Overproduction Of Extracellular Matrix Proteins Research Articles

Natural Products in Liver Fibrosis Management: A Five-year Review.

Liver fibrosis, characterized by the overproduction of extracellular matrix proteins within liver tissue, poses a rising global health concern. However, no approved antifibrotic drugs are currently available, highlighting the critical need for understanding the molecular mechanisms of liver fibrosis. This knowledge could not only aid in developing therapies but also enable early intervention, enhance disease prediction, and improve our understanding of the interaction between various underlying conditions and the liver. Notably, natural products used in traditional medicine systems worldwide and demonstrating diverse biochemical and pharmacological activities are increasingly recognized for their potential in treating liver fibrosis. This review aims to comprehensively understand liver fibrosis, emphasizing the molecular mechanisms and advancements in exploring natural products' antifibrotic potential over the past five years. It also acknowledges the challenges in their development and seeks to underscore their potency in enhancing patient prognosis and reducing the global burden of liver disease.

Activated Alpha 2-Macroglobulin Is a Novel Mediator of Mesangial Cell Profibrotic Signaling in Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is caused by the overproduction of extracellular matrix proteins (ECM) by glomerular mesangial cells (MCs). We previously showed that high glucose (HG) induces cell surface translocation of GRP78 (csGRP78), mediating PI3K/Akt activation and downstream ECM production. Activated alpha 2-macroglobulin (α2M*) is a ligand known to initiate this signaling cascade. Importantly, increased α2M was observed in diabetic patients’ serum, saliva, and glomeruli. Primary MCs were used to assess HG responses. The role of α2M* was assessed using siRNA, a neutralizing antibody and inhibitory peptide. Kidneys from type 1 diabetic Akita and CD1 mice and human DKD patients were stained for α2M/α2M*. α2M transcript and protein were significantly increased with HG in vitro and in vivo in diabetic kidneys. A similar increase in α2M* was seen in media and kidneys, where it localized to the mesangium. No appreciable α2M* was seen in normal kidneys. Knockdown or neutralization of α2M/α2M* inhibited HG-induced profibrotic signaling (Akt activation) and matrix/cytokine upregulation (collagen IV, fibronectin, CTGF, and TGFβ1). In patients with established DKD, urinary α2M* and TGFβ1 levels were correlated. These data reveal an important role for α2M* in the pathogenesis of DKD and support further investigation as a potential novel therapeutic target.

Inhibitor of MyoD family A increased fibronectin production by mesangial cells

Overproduction of extracellular matrix proteins, including fibronectin by glomerular mesangial cells (MCs) contributes to the development of diabetic nephropathy. Inhibitor of myogenic differentiation family isoform a (I‐mfa) is a multifunctional cytosolic protein, functioning as a transcription modulator or plasma membrane channel protein regulator. However, its renal effects are unknown. The aim of the present study was to determine if I‐mfa regulated fibronectin production by MCs and the mechanisms involved. In cultured human MCs, I‐mfa expression levels were upregulated and downregulated by transiently transfecting I‐mfa expression plasmids and siRNA against I‐mfa, respectively. Western blot analysis was conducted to determine the abundance of fibronectin protein in human MCs with different treatments and in kidney tissues from rats with and without diabetic nephropathy. RT‐PCR analysis was performed to assess mRNA levels of I‐mfa and fibronectin in human MCs with various treatments and in glomeruli from mice with and without diabetic nephropathy, respectively. We found that overexpression of I‐mfa significantly increased fibronectin protein abundance in human MCs. Silencing I‐mfa significantly reduced the expression level of fibronectin mRNA and blunted TGF‐β1‐stimulated production of fibronectin protein. We further found that high glucose, a central pathogenic stimulus in diabetes increased I‐mfa protein content in a time (≥ 48 h) and concentration (≥ 25 mM) dependent manner. Although high glucose exposure increased fibronectin at the protein level, it did not significantly elevate transcript levels of fibronectin in MCs. In agreement with the data from cultured cells, the abundance of I‐mfa protein was significantly greater in the renal cortex of rats with diabetic nephropathy compared to non‐diabetic rats. However, there was no significant difference in the level of glomerular I‐mfa mRNA between the mice with and without diabetic nephropathy. Moreover, hydrogen peroxide significantly increased I‐mfa protein abundance in a dose‐dependent manner (≥ 100 μM) in cultured human MCs. Antioxidants polyethylene glycol‐catalase, ammonium pyrrolidithiocarbamate and N‐acetyl cysteine significantly blunted high glucose‐induced increase of I‐mfa protein abundance. Taken together, our study suggests that an increased level of I‐mfa induced by high glucose/diabetes through the production of reactive oxygen species stimulates fibronectin production by MCs.Support or Funding InformationNIH/NIDDK (1RO1 DK115424); AHA Southwest Affiliate (16GRNT27780043); Harry S. Moss Heart Trust Award

Inhibitor of myogenic differentiation family isoform a, a new positive regulator of fibronectin production by glomerular mesangial cells.

Overproduction of extracellular matrix proteins, including fibronectin by mesangial cells (MCs), contributes to diabetic nephropathy. Inhibitor of myogenic differentiation family isoform a (I-mfa) is a multifunctional cytosolic protein functioning as a transcriptional modulator or plasma channel protein regulator. However, its renal effects are unknown. The present study was conducted to determine whether I-mfa regulated fibronectin production by glomerular MCs. In human MCs, overexpression of I-mfa significantly increased fibronectin abundance. Silencing I-mfa significantly reduced the level of fibronectin mRNA and blunted transforming growth factor-β1-stimulated production of fibronectin. We further found that high glucose increased I-mfa protein content in a time course (≥48 h) and concentration (≥25 mM)-dependent manner. Although high glucose exposure increased I-mfa at the protein level, it did not significantly alter transcripts of I-mfa in MCs. Furthermore, the abundance of I-mfa protein was significantly increased in the renal cortex of rats with diabetic nephropathy. The I-mfa protein level was also elevated in the glomerulus of mice with diabetic kidney disease. However, there was no significant difference in glomerular I-mfa mRNA levels between mice with and without diabetic nephropathy. Moreover, H2O2 significantly increased I-mfa protein abundance in a dose-dependent manner in cultured human MCs. The antioxidants polyethylene glycol-catalase, ammonium pyrrolidithiocarbamate, and N-acetylcysteine significantly blocked the high glucose-induced increase of I-mfa protein. Taken together, our results suggest that I-mfa, increased by high glucose/diabetes through the production of reactive oxygen species, stimulates fibronectin production by MCs.

Golgi Apparatus-Targeted Chondroitin-Modified Nanomicelles Suppress Hepatic Stellate Cell Activation for the Management of Liver Fibrosis.

Liver fibrosis is a serious liver disease associated with high morbidity and mortality. The activation of hepatic stellate cells (HSCs) and the overproduction of extracellular matrix proteins are key features during disease progression. In this work, chondroitin sulfate nanomicelles (CSmicelles) were developed as a delivery system targeting HSCs for the treatment of liver fibrosis. CS-deoxycholic acid conjugates (CS-DOCA) were synthesized via amide bond formation. Next, retinoic acid (RA) and doxorubicin (DOX) were encapsulated into CSmicells to afford a DOX+RA-CSmicelles codelivery system. CSmicelles were selectively taken up in activated HSCs and hepatoma (HepG2) cells other than in normal hepatocytes (LO2), the internalization of which was proven to be mediated by CD44 receptors. Interestingly, DOX+RA-CSmicelles preferentially accumulated in the Golgi apparatus, destroyed the Golgi structure, and ultimately downregulated collagen I production. Following tail-vein injection, DOX+RA-CSmicelles were delivered to the cirrhotic liver and showed synergistic antifibrosis effects in the CCl4-induced fibrotic rat model. Further, immunofluorescence staining of dissected liver tissues revealed CD44-specific delivery of CS derivatives to activated HSCs. Together, our results demonstrate the great potential of CS based carrier systems for the targeted treatment of chronic liver diseases.

Expression of TLR7, TLR9, JAK2, and STAT3 genes in peripheral blood mononuclear cells from patients with systemic sclerosis.

Systemic sclerosis (SSc) is a rare, chronic, multisystem autoimmune disease clinically characterized by progressive fibrosis of the skin and internal organs. The basic mechanism appears to involve endothelial cell injury, overproduction of extracellular matrix proteins, and aberrant immune activation. So far, there have been a few attempts to find genetic biomarkers for monitoring disease activity or for correlation with certain symptoms. In order to reveal reliable biomarkers, we analyzed the expression of four genes representing three important signaling pathways, TLR7, TLR9, and JAK2-STAT3. Using RT-qPCR technique, we analyzed the expression of TLR7, TLR9, JAK2, and STAT3 genes in peripheral blood mononuclear cells of 50 SSc patients and 13 healthy individuals. We detected significant upregulation of TLR7 gene expression in a group of SSc patients compared to non-SSc group. Receiver operating characteristic (ROC) analysis showed that TLR7 expression efficiently discriminates SSc cases from healthy individuals. High TLR7 expression positively correlated with the late form of disease, active SSc, and the presence of digital ulcers. Decreased levels of TLR9 and JAK2 mRNA were found in the patient's cohort in comparison to non-SSc individuals, but showed no correlation with specific clinical outcomes. The expression level of the STAT3 gene did not differ between the analyzed groups. This is the first study on the expression of TLR7, TLR9, and STAT3 genes in SSc patients. Our results show that TLR7, TLR9, and JAK2 genes are potential biomarkers for SSc. The results obtained in this study could contribute to better classification, monitoring, and outcome prediction of patients with SSc based on genetics.

Stimulation of Adenosine A2B Receptor Inhibits Endothelin-1-Induced Cardiac Fibroblast Proliferation and α-Smooth Muscle Actin Synthesis Through the cAMP/Epac/PI3K/Akt-Signaling Pathway.

Background and Purpose: Cardiac fibrosis is characterized by an increase in fibroblast proliferation, overproduction of extracellular matrix proteins, and the formation of myofibroblast that express α-smooth muscle actin (α-SMA). Endothelin-1 (ET-1) is involved in the pathogenesis of cardiac fibrosis. Overstimulation of endothelin receptors induced cell proliferation, collagen synthesis, and α-SMA expression in cardiac fibroblasts. Although adenosine was shown to have cardioprotective effects, the molecular mechanisms by which adenosine A2 receptor inhibit ET-1-induced fibroblast proliferation and α-SMA expression in cardiac fibroblasts are not clearly identified.Experimental Approach: This study aimed at evaluating the mechanisms of cardioprotective effects of adenosine receptor agonist in rat cardiac fibroblast by measurement of cell proliferation, and mRNA and protein levels of α-SMA.Key results: Stimulation of adenosine subtype 2B (A2B) receptor resulted in the inhibition of ET-1-induced fibroblast proliferation, and a reduction of ET-1-induced α-SMA expression that is dependent on cAMP/Epac/PI3K/Akt signaling pathways in cardiac fibroblasts. The data in this study confirm a critical role for Epac signaling on A2B receptor-mediated inhibition of ET-1-induced cardiac fibrosis via PI3K and Akt activation.Conclusion and Implications: This is the first work reporting a novel signaling pathway for the inhibition of ET-1-induced cardiac fibrosis mediated through the A2B receptor. Thus, A2B receptor agonists represent a promising perspective as therapeutic targets for the prevention of cardiac fibrosis.

The multiple biological action potential of melatonin - is melatonin, mitochondria and the ischemic/reperfusion injury relationship essential in the pathogenesis of obstructive nephropathy?

Melatonin is known to have a multiple biological action potential. Melatonin and several of its metabolites have been shown to act as an antioxidant and cytoprotective agent by reducing the oxidative stress by acting as a free radical scavenger [1], and also by activating several antioxidant systems [2]. Melatonin is originally defined as an agent which reduces lipid peroxidation and scavenges reactive oxygen species which usually initiate lipid peroxidation [3]. Furthermore, melatonin has potent antioxidant properties [4]. Melatonin stimulates antioxidant enzymes (e.g. glutathione peroxidase, glutathione reductase, etc.) and suppresses oxidative enzymes (lipoxygenase and nitric oxide synthase). It is believed that melatonin can protect the mitochondria and exert strong anti-apoptotic effects [5]. In a current study, Yildirim et al. showed that melatonin significantly reduces the apoptotic scores occurring after acute unilateral ureteral obstruction in a kidney rat model [6]. However, the authors did not observe a return to normal histological features in the obstructed kidneys. In general, obstructive uropathy leads to tubular damage (atrophy) due to apoptosis induction. Impaired urine flow can cause increased accumulation of reactive oxygen species and environmental changes favoring pro-apoptotic factors within the kidney. A wide range of intrarenal factors can stimulate intense fibrosis. Progressive fibrosis with overproduction of extracellular matrix proteins (e.g. collagen, fibronectin, etc.) is a key process in kidney function deterioration. These proteins are accumulated in the tubulointerstitium leading to further renal impairment. Urethral obstruction affects proper renal blood flow and glomerular filtration rate due to the increased pressure in the Bowman's capsules of the nephrons. In consequence, this leads to the overactivation of the renin-angiotensin system. Angiotensin II induces transforming growth factor-beta, which causes the overproduction of extracellular matrix proteins. Lipid peroxidation induced by free oxygen radicals leads to cell membrane damage via the degradation of polyunsaturated fatty acids of membranes and the loss of its integrity and cell lysis [7]. This fact may partly explain the lack of return to normal histological features in the obstructed kidneys presented in the Yildirim et al. study. Cell death has been divided into two types: programmed cell death (apoptosis) and accidental cell death (necrosis). Mitochondria are unique cell organelles which present with highly dynamic activity, in which they are constantly elongating and dividing to form a network. The dynamic nature of the mitochondrial networks is due to two opposing processes, mitochondrial fission and fusion, that operate concurrently. The fission and fusion processes are crucial for maintaining the mitochondrial function [8, 9]. Previous studies have shown that the mitochondria play a crucial role in the cell death process. The mitochondria release several proteins into the cytosol in response to the cell death which signal through the mitochondrial membrane permeabilization, triggering the activation of several cell death pathways. Mitochondria act as a node where cell death pathways are integrated and propagated, resulting in the release of pro-apoptotic factors from the mitochondria which induce massive activation of caspases and in turn activate a proteolitic cascade leading to cellular demise. Different factors which can activate the mitochondrial-dependent pathway of cell death (such a free oxygen radicals, DNA injury and gamma-radiation) are described [10]. Mitochondrial dysfunction is implicated in the etiology of various diseases (e.g neurodegenerative diseases, diabetes, cardiovascular disease, skeletal muscle disorders, etc.) [11]. Melatonin is a highly lipophilic substance that can cross cell membranes to easily reach subcellular compartments, including the mitochondria to where it seems to accumulate in high concentrations. Moreover, melatonin can interact with lipid bilayers and stabilize mitochondrial inner membranes [12]. As mentioned by Yildirim et al. in a current study, ischemia/reperfusion injuries in the kidneys are well known in the surgical era, but ischemia/reperfusion injury in obstructive nephropathy has not yet been extensively studied. It is well known, that the relationship between melatonin, mitochondria and ischemia/reperfusion is a common problem encountered in the clinic (e.g. liver surgery, liver transplantation, etc.). The pathogenesis of ischemia/reperfusion injury is multifactorial and includes the overproduction of free oxygen radicals. For example, the liver ischemia/reperfusion leads to the impairment of the hepatic mitochondrial function and energy metabolism. On the other hand, melatonin administration prior to the injury protects against mitochondrial dysfunction in the liver induced by the ischemia/reperfusion in the rat model [13]. In conclusion, further research is needed to clarify the relationship between the mitochondria, ischemia/reperfusion injury, melatonin and renal function. In the case of urine flow obstruction, this relationship will aid in better understanding the complex pathogenesis of obstructive nephropathy.

Serum levels of anti-Fcγ receptor IIB/C antibodies are increased in patients with systemic sclerosis.

Systemic sclerosis (SSc) is a systemic autoimmune disorder that results in fibrosis of the skin and multiple internal organs. Although the precise mechanism is unknown, it appears to result from the overproduction of extracellular matrix proteins and aberrant immune activations. Receptors for the Fc region of immunoglobulin (Ig)G (FcγR) are members of the Ig superfamily that modulate both activation and inhibition of immune responses. FcγRIIB is the sole inhibitory member, which has an intrinsic cytoplasmic immunoreceptor tyrosine-based inhibitory motif. The present study was undertaken to investigate the circulating concentrations of anti-FcγRIIB/C antibodies (Ab) in patients with SSc. Serum levels of anti-FcγRIIB/C Ab were significantly increased in patients with SSc compared to those in controls and in patients with localized scleroderma. Serum levels of anti-FcγRIIB/C Ab in patients with limited cutaneous SSc were similar to those in patients with diffuse cutaneous SSc. Among SSc patients, serum levels of anti-FcγRIIB/C Ab were increased in those with nail-fold bleeding and decreased in those with diffuse pigmentation and calcinosis. These findings support the notion that increased serum anti-FcγRIIB/C Ab levels are involved in aberrant immune responses in SSc.

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Serum levels of interleukin‐1α in patients with systemic sclerosis

Systemic sclerosis (SSc) is an autoimmune systemic connective tissue disorder characterized by sclerotic change of the skin and multiple internal organs. Although the pathogenesis of this disorder is still unknown, overproduction of extracellular matrix proteins, including collagens and fibronectin, and aberrant immune activation may be involved in the mechanism. Interleukin (IL)-1 is one of the key regulators of inflammatory response. IL-1 is also involved in regulating connective tissue remodeling and cellular differentiation of epithelial and ectodermal cells. There are three major members of the IL-1 family: IL-1α, IL-1β and IL-1 receptor antagonist. IL-1α was first described as a factor derived from keratinocytes that stimulates thymocyte proliferation. IL-1α plays a crucial role in procollagen production by fibroblasts derived from patients with SSc. The present study was undertaken to investigate the serum levels of IL-1α in patients with SSc. Serum samples were obtained from 66 Japanese patients with SSc and 19 healthy controls. Levels of serum IL-1α were measured with a specific enzyme-linked immunoassay kit. Mean serum levels were significantly higher in SSc patients than in those healthy control subjects. Moreover, contracture of phalanges was found at a significantly lower prevalence in SSc patients with elevated serum IL-1α levels than those with normal levels. These results suggest that IL-1α may play a role in the pathogenesis of SSc.

The antioxidant effect of β-caryophyllene protects rat liver from carbon tetrachloride-induced fibrosis by inhibiting hepatic stellate cell activation

Plant-based whole foods provide thousands of bioactive metabolites to the human diet that reduce the risk of developing chronic diseases. β-Caryophyllene (CAR) is a common constituent of the essential oil of numerous plants, vegetables, fruits and medicinal herbs, and has been used as a flavouring agent since the 1930 s. Here, we report the antioxidant activity of CAR, its protective effect on liver fibrosis and its inhibitory capacity on hepatic stellate cell (HSC) activation. CAR was tested for the inhibition of lipid peroxidation and as a free radical scavenger. CAR had higher inhibitory capacity on lipid peroxidation than probucol, α-humulene and α-tocopherol. Also, CAR showed high scavenging activities against hydroxyl radical and superoxide anion. The activity of 5-lipoxygenase, an enzyme that actively participates in fibrogenesis, was significantly inhibited by CAR. Carbon tetrachloride-treated rats received CAR at 2, 20 and 200 mg/kg. CAR significantly improved liver structure, and reduced fibrosis and the expression of Col1a1, Tgfb1 and Timp1 genes. Oxidative stress was used to establish a model of HSC activation with overproduction of extracellular matrix proteins. CAR (1 and 10 μm) increased cell viability and significantly reduced the expression of fibrotic marker genes. CAR, a sesquiterpene present in numerous plants and foods, is as a natural antioxidant that reduces carbon tetrachloride-mediated liver fibrosis and inhibits hepatic cell activation.

Serum levels of soluble CD21 in patients with systemic sclerosis

Systemic sclerosis (SSc) is a systemic disorder that typically results in fibrosis of the skin and multiple internal organ systems. Although the precise mechanism is unknown, overproduction of extracellular matrix proteins, including collagens and fibronectins, and aberrant immune activation might be involved in the pathogenesis. The soluble cluster of differentiation 21 (sCD21) represents the extracellular portion of the CD21 glycoprotein that is released by shedding from the cell surfaces into plasma. sCD21 binds complement fragments and activates monocytes through binding to membrane CD23. The present study was undertaken to investigate the serum levels of sCD21 in patients with SSc. Serum sCD21 levels were reduced with age both in patients with SSc and normal controls. Serum sCD21 levels in patients with SSc were significantly decreased compared to those in control subjects. When we divided patients with SSc into limited cutaneous SSc (lcSSc) and diffuse cutaneous SSc (dcSSc), patients with lcSSc had lower levels of serum sCD21 than those with dcSSc. Moreover, the prevalence of pulmonary fibrosis in the patients with dcSSc inversely correlated with serum sCD21 levels. Our finding may support the notion that B-cell activation is involved in the mechanism for pulmonary fibrosis and skin sclerosis.

CD38-mediated Ca2+ Signaling Contributes to Angiotensin II-induced Activation of Hepatic Stellate Cells: ATTENUATION OF HEPATIC FIBROSIS BY CD38 ABLATION

CD38 is a type II glycoprotein that is responsible for the synthesis and hydrolysis of cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), Ca(2+)-mobilizing second messengers. The activation of hepatic stellate cells (HSCs) is a critical event in hepatic fibrosis because these cells are the main producers of extracellular matrix proteins in the liver. Recent evidence indicates that the renin-angiotensin system plays a major role in liver fibrosis. In this study, we showed that angiotensin II (Ang II) evoked long lasting Ca(2+) rises and induced NAADP or cADPR productions via CD38 in HSCs. Inositol 1,4,5-trisphosphate as well as NAADP-induced initial Ca(2+) transients were prerequisite for the production of cADPR, which was responsible for later sustained Ca(2+) rises in the Ang II-treated HSCs. Ang II-mediated inositol 1,4,5-trisphosphate- and NAADP-stimulated Ca(2+) signals cross-talked in a dependent manner with each other. We also demonstrated that CD38 plays an important role in Ang II-induced proliferation and overproduction of extracellular matrix proteins in HSCs, which were reduced by an antagonistic cADPR analog, 8-bromo-cADPR, or in CD38(-/-) HSCs. Moreover, we presented evidence to implicate CD38 in the bile duct ligation-induced liver fibrogenesis; infiltration of inflammatory cells and expressions of alpha-smooth muscle actin, transforming growth factor-beta1, collagen alphaI(1), and fibronectin were reduced in CD38(-/-) mice compared with those in CD38(+/+) mice. These results demonstrate that CD38-mediated Ca(2+) signals contribute to liver fibrosis via HSCs activation, suggesting that intervention of CD38 activation may help prevent hepatic fibrosis.

Effect of imatinib mesylate (IM) on endothelial cells (EC) functions

Objective Systemic sclerosis (SSc) is systemic autoimmune disease that is characterized by microangiopathy with progressive loss of capillaries, sparse inflammation and an overproduction of extracellular matrix proteins by SSc fibroblasts leading to tissue fibrosis. IM is a tyrosine kinase inhibitor that is widely used for the treatment of CML and GIST tumors and that targets selectively abl and PDGF receptor. We have demonstrated recently, the IM inhibits potently the productions of extracellular matrix by SSc fibroblasts and prevents experimental dermal fibrosis. The aim of the present study was to exclude that the antifibrotic effects of SSc are accompanied by antiangiogenic side-effects, which might augment vascular disease in SSc. Methods and results EC were incubated with IM in concentrations from 0.01 to 1.0 μg/ml IM, which corresponds to physiologically relevant doses. The expression of VEGF was analyzed in microvascular EC after incubation with IM. IM did not alter the expression of mRNA of VEGF as analyzed by real-time PCR. Cell viability was analyzed by caspase3 assays and by annexin V/ propidium iodide staining. IM did not increase the percentage of PI-positive and annexin V–positive cells. Consistently, the activity of caspase3 did not increase upon incubation of EC with IM. IM also did not alter the rate of proliferating cells or metabolic capacity of EC as assessed with the MTT assay neither after 24 h nor after long term incubation for 4 d. Cell migration was analyzed by scratch and migration assay. IM did not reduce the migratory capacity of EC. IM did also not alter the ability of endothelial cells to form tubes upon stimulation with VEGF as analyzed by capillary morphogenesis assay. The mouse model of bleomycin-induced dermal fibrosis was used to assess the effect of IM on markers of endothelial apoptosis in vivo. Consistent with the in vitro data, no differences in the number of apoptotic endothelial cells was observed in vivo with TUNEL assay. Conclusion IM does not inhibit proliferation, migration or tube forming in endothelial cells or induce apoptosis in vitro or in vivo, suggesting that IM has no direct inhibitory effects on endothelial cells.

Role of cytokines and proteases in murine scleroderma.

Scleroderma is a fibrotic condition characterized by immunological abnormalities, vascular injury and increased accumulation of extracellular matrix proteins in the skin. Although the etiology of scleroderma has not been fully elucidated, a growing body of evidence suggests that the overproduction of extracellular matrix proteins by activated fibroblasts results from an imbalance between synthesis and degradation of connective tissues. A number of mediators, cytokines, chemokines and growth factors secreted by inflammatory cells and mesenchymal cells (fibroblasts and myofibroblasts) play an important role in the fibrotic process of scleroderma. In this article, we describe recent advances concerning immunological aspects in the pathogenesis of bleomycin-induced murine scleroderma, laying stress on the involvement of interleukin-13 (IL-13) and plasminogen activator inhibitor-1 (PAI-1).

Group IVA phospholipase A2-mediated production of fibronectin by oxidized LDL in mesangial cells

The deposition of atherogenic lipoproteins such as oxidized low-density lipoprotein (oxLDL) within the mesangium is involved in the overproduction of extracellular matrix proteins, a key event in the progression of glomerular diseases including glomerulosclerosis. To clarify the mechanisms underlying the oxLDL-induced production of extracellular matrix proteins, we examined the possible involvement of group IVA phospholipase A(2) (PLA(2)) using human mesangial cells and group IVA PLA(2)-deficient mouse mesangial cells. oxLDL accelerated the production of fibronectin and collagen (type IV), components of extracellular matrix proteins, with the preceding release of arachidonic acid. Methyl arachidonyl fluorophosphonate (MAFP), known as an inhibitor of group IVA PLA(2), markedly suppressed the oxLDL-induced production of fibronectin as well as the release of arachidonic acid, whereas it did not inhibit the production of collagen. The inhibitory effect of MAFP on the production of fibronectin was reversed by adding arachidonic acid and 12-hydroxyeicosatetraenoic acid. Furthermore, we found that in group IVA PLA(2)-deficient mouse mesangial cells, the production of fibronectin in response to oxLDL was weak as compared with that in wild-type cells. However, the production by oxLDL of collagen was not suppressed in the group IVA PLA(2)-deficient cells. These findings suggest that group IVA PLA(2) is involved in the production of fibronectin in oxLDL-stimulated mesangial cells.

N-Acetyl-Seryl-Aspartyl-Lysyl-Proline Prevents Renal Insufficiency and Mesangial Matrix Expansion in Diabetic db/db Mice

We have previously reported that N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), which is a tetrapeptide hydrolyzed by ACE, inhibits the transforming growth factor-beta (TGF-beta)-induced expression of extracellular matrix proteins via inhibition of the Smad signaling in human mesangial cells. To test in vivo the antifibrotic efficacy of Ac-SDKP, we examined whether long-term Ac-SDKP treatment can prevent renal insufficiency and glomerulosclerosis in diabetic db/db mice. Diabetic db/db mice or nondiabetic db/m mice were treated with Ac-SDKP for 8 weeks using osmotic minipumps. The treatment with Ac-SDKP increased plasma Ac-SDKP concentrations by approximately threefold in both groups but did not affect the blood glucose levels. Histologically, the increased glomerular surface area, mesangial matrix expansion, and overproduction of extracellular matrix proteins in db/db mice were significantly inhibited by Ac-SDKP. Furthermore, Ac-SDKP treatment normalized the increased plasma creatinine value in db/db mice, whereas the albuminuria in Ac-SDKP-treated db/db mice was somewhat decreased as compared with nontreated db/db mice, although the difference was not statistically significant. In addition, the nuclear translocation of Smad3 was inhibited by Ac-SDKP. These results demonstrate that long-term Ac-SDKP treatment ameliorates renal insufficiency and glomerulosclerosis in db/db mice via inhibition of TGF-beta/Smad pathway, suggesting that Ac-SDKP could be useful in the treatment of diabetic nephropathy.