Overhydroxylation of lysyl residues is the initial step for altered collagen cross-links and fibril architecture in fibrotic skin.
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In fibrotic skin of lipodermatosclerosis a substantial increase of the cross-link hydroxylysylpyridinoline is observed. Hydroxylysylpyridinoline is a typical cross-link of skeletal tissue and is thought to play a major part in the hardening of sclerotic tissue. We investigated whether the increase in hydroxylysylpyridinoline is due to overhydroxylation of lysyl residues in the collagen molecule, which may also be associated with an increase of glycosylated hydroxylysine residues. Furthermore, we determined whether the collagen fibrils in lipodermatosclerosis showed a decrease of the diameter in the tissue as well as in vitro after fibrillogenesis of pepsin-solubilized collagens. Isolated alpha-chains of pepsin solubilized collagen I showed an increase in lysyl hydroxylation (hyl/(hyl + lys)) as compared with normal control [alpha1(I): lipodermatosclerosis 0.18 +/- 0.01; control 0.12 +/- 0.01; alpha2(I): lipodermatosclerosis 0.36 +/- 0.02; control 0. 25 +/- 0.03, p < 0.001]. Furthermore, the content of enzymatic glycosylated hydroxlysine residues increased. This increase is associated with a decrease of fibril diameter of both tissue and fibrils formed in vitro of pepsin-solubilized collagens. In the same pool of collagens an increase in collagen III content was observed as compared with controls (lipodermatosclerosis 14.5% +/- 1.6, control 10.3% +/- 1.6, p < 0.001). Our results showed that the overhydroxylation of lysyl residues, which is required for the generation of hydroxylysylpyridinoline, is not only restricted to the telopeptides but also affects the helical part of the molecule. This process is further associated with an increase of glycosylated hydroxylysyl residues. These changes along with the increase in collagen III content seem to be responsible for the observed alteration in the architecture of collagen fibrils in sclerotic skin. (+info)
Factors produced by activated leukocytes alter renal epithelial cell differentiation.
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The development of tubulointerstitial fibrosis in inflammatory renal diseases has been linked to disease progression to end-stage renal failure. Understanding the interactions of the factors influencing inflammation and activating the fibrotic process, that is, the inflammatory infiltrate and the resident proximal tubular cells, may lead to a determination of the mechanisms that regulate tubulointerstitial fibrosis. We used an in vitro model of human proximal tubule cells that were stimulated with supernatant from activated peripheral blood mononuclear cells (leukocytes) to study the alterations in cellular phenotype, and examined the signaling pathways mediating epithelial-fibroblast like transdifferentiation. Our hypothesis of the proposed sequence of events leading to tubulointerstitial fibrosis is explained. (+info)
Angiotensin-converting enzyme in non-neoplastic kidney diseases.
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BACKGROUND: The angiotensin I-converting enzyme (ACE, CD143, kininase II) plays a critical role in controlling the level of vasoactive peptides such as angiotensins and kinins in the local circulations and tissue interstitium. Because recent work has documented a vessel-, organ-, and species-specific pattern of endothelial ACE expression in the vascular system, we have analyzed whether or not changes of this pattern occur in vessels, tubules, and interstitium of the human kidney that is affected by different non-neoplastic diseases. METHODS: Using a set of well-characterized monoclonal antibodies (mAbs), ACE was assessed on renal tissue of 135 patients by immunohistochemistry, including an additional analysis at the ultrastructural level. A semiquantitative evaluation allowed the estimation and comparison of ACE content in different renal compartments. These data were compared with several clinical findings, diagnosis, therapeutic modalities, and histological features. RESULTS: In contrast to the normal human kidney, where ACE is abundant in the brush border of the proximal tubule but is usually absent in endothelial cells of any vessel type, an endothelial neoexpression of ACE was observed in different diseases. In general, this neoexpression was associated with histological sites of interstitial fibrosis and showed some selectivity for glomerular endothelial cells in diabetes mellitus and chronic arterial hypertension. There was also a loss of epithelial ACE in the proximal tubule in certain pathological conditions, for example, in chronic fibroplastic processes, acute pyelonephritis, and different stages of acute renal failure. CONCLUSIONS: Neoexpression of ACE by renal endothelial cells, as well as changes of the tubular ACE content, is a common finding in diseased human kidneys. As associated with certain tissue sites, clinical and/or morphological features, these changes may be involved in parenchymal remodeling and renal pathophysiology. (+info)
Transglutaminase transcription and antigen translocation in experimental renal scarring.
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It was recently demonstrated that renal tissue transglutaminase (tTg) protein and its catalytic product the epsilon(gamma-glutamyl) lysine protein cross-link are significantly increased in the subtotal (5/6) nephrectomy model (SNx) of renal fibrosis in rats. It was proposed that the enzyme had two important physiologic functions in disease development; one of stabilizing the increased extracellular matrix (ECM) by protein cross-linking, the other in a novel form of tubular cell death. This study, using the same rat SNx model, demonstrates first by Northern blotting that expression of tTg mRNA when compared with controls is increased by day 15 (+70% increase, P < 0.05), then rises steadily, peaking at day 90 (+391%, P < 0.01), and remains elevated at 120 d (+205%, P < 0.05) when compared with controls. In situ hybridization histochemistry demonstrated that the tubular cells were the major site of the additional tTg synthesis. Immunohistochemistry on cryostat sections revealed a sixfold increase (P < 0.001) in ECM-bound tTg antigen at 90-d post-SNx, whereas in situ transglutaminase activity demonstrated by the incorporation of fluorescein cadaverine into cryostat sections indicated a 750% increase (P < 0.001) on day 90 in SNx animals. This increased activity was extracellular and predominantly found in the peritubular region. These results indicate that increased tTg gene transcription by tubular cells underlies the major changes in renal tTg protein reported previously in SNx rats, and that the presence of the epsilon(gamma-glutamyl) lysine cross-links in the extracellular environment is the result of the extracellular action of tTg. These changes may be in response to tubular cell injury during the scarring process and are likely to contribute to the progressive expansion of the ECM in renal fibrosis. (+info)
Connective tissue growth factor (CTGF) is a cysteine-rich peptide synthesized and secreted by fibroblastic cells after activation with transforming growth factor beta (TGF-beta) that acts as a downstream mediator of TGF-beta-induced fibroblast proliferation. We performed in vitro and in vivo studies to determine whether CTGF is also essential for TGF-beta-induced fibroblast collagen synthesis. In vitro studies with normal rat kidney (NRK) fibroblasts demonstrated CTGF potently induces collagen synthesis and transfection with an antisense CTGF gene blocked TGF-beta stimulated collagen synthesis. Moreover, TGF-beta-induced collagen synthesis in both NRK and human foreskin fibroblasts was effectively blocked with specific anti-CTGF antibodies and by suppressing TGF-beta-induced CTGF gene expression by elevating intracellular cAMP levels with either membrane-permeable 8-Br-cAMP or an adenylyl cyclase activator, cholera toxin (CTX). cAMP also inhibited collagen synthesis induced by CTGF itself, in contrast to its previously reported lack of effect on CTGF-induced DNA synthesis. In animal assays, CTX injected intradermally in transgenic mice suppressed TGF-beta activation of a human CTGF promoter/lacZ reporter transgene. Both 8-Br-cAMP and CTX blocked TGF-beta-induced collagen deposition in a wound chamber model of fibrosis in rats. CTX also reduced dermal granulation tissue fibroblast population increases induced by TGF-beta in neonatal mice, but not increases induced by CTGF or TGF-beta combined with CTGF. Our data indicate that CTGF mediates TGF-beta-induced fibroblast collagen synthesis and that in vivo blockade of CTGF synthesis or action reduces TGF-beta-induced granulation tissue formation by inhibiting both collagen synthesis and fibroblast accumulation. (+info)
Activation of pancreatic stellate cells in human and experimental pancreatic fibrosis.
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The mechanisms of pancreatic fibrosis are poorly understood. In the liver, stellate cells play an important role in fibrogenesis. Similar cells have recently been isolated from the pancreas and are termed pancreatic stellate cells. The aim of this study was to determine whether pancreatic stellate cell activation occurs during experimental and human pancreatic fibrosis. Pancreatic fibrosis was induced in rats (n = 24) by infusion of trinitrobenzene sulfonic acid (TNBS) into the pancreatic duct. Surgical specimens were obtained from patients with chronic pancreatitis (n = 6). Pancreatic fibrosis was assessed using the Sirius Red stain and immunohistochemistry for collagen type I. Pancreatic stellate cell activation was assessed by staining for alpha-smooth muscle actin (alphaSMA), desmin, and platelet-derived growth factor receptor type beta (PDGFRbeta). The relationship of fibrosis to stellate cell activation was studied by staining of serial sections for alphaSMA, desmin, PDGFRbeta, and collagen, and by dual-staining for alphaSMA plus either Sirius Red or in situ hybridization for procollagen alpha(1) (I) mRNA. The cellular source of TGFbeta was examined by immunohistochemistry. The histological appearances in the TNBS model resembled those found in human chronic pancreatitis. Areas of pancreatic fibrosis stained positively for Sirius Red and collagen type I. Sirius Red staining was associated with alphaSMA-positive cells. alphaSMA staining colocalized with procollagen alpha(1) (I) mRNA expression. In the rat model, desmin staining was associated with PDGFRbeta in areas of fibrosis. TGFbeta was maximal in acinar cells adjacent to areas of fibrosis and spindle cells within fibrotic bands. Pancreatic stellate cell activation is associated with fibrosis in both human pancreas and in an animal model. These cells appear to play an important role in pancreatic fibrogenesis. (+info)
A rat model of progressive chronic renal failure produced by microembolism.
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We report a new model of chronic progressive renal failure in rats, produced by a single injection of microspheres (20 to 30 micrometer in diameter) into the left renal artery after right nephrectomy. Significant proteinuria appeared after 4 weeks, followed by hypoalbuminemia and hypercholesterolemia, in rats that received approximately 5 x 10(5) microspheres (0.8 mg). Renal function partially recovered by 4 weeks after nephrectomy and injection from postoperative dysfunction, but deteriorated again 12 weeks after operation. In the early stage, histologic examination showed tubules with cuff-like thickening of basement membranes scattered among apparently intact tubules. Many epithelial cells in the atrophic tubuli were immunoreactive for proliferating cell nuclear antigen (PCNA). Dilated tubules became apparent several weeks after development of tubular atrophy, most likely representing distal tubules. Dilated tubuli were mostly negative for the proliferation marker. These results showed similarity to findings in human chronic renal failure and strongly suggested that tubular atrophy and dilation in chronic tubulointerstitial lesions differ in pathogenesis. This new model of renal failure induced by microembolism should be useful for studying the interaction between normal and diseased tissue elements in histologically heterogenous lesions as well as the pathogenesis of interstitial fibrosis in disturbance of microcirculation. (+info)
BACKGROUND: Mounting experimental evidence suggests that estrogen treatment protects against neointima formation in response to vascular injury in vivo. Previous studies have suggested that this process includes the activation and migration of adventitial fibroblasts. The present in vitro study was designed to establish a mechanism whereby estrogen attenuates migration of adventitial fibroblasts. METHDS AND RESULTS: Primary cultures of vascular smooth muscle cells (VSMCs) and adventitial fibroblasts were derived from female Sprague-Dawley rats. Reverse transcriptase-polymerase chain reaction and Western blotting were used to determine that expression of the estrogen receptor (ER) was restricted to early-passage VSMCs. Migration of transduced (retrovirally mediated) fibroblasts was determined by counting the number of blue lacZ-expressing cells attached to Boyden-type chambers preconditioned under defined experimental conditions. Compared with growth medium alone, chambers treated with medium conditioned by VSMCs demonstrated a 2-fold increase in fibroblast migration, suggesting that VSMCs release soluble factor(s) competent to bind the Transwell membrane and promote fibroblast migration. In contrast, treatment of VSMCs with 17beta-estradiol (10(-9) to 10(-7) mol/L) before preconditioning of the chamber induced a dose-dependent inhibition of fibroblast migration. Cotreatment of VSMCs with 17beta-estradiol and the ER antagonist ICI-182780 (10(-7) mol/L) blocked the inhibitory effect of estrogen on fibroblast migration. CONCLUSIONS: These observations suggest a novel mechanism of hormonal vasoprotection by which estrogen directly modulates VSMC expression of factor(s) controlling migration of adventitial fibroblasts via an ER-dependent mechanism. (+info)