Inhibition of the mitogen activated protein kinase, p38 alpha, prevents proinflammatory cytokine induction by human adherent mononuclear leukocytes in response to lipid loading.

Macrophage infiltration, inflammatory processes and oxidatively modified low density lipoprotein (LDL) are known contributing factors in the formation of the atherosclerotic plaque. To determine whether a direct link might exist between these factors, we examined the effect of oxidized LDL upon proinflammatory cytokine production in adherent human peripheral blood mononuclear leukocytes. Oxidized LDL, as well as a combination of cholesterol and 25-hydroxycholesterol, induced tumor necrosis factor-alpha (TNFalpha) and interleukin-1 beta (IL-1 beta) mRNA as measured by quantitative real time PCR, by a maximum of two- to fourfold following a 24-h incubation. Analysis of cell culture supernatants revealed a concomitant stimulation of TNFalpha and IL-1 beta secreted protein as determined by ELISA. Treatment of human peripheral blood mononuclear leukocytes with oxidized LDL or the combination of cholesterol and 25-hydroxycholesterol caused activation of p38 alpha as determined by the ability of immunoprecipitated p38 to phosphorylate an ATF-2 fusion protein, a surrogate substrate of p38 alpha. VK-19911 (Pyridine, 4-[4-(4-fluorophenyl)-1-(4-piperidinyl)-1H-imidazol-5-yl]-dihydrochloride), a specific inhibitor of p38 alpha, prevented the induction of TNFalpha and IL-1 beta by oxidized LDL in a dose-dependent manner. Activated p38 alpha is known to be involved in the stabilization of cyclooxygenase-2 mRNA in response to stimuli such as lipopolysaccharide; however, in the setting of oxidized LDL-induced p38 alpha activation, COX-2 mRNA levels were not affected. Taken together, the data imply a potential role for p38 alpha activation in lipid-associated inflammatory processes.

Advanced glycosylation end products up-regulate connective tissue growth factor (insulin-like growth factor-binding protein-related protein 2) in human fibroblasts: a potential mechanism for expansion of extracellular matrix in diabetes mellitus.

Expansion of extracellular matrix with fibrosis occurs in many tissues as part of the end-organ complications in diabetes, and advanced glycosylation end products (AGE) are implicated as one causative factor in diabetic tissue fibrosis. Connective tissue growth factor (CTGF), also known as insulin-like growth factor-binding protein-related protein-2 (IGFBP-rP2), is a potent inducer of extracellular matrix synthesis and angiogenesis and is increased in tissues from rodent models of diabetes. The aim of this study was to determine whether CTGF is up-regulated by AGE in vitro and to explore the cellular mechanisms involved. AGE treatment of primary cultures of nonfetal human dermal fibroblasts in confluent monolayer increased CTGF steady state messenger RNA (mRNA) levels in a time- and dose-dependent manner. In contrast, mRNAs for other IGFBP superfamily members, IGFBP-rP1 (mac 25) and IGFBP-3, were not up-regulated by AGE. The effect of the AGE BSA reagent on CTGF mRNA was due to nonenzymatic glycosylation of BSA and, using neutralizing antisera to AGE and to the receptor for AGE, termed RAGE, was seen to be due to late products of nonenzymatic glycosylation and was partly mediated by RAGE. Reactive oxygen species as well as endogenous transforming growth factor-beta1 could not explain the AGE effect on CTGF mRNA. AGE also increased CTGF protein in the conditioned medium and cell-associated CTGF. Thus, AGE up-regulates the profibrotic and proangiogenic protein CTGF (IGFBP-rP2), a finding that may have significance in the development of diabetic complications.

CTGF expression is induced by TGF- beta in cardiac fibroblasts and cardiac myocytes: a potential role in heart fibrosis.

Connective tissue growth factor (CTGF) is a cysteine-rich protein induced by transforming growth factor beta (TGF- beta) in connective tissue cells. CTGF can trigger many of the cellular processes underlying fibrosis, such as cell proliferation, adhesion, migration and the synthesis of extracellular matrix; however, its role in acute and chronic cardiac injury is not fully understood. Here, we show that TGF- beta is a specific inducer of CTGF expression in both cardiac fibroblasts and cardiac myocytes. The activity of a CTGF promoter-based reporter construct correlated with endogenous CTGF expression, suggesting that TGF- beta induces CTGF expression most likely by activating its promoter. Upregulation of CTGF coincided with an increase in fibronectin, collagen type I and plasminogen activator inhibitor-1 production. Forskolin, a stimulator of cyclic AMP, blocked TGF- beta induced CTGF expression and reduced the basal level of CTGF, whereas an inhibitor that blocks the MAP kinase signaling pathway (PD 98059) significantly enhanced TGF- beta induced CTGF expression. Furthermore, we found that both TGF- beta and CTGF mRNAs were significantly elevated in the left ventricles and septa of rat hearts 2-16 weeks following myocardial infarction. This correlated well with concomitant increases in fibronectin, and type I and type III collagen mRNA levels in these animal hearts. Significant upregulation of CTGF was also detected in human heart samples derived from patients diagnosed with cardiac ischemia. Based on these findings, we propose that CTGF is an important mediator of TGF- beta signaling in the heart and abnormal expression of this gene could be used as a diagnostic marker for cardiac fibrosis.

Insulin-like growth factor-binding protein-3 induces fetalization in neonatal rat cardiomyocytes.

We employed cDNA microarrays representing 4000 distinct sequences to profile changes in gene expression in a rodent model of heart disease, namely, progression to heart failure after myocardial infarction. Differential gene expression in the left ventricle was examined at 4-week intervals over a 12-week period after coronary artery ligation in rats. Over this time course, insulin-like growth factor-binding protein-3 (IGFBP-3) was found to have a greater expression than in nondiseased tissues. We then employed quantitative real-time PCR to analyze gene expression in neonatal rat cardiac myocytes that had been treated with recombinantly expressed IGFBP-3 to examine a number of transcriptional responses designed to reflect the heart failure phenotype. The IGFBP-3 protein was shown to induce transcription of atrial natriuretic factor (ANF) and beta-myosin heavy chain (B-MHC). Analysis of conditioned media taken from IGFBP-3-treated cardiac myocyte cultures demonstrated an increase in ANF protein as well as in protein synthesis, as determined by metabolic incorporation of a radiolabeled amino acid. However, transcriptional changes of troponin-1, endothelin-1, or angiotensin-II by IGFBP-3 were not observed.