiferous tubules

We have used in situ hybridization and Northern blot analysis with oligonucleotide probe to characterize the site of pituitary adenylate cyclase-activating polypeptide (PACAP) synthesis in the rat testis. We observed strong hybridization signal in one third of the cross-sections of the seminiferous tubules, whereas some tubules were devoid of hybridization signal, thus suggesting that PACAP mRNA is expressed in a stage-specific manner. More detailed analysis showed that PACAP mRNA was present in round spermatids at stages III-VII of the cycle. Northern blot hybridization to RNAs extracted from samples of seminiferous tubules at different stages of the epithelial cycle confirmed that expression of PACAP mRNA is restricted to specific stages of the cycle. The highest amount of PACAP mRNA was detected at stages V to early -VII of the cycle, whereas very low levels of mRNA were present at stages I-II and IX-XIV. The present results demonstrate that PACAP mRNA is expressed in the developing germ cells. This suggests that PACAP may function as a paracrine or autocrine regulatory factor for the Sertoli and germ cells, with a specific function during early spermiogenesis, shortly before the onset of nuclear elongation, at the last period of haploid gene activity.     

Use of the Taylor Visagraph II system to evaluate eye movements made during reading

The clinical utility of transjugular intrahepatic portasystemic shunts (TIPS) is frequently complicated by the ingrowth of tissue into the stent lumen, causing stent stenosis. These studies were undertaken to define the cellular and matrix components of the pseudointima, define the phenotype and function of the mesenchymal cells in the pseudointima and maintain them in culture, and to study the differences between stenotic and nonstenosed stents. A total of 35 stents were evaluated. TIPS pseudointima were examined histologically, by immunohistochemistry and in situ hybridization to determine the cellular and connective tissue constituents. Mesenchymal cells were grown from tissue within the TIPS and around it, and their phenotype was studied and compared with control smooth muscle cells and fibroblasts. Masson's trichrome staining of histological sections demonstrated that TIPS tissue was composed of collagen and palisades of mesenchymal cells and was lined by an endothelium. Immunostaining demonstrated strong and uniform alpha-smooth muscle staining in TIPS mesenchymal cells and peri-TIPS cells. Type I procollagen mRNA expression was demonstrated in mesenchymal cells in and around the stent by in situ hybridization. TIPS mesenchymal cells secreted less radiolabeled fibronectin, and far more type III, relative to type I, collagen compared with peri-TIPS cells. TIPS cells also expressed high levels of type III procollagen mRNA compared with peri-TIPS cells. There was no difference between stenotic stents and nonstenosed stents with respect to clinical features, time from stenting, gross morphology, histology, presence of bile fistulae, and cell phenotype. However, smooth muscle cells (SMC) from stenotic stents demonstrated both greater cell proliferation and collagen I and III secretion compared with those from nonstenosed stents. These data demonstrate that TIPS stenosis results from an accumulation of collagen and proliferation of SMC within the stent lumen.     

Renal expression of transforming growth factor-beta inducible gene-h3 (beta ig-h3) in normal and diabetic rats

BACKGROUND: Transforming growth factor-beta (TGF-beta) has been implicated in the pathogenesis of a number of kidney diseases characterized by glomerulosclerosis and tubulointerstitial fibrosis. TGF-beta is secreted in a latent form requiring extracellular modification to become biologically active. TGF-beta inducible gene-h3 (beta ig-h3) is a recently identified TGF-beta-induced gene product. The present study sought to examine beta ig-h3 expression in normal and diabetic rats. METHODS: Beta ig-h3, TGF-beta1 and alpha1 (IV) collagen gene expression were assessed by Northern blot analysis and in situ hybridization in 20 Sprague Dawley rats, randomly assigned to receive streptozotocin (diabetic, N = 11) or citrate buffer alone (control, N = 9) and sacrificed eight months later. The effect of exogenous TGF-beta1 on beta ig-h3 expression was also assessed in cultured proximal tubular cells. RESULTS: In situ hybridization localized beta ig-h3 gene expression to the juxtaglomerular apparatus and the pars recta (S3 segment) of proximal tubules in both control and diabetic animals. Kidney TGF-beta 1, beta ig-h3 and alpha1 (IV) collagen mRNA from diabetic rats were increased two- to threefold compared with controls (P < 0.01). There was a significant correlation between TGF-beta1 and beta ig-h3 gene expression in kidneys from diabetic rats (r = 0.73, P = 0.01). In addition, beta ig-h3 mRNA increased in response to exogenous TGF-beta1 in a dose-dependent fashion in cultured proximal tubular cells. CONCLUSION: These findings support the hypothesis that biologically active TGF-beta plays a pathogenetic role in diabetic kidney disease and suggest that beta ig-h3 may be a useful index of TGF-beta1 bioactivity in the kidney.     

Primary structure, developmental expression, and immunolocalization of the murine laminin alpha4 chain

The complete primary structure of the mouse laminin alpha4 chain was derived from cDNA clones. The translation product contains a 24-residue signal peptide preceding the mature alpha4 chain of 1,792 residues. Northern analysis on whole mouse embryos revealed that the expression was weak at day 7, but it later increased and peaked at day 15. In adult tissues the strongest expression was observed in lung and cardiac and skeletal muscles. Weak expression was also seen in other adult tissues such as brain, spleen, liver, kidney, and testis. By in situ hybridization of fetal and newborn tissues, expression of the laminin alpha4 chain was mainly localized to mesenchymal cells. Strong expression was seen in the villi and submucosa of the developing intestine, the mesenchymal stroma surrounding the branching lung epithelia, and the external root sheath of vibrissae follicles, as well as in cardiac and skeletal muscle fibers. In the developing kidney, intense but transient expression was associated with the differentiation of epithelial kidney tubules from the nephrogenic mesenchyme. Immunohistologic staining with affinity-purified IgG localized the laminin alpha4 chain primarily to lung septa, heart, and skeletal muscle, capillaries, and perineurium.     

Spatial and temporal expression of fibril-forming minor collagen genes (types V and XI) during fracture healing

Skeletal development involves the coordinated participation of several types of collagen, including both major and minor fibrillar collagens. Although much is known about the major fibrillar collagens, such as types I and II, less is known about the minor fibrillar collagens, and their role in the repair and regeneration of bone has not been extensively studied. To clarify the role of minor fibrillar collagens in fracture repair, we examined the spatial and temporal expression of mRNAs for pro-alpha 2(V) collagen and pro-alpha 1(XI) collagen in healing fractures in the rat by in situ hybridization and compared their patterns of expression with those of mRNAs for pro-alpha 1(I) collagen, pro-alpha 1(II) collagen, and osteocalcin. A strong signal for pro-alpha 2(V) was detected in the periosteal osteoprogenitor cells, whereas osteocalcin mRNA was strongly expressed only in the deep layers of the hard callus. The distribution of the pro-alpha 2(V) signal was correlated with that of pro-alpha 1(I) but was mutually exclusive of that of pro-alpha 1(II). The expression of pro-alpha 1(XI) mRNA was synchronously regulated with that of pro-alpha 1(II) during chondrogenesis in the soft callus. In the hard callus, pro-alpha 1(XI) signal was found in osteoblastic cells at the site of intramembranous and endochondral ossification. These cells simultaneously expressed pro-alpha 2(V), although they were negative for pro-alpha 1(II). These findings suggest that the alpha 2(V) collagen chain participates in the formation of the noncartilaginous fibrillar network in the hard callus and preferentially contributes to the initial stage of the intramembranous bone formation. Recent reports have revealed that type-XI collagen, which had been classified as a cartilage-type collagen, is not necessarily specific for cartilage. The present results advanced this recognition and demonstrated a coexpression of alpha 1(XI) mRNA and alpha 2(V) mRNA in the noncartilaginous tissues in the fracture callus; this suggests the presence of tissue-specific and stage-specific heterotrimers consisting of alpha 1(XI) and alpha 2(V) collagen chains and the association of such hybrid trimers with the major fibrillar collagens in the process of fracture healing.