Extracellular Matrix Upregulation Research Articles

Ablation of the gene encoding p66Shc protects mice against AGE-induced glomerulopathy by preventing oxidant-dependent tissue injury and further AGE accumulation

AGEs have been implicated in renal disease associated with ageing, diabetes and other age-related disorders. Reactive oxygen species (ROS) promote formation of AGEs, which cause AGE-receptor-mediated ROS generation with activation of signalling pathways leading to tissue injury and further AGE accumulation. ROS generation is regulated by the Src homology 2 domain-containing transforming protein C1 (Shc1) isoform p66(Shc), whose deletion has been shown to protect from tissue injury induced by ageing, diabetes, hyperlipidaemia and ischaemia-reperfusion by preventing oxidative stress. This study was aimed at assessing the role of p66(Shc) in the modulation of oxidative stress and oxidant-dependent renal injury induced by AGEs. For 10 weeks, male p66 (shc) knockout (KO) and wild-type (WT) mice were injected with 60 microg/day albumin modified or unmodified by N epsilon-(carboxymethyl) lysine (CML). Mice were then killed for the assessment of renal function and structure, as well as systemic and renal tissue oxidative stress. Upon CML injection, KO mice, in contrast to WT mice, showed no or only mild forms of proteinuria, glomerular hypertrophy, mesangial expansion, glomerular sclerosis, renal/glomerular cell apoptosis and extracellular matrix upregulation. Moreover, KO mice had lower circulating and tissue AGEs than WT mice and unchanged plasma isoprostane 8-epi-prostaglandin-F(2alpha) levels, renal/glomerular CML, 4-hydroxy-2-nonenal, AGE receptor and NAD(P)H oxidase 4 (NOX4) content (and expression of the corresponding genes), and nuclear factor kappaB activation (NFkappaB). Mesangial cells from KO mice exposed to CML showed no or slight increase in ROS levels and NFkappaB activation, again at variance with WT cells. These data indicate that p66(Shc) participates in the pathogenesis of AGE-dependent glomerulopathy by mediating AGE-induced tissue injury and further AGE formation through ROS-dependent mechanisms involving NFkappaB activation and upregulation of Nox4 expression and NOX4 production.

Transforming Growth Factor-β Receptor Type I-dependent Fibrogenic Gene Program Is Mediated via Activation of Smad1 and ERK1/2 Pathways

The transforming growth factor (TGF)-beta/Smad3 signaling pathway is considered a central mediator of pathological organ fibrosis; however, contribution of Smad2/3-independent TGF-beta signaling has not been fully explored. The present study utilized previously a described model of scleroderma (SSc) fibrosis based on forced expression of the TGF-betaRI (ALK5) (Pannu, J., Gardner, H., Shearstone, J. R., Smith, E., and Trojanowska, M. (2006) Arthritis Rheum. 54, 3011-3021). This study was aimed at determining the molecular mechanisms underlying the profibrotic program in this model. We demonstrate that the TGF-betaRI-dependent up-regulation of collagen and CCN2 (CTGF) does not involve Smad2/3 activation but is mediated by ALK1/Smad1 and ERK1/2 pathways. The following findings support this conclusion: (i) Smad2 and -3 were not phosphorylated in response to TGF-betaRI, (ii) a TGF-betaRI mutant defective in Smad2/3 activation, ALK5(3A), potently stimulated collagen production, (iii) elevation of TGF-betaRI triggered sustained association of ALK5 with ALK1 and high levels of Smad1 phosphorylation, (iv) blockade of Smad1 via small interfering RNA abrogated collagen and CCN2 up-regulation in this model, (v) elevated TGF-betaRI led to a prolonged activation of ERK1/2, (vi) the pharmacologic inhibitor of ERK1/2 inhibited Smad1 phosphorylation and abrogated profibrotic effects of elevated TGFbeta-RI. Additional experiments demonstrated that a GC-rich response element located -6 to -16 (upstream of the transcription start site) in the CCN2 promoter mediated Smad1-dependent increased promoter activity in this model. This element was shown previously to mediate up-regulation of the CCN2 promoter in SSc fibroblasts. In conclusion, this study defines a novel ALK1/Smad1- and ERK1/2-dependent, Smad3-independent mode of TGF-beta signaling that may operate during chronic stages of fibrosis in SSc.

Purinergic modulation of mesangial extracellular matrix production: Role in diabetic and other glomerular diseases

Extracellular adenosine triphosphate (ATP) (eATP) mediates several biologic activities via purinergic P2 receptors (P2Rs). This study aimed at (1) evaluating the role of the purinergic system in modulating mesangial extracellular matrix (ECM) and transforming growth factor-beta (TGF-beta) production and (2) its contribution to diabetes-induced mesangial ECM accumulation. Rat mesangial cells were grown in normal glucose (5.5 mmol/L) or high glucose (30 mmol/L) containing media and probed with purinergic agonists and antagonists for the assessment of the expression pattern and function of P2Rs; release of ATP and activity of ectoATPases; and changes in ECM and TGF-beta expression. Cells cultured in normal glucose and high glucose expressed similar amounts of functional P2Rs of the P2X(2), P2X(3), P2X(4), P2X(5), P2X(7), P2Y(1), P2Y(2), P2Y(4), and P2Y(6) subtypes. Levels of eATP were higher in high glucose vs. normal glucose, with unchanged ectoATPase activity. The ATP-hydrolyzing enzymes hexokinase or apyrase reduced ECM and TGF-beta production from cells grown in high glucose, but not normal glucose. Under both normal glucose and high glucose conditions, ATP and the P2X(7) agonist benzoylbenzoylATP increased dose-dependently ECM and TGF-beta production, whereas the P2Y agonist uridine triphosphate (UTP) produced the opposite effect. The P2X(7) inhibitor oxidized ATP attenuated the ECM and TGF-beta up-regulation induced by ATP and, to a lesser extent, that caused by high glucose. A TGF-beta neutralizing antibody also prevented ATP-induced ECM up-regulation. These data indicate a role for eATP in regulating ECM production via TGF-beta and suggest that P2XRs and P2YRs differentially modulate this process. An increased ATP release induced by hyperglycemia might contribute to mesangial matrix expansion occurring in diabetes.

Emergence of metastatic hormone-refractory disease in prostate cancer after anti-androgen therapy.

The anti-androgens used in prostate cancer therapy have been designed to interfere with the normal androgen receptor (AR)-mediated processes that ensure prostate cell survival, triggering tumor cells to undergo programmed cell death. While anti-androgens were originally designed to treat advanced disease, they have recently been used to debulk organ-confined prostate tumors, to improve positive margins prior to surgery, and for chemoprevention in patients at high risk for prostate cancer. However, tumors treated with anti-androgens frequently become hormone refractory and acquire a more aggressive phenotype. Progression toward metastatic hormone-refractory disease has often been regarded as the outgrowth of a small number of hormone-independent cells that emerge from a hormone-dependent tumor during anti-androgen treatment by natural selection. While a number of selective advantages have recently been identified, there is also considerable evidence suggesting that the progression toward metastatic hormone-refractory disease is an dynamic process which involves abrogation of programmed cell death as a result of the attenuation of DNA fragmentation and maintenance of mitochondrial membrane potential in tumor cells; the upregulation of stromal-mediated growth factor signaling pathways; and the upregulation of extracellular matrix (ECM) protease expression.

Insulin-like Growth Factor-I Induces Renal Cell Hypertrophy via a Calcineurin-dependent Mechanism

Insulin-like growth factor-I (IGF-I) may play an important role in the development of renal hypertrophy. In this study we determined the effect of IGF-I on cultured mesangial cells (MCs) and examined activation of key signaling pathways. IGF-I induced hypertrophy as determined by an increase in cell size and an increase in protein to DNA ratio and increased accumulation of extracellular matrix (ECM) proteins. IGF-I also activated both Erk1/Erk2 MAPK and phosphatidylinositol 3-kinase (PI3K) in MCs. Inhibition of either MAPK or PI3K, however, had no effect on IGF-I-induced hypertrophy or ECM production. Next, we examined the effect of IGF-I on activation of the calcium-dependent phosphatase calcineurin. IGF-I treatment stimulated calcineurin activity and increased the protein levels of calcineurin and the calcineurin binding protein, calmodulin. Cyclosporin A, an inhibitor of calcineurin, blocked both IGF-I-mediated hypertrophy and up-regulation of ECM. In addition, calcineurin resulted in sustained Akt activation, indicating possible cross-talk with other signaling pathways. Finally, IGF-I treatment resulted in the calcineurindependent nuclear localization of NFATc1. Therefore, IGF-I induces hypertrophy and increases ECM accumulation in MCs. IGF-I-mediated hypertrophy is associated with activation of Erk1/Erk2 MAPK and PI3K but does not require either of these pathways. Instead, IGF-I mediates hypertrophy via a calcineurin-dependent pathway.

TGFβ1 in liver fibrosis: time to change paradigms?

TGFβ1 in liver fibrosis: time to change paradigms?

THE EXPRESSION OF EXTRACELLULAR MATRIX PROTEINS DURING ACCELERATED REJECTION OF CARDIAC ALLOGRAFTS IN SENSITIZED RATS

Extracellular matrix (ECM) proteins exert comitogenic effects on T cell activation in vitro, and may play a role in selective lymphocyte migration and tissue positioning in vivo. However, putative roles of ECM in host immune responses leading to graft rejection remain largely obscure. The present study evaluates intragraft expression of prominent ECM components, laminin (LN) and fibronectin (FN), and analyzes their interactions with host cellular repertoire in a well-defined cardiac allograft model in sensitized rats. (LEW x BN)F1 cardiac allografts are rejected within 24 hr in sensitized LEW rats. Immunohistochemical analysis has revealed that this brisk rejection response was associated with an early increase in the intermyocyte and endothelial deposition of LN and FN at the graft site, with their peak at 6 hr after transplantation. The upregulation of ECM preceded intragraft cellular infiltration, which at 6 hr consisted primarily of monocytes and macrophages. The infiltrating cells localized selectively in FN-rich cardiac interstitial and perivascular areas, as documented by two-color staining and laser scanning confocal microscopy. Marginal and transient increase in ECM expression was noted within control isografts. Next, we tested the role of ECM proteins in systemic lymphocyte recirculation. Specifically sensitized lymph node lymphocytes (LNL) were labelled in vitro with a DNA-binding fluorochrome, H33342, and injected i.v. into secondary engrafted recipients, which were sacrificed 6 hr later. These LNL were detected within host cervical lymph nodes in close association with FN deposits. Moreover, LNL labeled in vitro with a cationic membrane-binding fluorochrome, Di "I", were traced in hosts the lymph nodes of which were analyzed for simultaneous detection of LN and FN. Again, transferred LNL migrated selectively to FN-containing compartments, as shown by laser scanning confocal microscopy. Thus, ECM proteins should be regarded as active and important participants in host immune responses leading to graft rejection. FN may act as an ECM component "signal" for graft-infiltrating cells, and may play a key role in selective homing and specific tissue positioning of recirculating specifically sensitized lymphocytes in host peripheral lymphoid tissues.

THE EXPRESSION OF EXTRACELLULAR MATRIX PROTEINS DURING ACCELERATED REJECTION OF CARDIAC ALLOGRAFTS IN SENSITIZED RATS

Extracellular matrix (ECM) proteins exert comito-genic effects on T cell activation in vitro, and may play a role in selective lymphocyte migration and tissue positioning in vivo. However, putative roles of ECM in host immune responses leading to graft rejection remain largely obscure. The present study evaluates intragraft expression of prominent ECM components, laminin (LN) and fibronectin (FN), and analyzes their interactions with host cellular repertoire in a well-defined cardiac allograft model in sensitized rats. (LEWxBN)F1 cardiac allografts are rejected within 24 hr in sensitized LEW rats. Immunohistochemical analysis has revealed that this brisk rejection response was associated with an early increase in the intermyocyte and endothelial deposition of LN and FN at the graft site, with their peak at 6 hr after transplantation. The upregulation of ECM preceded intra-graft cellular infiltration, which at 6 hr consisted primarily of monocytes and macrophages. The infiltrating cells localized selectively in FN-rich cardiac interstitial and perivascular areas, as documented by two-color staining and laser scanning confocal microscopy. Marginal and transient increase in ECM expression was noted within control isografts. Next, we tested the role of ECM proteins in systemic lymphocyte recirculation. Specifically sensitized lymph node lymphocytes (LNL) were labelled in vitro with a DNA-binding fluorochrome, H33342, and injected i.v. into secondary engrafted recipients, which were sacrificed 6 hr later. These LNL were detected within host cervical lymph nodes in close association with FN deposits. Moreover, LNL labeled in vitro with a cationic membrane-binding fluorochrome, Di “I”, were traced in hosts the lymph nodes of which were analyzed for simultaneous detection of LN and FN. Again, transferred LNL migrated selectively to FN-containing compartments, as shown by laser scanning confocal microscopy. Thus, ECM proteins should be regarded as active and important participants in host immune responses leading to graft rejection. FN may act as an ECM component “signal” for graft-infiltrating cells, and may play a key role in selective homing and specific tissue positioning of recirculating specifically sensitized lymphocytes in host peripheral lymphoid tissues.