Human mast cells activate fibroblasts: tryptase is a fibrogenic factor stimulating collagen messenger ribonucleic acid synthesis and fibroblast chemotaxis

The effect of human mast cells on fibroblast activity was studied using an organotypic skin-equivalent culture system. Human mast cell-1 (HMC-1) cells were embedded in a collagen gel with neonatal dermal fibroblasts at a ratio of 1:4; keratinocytes then were allowed to stratify above this composite culture. Analysis of type a1(I) procollagen mRNA synthesis by in situ hybridization revealed a substantial increase in mRNA levels in the presence of mast cells and especially following degranulation, induced by calcium ionophore A23187. Tryptase, a major product of human mast cells, could substitute for mast cells in this culture system, up-regulating procollagen mRNA synthesis. Tryptase pretreated with the specific protease inhibitor bis(5-amidino-2-benzimidazo-lyl)methane (BABIM) markedly attenuated the collagen mRNA up-regulation. Further studies revealed HMC-1 cell sonicates stimulated fibroblast chemotaxis and procollagen mRNA synthesis. Inhibition of HMC-1 sonicates with either BABIM or a neutralizing mAb against tryptase resulted in significant reduction of fibroblast chemotaxis and procollagen mRNA, implying that tryptase accounted for the majority of HMC-1 sonicate activity. Tryptase directly stimulated fibroblast chemotaxis with optimal concentrations between 10 pM and 1 nM. The maximal response of optimal concentrations of tryptase was comparable with the known fibrogenic factor, TGF-beta. Inhibition of tryptase with BABIM resulted in approximately 50% reduction in chemotactic activity. Additional studies revealed that tryptase (0.3-3 nM) stimulated procollagen mRNA synthesis in confluent monolayers of dermal fibroblasts.     

Inhibition of type I collagen production by dermal fibroblasts upon contact with activated T cells: different sensitivity to inhibition between systemic sclerosis and control fibroblasts

OBJECTIVE: To assess the role of T lymphocyte-fibroblast contact in type I collagen production by cultured dermal fibroblasts from normal individuals and from patients with diffuse systemic sclerosis (SSc). METHODS: Cell membranes were prepared from activated CD4+ and CD8+ T cells, or type 1 T helper (Th1) clones, and added to confluent fibroblast monolayers. Type I collagen production was measured in culture supernatants, and messenger RNA (mRNA) levels of type I procollagen alpha1 (pro alpha1[I]) and matrix metalloproteinase 1 (MMP-1) were evaluated by Northern hybridization analysis. RESULTS: Dose-dependent inhibition of type I collagen production was observed with CD4+ and CD8+ T cells from both SSc patients and controls. Inhibition of type I collagen was significantly less pronounced in fibroblasts from SSc patients than in fibroblasts from controls (P < 0.02). Inhibition was not reversed by the addition of exogenous transforming growth factor beta, interleukin-4, interleukin-1 receptor antagonist, anti-tumor necrosis factor, anti-CD40, or indomethacin, whereas anti-interferon-gamma (IFNgamma) reversed Th1-mediated inhibition. This inhibitory activity was specific for type I collagen, since mRNA levels of pro alpha1(I) were decreased, whereas mRNA levels of MMP-1 were strongly increased. CONCLUSION: The production of type I collagen by skin fibroblasts is specifically down-regulated by membranes from activated T cells. The contact-dependent regulatory activity exerted by T cells on fibroblasts depends, at least in part, on the presence of membrane-associated IFNgamma. However, SSc fibroblasts are more resistant to inhibition than are fibroblasts from normal individuals.     

A chemically modified tetracycline inhibits streptozotocin-induced diabetic depression of skin collagen synthesis and steady-state type I procollagen mRNA

Wasting of connective tissues including skin, bone, and cartilage have been closely associated with elevated matrix metalloproteinase (MMP) activity and depressed collagen content in the streptozotocin (STZ)-induced diabetic rat, while tetracyclines have been reported to normalize total body weight, skin hydroxyproline and collagen content in this model, in part through inhibition of MMPs. In the present study, we report the effect of CMT-1, a chemically modified tetracycline that lacks antimicrobial properties but retains divalent cation binding and MMP inhibitory activity, on diabetic skin collagen synthesis and steady-state levels of procollagen alpha 1(I) mRNA. Male, 4-month old Sprague-Dawley rats received a single injection of 75 mg/kg STZ or citrate vehicle alone and diabetic status was confirmed by positive glucosuria. Some diabetic animals received 10 mg/day of CMT-1 by oral gavage and, 28 days after STZ treatment, body weight, blood glucose values and the in vivo rates of skin collagen production were measured using the pool-expansion technique. Steady-state levels of procollagen alpha 1(I) mRNA were analyzed 21 days after STZ treatment by hybridization of total RNA with a 32P labelled cDNA to rat type I procollagen alpha 1(I) mRNA in a dot-blot assay. STZ treatment was found to significantly depress body weight, skin collagen hydroxyproline content, the in vivo rate of collagen production, and hybridizable levels of type I procollagen alpha 1(I) mRNA. CMT-1 administered daily to STZ-treated rats inhibited the diabetic depression of these parameters but had little or no effect on non-diabetic controls or on STZ-induced hyperglycemia. Thus, in addition to the inhibition of MMP mediated extracellular collagen degradation, these results suggest CMT-1 also acts to inhibit diabetic connective tissue breakdown in STZ-induced diabetes by increasing both steady-state levels of type I procollagen mRNA and collagen synthesis through mechanism(s) that are independent of the antibacterial properties of tetracyclines.     

Expression of collagen, interstitial collagenase, and tissue inhibitor of metalloproteinases-1 in restenosis after carotid endarterectomy

Extracellular matrix is the principal component of the fibrous caps of atherosclerotic plaques and intimal hyperplastic lesions of reconstructed arteries. Interstitial collagen form an important part of the matrix, and the balance between collagen synthesis and degradation by interstitial collagenase (matrix metalloproteinase-1, MMP-1) may determine whether plaques rupture or vessels develop stenosis. We examined type I procollagen gene expression in human atherosclerotic and restenotic carotid arteries using in situ messenger RNA (mRNA) hybridization and the expression of MMP-1 and its endogenous inhibitor (tissue inhibitor of metalloproteinases-1, TIMP-1) by immunohistochemistry. Compared with normal arteries, atherosclerotic plaques bed increased expression of immunoreactive MMP-1 and TIMP-1 with modest increase of type 1 procollagen mRNA. Early restenotic lesions (< 1.5 years) contained abundant type I procollagen mRNA but little immunoreactive MMP-1 and TIMP-1. Late restenotic lesions (> 4 years) resembled atheroma and exhibited increased immunoreactive MMP-1 and TIMP-1 as well as abundant type I procollagen mRNA. Compared with atherosclerotic plaques, type I procollagen is increased and MMP-1 is decreased in early restenotic lesions. MMP-1 and TIMP-1 expressions are upregulated in lesions with a clear atheroma. These findings suggest that the balance between proteolysis and matrix synthesis may influence both the stability of atheromatous plaques and the development of restenotic lesions.     

Exogenous non-crosslinked collagen enhances granulation tissue formation in dermal excision wounds in guinea pigs

Based on previous observations indicating a role for collagen peptides in eliciting a positive feedback for collagen biosynthesis, this study was initiated to elucidate the effect of non-crosslinked collagen on granulation tissue formation in dermal excision wounds. The wounds were treated with either non-crosslinked or crosslinked native collagen, or left untreated as controls. Granulation tissue was analyzed for collagen type I mRNA, for levels of interstitial collagen and for the number of blood vessels. The results indicated significant increases in procollagen type I mRNA, in interstitial collagen, in the number of blood vessels and in epithelial advance in the non-crosslinked collagen-treated wounds relative to the untreated controls. It is assumed that the presence of non-crosslinked collagen in a healing wound enhances both procollagen type I biosynthesis and the repair process of dermal wounds, due to the more readily released collagen peptides derived from this exogenous collagen dressing.     

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.     

Ontogeny of estrogen receptor-beta expression in rat testis

The recently discovered estrogen receptor-beta (ERbeta) is expressed in rodent and human testes. To obtain insight in the physiological role of ERbeta we have investigated the cell type-specific expression pattern of ERbeta messenger RNA (mRNA) and protein in the testis of rats of various ages by in situ hybridization and immunohistochemistry. In fetal testes of rats 16 days postcoitum and testes of 4-day-old animals, fetal germ cells (gonocytes) reveal the ERbeta mRNA in their cytoplasm and the ERbeta protein in their nucleus. In testes of 11- and 15-day-old rats, ERbeta mRNA and protein were detected in Sertoli cells and type A spermatogonia. No signal was found in other types of germ cells. In the adult testes, expression of ERbeta mRNA as well as ERbeta protein was found in pachytene spermatocytes from epithelial stages VII-XIV and in round spermatids from stages I-VIII. Low ERbeta expression was observed in all type A spermatogonia, including undifferentiated A spermatogonia, whereas no expression was found in In and type B spermatogonia and early spermatocytes. At all ages, Sertoli cells showed a weak hybridization signal as well as weak immunoreactivity for ERbeta. In adult testes, no ERbeta mRNA or protein was detected in the interstitial tissue, indicating that Leydig cells and peritubular myoid cells do not express ERbeta. The expression of ERbeta in fetal and late male germ cells as well as in Sertoli cells suggests that estrogens directly affect germ cells during testicular development and spermatogenesis.     

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.     

Survey of echinococcosis in Hokkaido and measure against it

Exposure to hyperoxia results in lung injury and a decrease in lung collagen. Retinol is known to influence collagen gene expression, and retinol deficiency has been shown to potentiate hyperoxic lung injury. To investigate the combined effects of retinol deficiency and hyperoxia on lung collagen expression, retinol-deficient rats were exposed to acute hyperoxia, and expression of the alpha-1 chains of type I procollagen [pro alpha 1 (I)] and type III procollagen [pro alpha 1 (III)] were determined using Northern hybridization analyses and immunohistochemical staining. Hyperoxia alone reduced pro alpha 1 (I) mRNA by 60 +/- 4% (p < .05) and pro alpha 1 (III) mRNA by 30 +/- 5% (p < .05), and retinol deficiency alone reduced pro alpha 1 (I) mRNA abundance by 49 +/- 8.8% (p < .05) and pro alpha 1 (III) mRNA abundance by 14 +/- 7.5% (p = not significant), respectively. Retinol deficiency plus hyperoxia did not cause any further reduction in procollagen mRNA than that seen with oxygen exposure alone. Immunohistochemical staining demonstrated decreased staining for type I collagen in retinol-deficient animals. Hyperoxic exposure resulted in decreased connective tissue staining and increased alveolar wall staining for type I collagen. Retinol deficiency and hyperoxia together resulted in a marked increase in alveolar exudates staining for type I collagen. No changes in type III collagen staining were seen. These findings demonstrate that while retinol deficiency does not potentiate hyperoxia-induced reductions in procollagen mRNA, it is associated with alterations in collagen staining in distal lung and immunohistologic evidence of collagen fragments in alveolar exudates.     

2-Iminothiazolidine-4-carboxylic acid produces hippocampal CA1 lesions independent of seizure excitation and glutamate receptor activation

Impaired wound healing is a common complication of diabetes mellitus. The underlying pathophysiology of diabetes-impaired healing is poorly understood. In the present study we have compared cell proliferation rates, apoptosis (programmed cell death), the myofibroblast marker alpha-smooth muscle actin and procollagen I mRNA expression, between diabetic and control mice. Full-thickness skin wounds were made in non-obese diabetic (NOD) mice and C57B6 controls. NOD mice showed a marked retardation of wound healing at both 7 and 14 days after wounding. Comparison of cell proliferation rates 7 days after wounding, using 5-bromo-2'-deoxy-Uridine incorporation, showed higher rates of cell proliferation in controls (88.1 +/- 12.8) than in NOD wounds (52.1 +/- 9.9, p < 0.02, n = 4). Immunohistochemical detection of alpha-smooth muscle actin, showed a later onset in diabetic wounds, suggesting that wound contraction may be delayed in the diabetic animals. In situ hybridisation for alpha 1 (I) procollagen mRNA expression, showed reduced procollagen I expression in the diabetic wounds when compared with controls. Lastly, there appeared to be higher levels of apoptosis in diabetic wounds, shown by the terminal transferase mediated UTP nick end-labelling technique. Apoptotic cells were rare in control wounds confirming previous studies, which showed that apoptosis occurs late in normal wound healing as the wound matures into scar tissue. In conclusion, we hypothesize that reduced cell proliferation, retarded onset of the myofibroblast phenotype, reduced procollagen I mRNA expression and aberrant control of apoptotic cell death may contribute to impaired wound healing seen in this diabetic model.     

Hexokinase, glucose-6-phosphate dehydrogenase and antioxidant enzymes in diabetic reticulocytes: effects of insulin and vanadate

Growth factors of the transforming growth factor-beta superfamily are involved in cutaneous wound healing. In this study we analyze the expression of the bone morphogenetic protein-6 (BMP-6) gene, a transforming growth factor-beta related gene, in skin wounds. In normal mouse skin high levels of BMP-6 mRNA and protein are expressed by postmitotic keratinocytes of stratified epidermis until day 6 after birth. BMP-6 expression is strongly reduced in adult epidermis with diminished mitotic activity. After skin injury we found large induction of BMP-6-specific RNA and protein in keratinocytes at the wound edge and keratinocytes of the newly formed epithelium as well as in fibroblast shaped cells in the wound bed. BMP-6-specific RNA was induced within 24 h after injury, whereas significant upregulation of BMP-6 on the protein level was detected only 2-3 d after injury. Protein was confined to outermost suprabasal epidermal layers, whereas BMP-6-specific RNA was distributed throughout all epidermal layers including basal keratinocytes and the leading edge of the migrating keratinocytes. We also detected high levels of BMP-6-specific RNA and protein in chronic human wounds of different etiology. In contrast to the overall distribution pattern of BMP-6-specific RNA, the protein was not detected in keratinocytes directly bordering the wound. In order to test the influence of BMP-6 abundance on the progress of wound healing, we analyzed the wound response of transgenic mice overexpressing BMP-6 in the epidermis. In these mice, reepitheliazation of skin wounds was significantly delayed, suggesting that strict spatial and temporal regulation of BMP-6 expression is necessary not only for formation but also for reestablishment of a fully differentiated epidermis.     

Increase in the expression of biglycan mRNA expression Co-localized closely with that of type I collagen mRNA in the infarct zone after experimentally-induced myocardial infarction in rats

Biglycan, a small dermatan sulphate proteoglycan, has been postulated to interact with other components of the extracellular matrix (ECM), specifically collagens. We hypothesized that biglycan messenger ribonucleic acid (mRNA) is increased in the myocardial infarct zone. Biglycan mRNA expression after acute myocardial infarction (AMI) in rats was determined with the use of Northern blotting and in situ hybridization, and its expression pattern was compared to that of type I collagen mRNA [alpha1(I) collagen]. The left coronary artery was ligated in male Sprague-Dawley rats, and the hearts were excised on days 2 and 7. The Northern blot analysis demonstrated that expression of biglycan mRNA in the infarct on days 2 and 7 were 4.0- and 6.8-fold higher, respectively, compared to the sham-operated hearts. The in situ hybridization revealed intense signals for both biglycan and alpha1(I) collagen mRNA on day 2 in the spindle-shaped mesenchymal cells located between the surviving myocytes in the infarct peripheral zone. On day 7, biglycan mRNA signals were observed in the interior of the infarct around the infarct granulation tissue, a distribution that was essentially the same as that of alpha1(I) collagen. These results demonstrated that the increases in the infarct biglycan mRNA expression produced by mesenchymal cells (presumably myofibroblasts and fibroblasts) was closely co-localized with that of type I collagen mRNA, indicating that biglycan contributes to the infarct healing processes.     

SP-A2 gene expression in human fetal lung airways

In the present study, we characterized surfactant protein (SP)-A messenger RNA (mRNA) in mid-trimester human fetal trachea and bronchi. SP-A protein was localized by immunocytochemistry to scattered epithelial cells in the airway surface epithelium and in submucosal glands of the fetal trachea and bronchi. SP-A mRNA (2.2 kb) was detected by Northern blot analysis in human fetal trachea, as well as in primary and more distal bronchi. The levels of detectable SP-A mRNA were highest in the upper airways and were decreased in smaller bronchi in comparison. SP-A mRNA was barely detectable in the distal fetal lung tissue. In contrast, SP-A mRNA was abundant in cultured explants of distal human fetal lung tissue. SP-A1 and SP-A2 mRNA were detected by primer extension analysis in adult human lung tissue and in cultured human fetal lung explants. Only SP-A2 mRNA was detected in RNA isolated from human fetal trachea and bronchi. SP-A mRNA was localized by in situ hybridization in the fetal trachea and bronchi in scattered cells in the surface epithelium and, most prominently, in submucosal glands. Our results suggest that SP-A2, and not SP-A1, is produced in the human fetal tracheal and bronchial epithelium and in submucosal glands.     

Malingering behavior in private medical practice

OBJECTIVE: This study investigated the influence of the fetal environment on the healing characteristics of adult skin. SUMMARY BACKGROUND DATA: The remarkable ability of the fetus to heal without scarring is poorly understood. The unique qualities of fetal wound healing may be caused by the fetal environment, the fetal tissues, or a combination of both. There are numerous differences between the prenatal and postnatal environments that may play a role in the unique fetal response to injury. METHODS: Full-thickness adult sheep skin was transplanted onto the backs of 60-day-gestation fetal lambs (term, 145 days of gestation). The adult skin grafts were thus perfused by fetal blood and bathed in amniotic fluid. Previous work has demonstrated that, before midgestation, fetal lambs do not reject allogenic skin grafts. Forty days later (100 days of gestation), incisional wounds were made on both the adult skin graft and the adjacent fetal skin. The wounds were harvested 14 days postwounding and analyzed by both light microscopy and immunohistochemical testing using antibodies to collagen types I, III, and VI. RESULTS: The wounds in the adult skin grafts healed with scar formation. This observation contrasts strongly with the scarless healing of the incisional fetal skin wounds. CONCLUSIONS: This study suggests that scarless fetal skin healing properties are intrinsic to fetal skin and are not primarily the result of the fetal environment.     

Collagen synthesis by liver stellate cells is released from its normal feedback regulation by acetaldehyde-induced modification of the carboxyl-terminal propeptide of procollagen

Acetaldehyde stimulates collagen synthesis in stellate cells and forms adducts with procollagen in the liver of alcoholics. To assess the possibility that modification of the carboxyl-terminal propeptide by acetaldehyde affects its capacity to exert a feedback inhibition of collagen synthesis after splitting from procollagen, the propeptide was prepared by gel filtration of the bacterial collagenase digests of procollagen type I (obtained from 10(9) calvaria fibroblasts of newborn rats) and reacted with either 250 mM acetaldehyde and 100 mM CNBH3 or with 170 microM acetaldehyde without reducing agents, to mimick in vivo conditions. The unmodified propeptide produced a concentration-dependent inhibition of collagen synthesis by Ito cells. By contrast, the acetaldehyde-modified propeptide produced a lesser inhibition of procollagen synthesis in the cells, associated with a greater accumulation of collagen in the media. The incubation with 170 microM acetaldehyde and, to a lesser extent, 50 mM ethanol produced collagenase-digestible adducts in stellate cells. Thus, the formation of acetaldehyde adducts with the carboxyl-terminal propeptide of procollagen may account, at least in part, for the stimulatory effect of acetaldehyde on collagen synthesis by stellate cells and may lead to collagen accumulation through a decrease of the normal feedback regulation of collagen synthesis by the propeptide.     

Increased in vivo collagen synthesis and in vitro cell proliferative effect of glycolic acid

BACKGROUND: Glycolic acid treatment is believed to reverse the photoaging process by increasing collagen synthesis in the skin. However, this effect has not been clearly defined even though alpha hydroxy acid products are used extensively. OBJECTIVE: This study aimed to define the primary effect of glycolic acid on collagen synthesis that may be achieved by functional activation or proliferation of fibroblasts. METHODS: Glycolic acid treatment was compared in vivo with lactic acid (hairless mice) and in vitro to malic acid (normal human skin fibroblast culture) with controls. To find the functional activation of fibroblasts, Northern blot assay for type I collagen synthesis with histometric analysis (in vivo) was performed. Cell proliferation assay (MTT) with procollagen type I C-peptide (PICP) enzyme immunoassay and radioisotope ([3H]proline) incorporated collagen production from cultured fibroblasts were determined. RESULTS: The in vivo collagen mRNA expression with histometric analysis revealed greater collagen synthesis by glycolic acid compared with lactic acid and control. In vitro cell proliferative effect of glycolic and greater amount of collagen production showed a steady increase in a dose-dependent manner. CONCLUSION: Both in vivo and in vitro, glycolic acid treatment increased the production of collagen and fibroblast proliferation. These effects may be the mechanism by which glycolic acid reverses the process of photoaging.