Hypoxia has been shown to promote tumor metastasis and lead to therapy resistance. Recent work has demonstrated that hypoxia represses E-cadherin expression, a hallmark of epithelial to mesenchymal transition, which is believed to amplify tumor aggressiveness. The molecular mechanism of E-cadherin repression is unknown, yet lysyl oxidases have been implicated to be involved. Gene expression of lysyl oxidase (LOX) and the related LOX-like 2 (LOXL2) is strongly induced by hypoxia. In addition to the previously demonstrated LOX, we characterize LOXL2 as a direct transcriptional target of HIF-1. We demonstrate that activation of lysyl oxidases is required and sufficient for hypoxic repression of E-cadherin, which mediates cellular transformation and takes effect in cellular invasion assays. Our data support a molecular pathway from hypoxia to cellular transformation. It includes up-regulation of HIF and subsequent transcriptional induction of LOX and LOXL2, which repress E-cadherin and induce epithelial to mesenchymal transition. Lysyl oxidases could be an attractive molecular target for cancers of epithelial origin, in particular because they are partly extracellular.Constant availability of molecular oxygen is crucial for the structure and function of any mammalian cell. Therefore, cellular responses to reduced oxygen tensions (hypoxia) play an important role in development and many aspects of physiological homeostasis. Many important disease processes, including ischemic vascular diseases and cancer, involve reduced tissue oxygenation, and cellular adaptation to this is implicated in disease progression and clinical outcome. The hypoxia-inducible transcription factor (HIF) 2 is a central mechanism responding to low cellular oxygenation and mediates a variety of systemic and local adaptive responses, including the control of red cell production, regulation of angiogenesis, modulation of vascular tone, enhancement of glycolysis, and cellular glucose uptake (for a review, see Refs. 1-3). HIF consists of a heterodimer of ␣-and ␤-subunits, both being basic helix-loop-helix-Per Arnt Sim domain proteins. Whereas HIF␤ is constitutively expressed, HIF␣ subunits are unstable and inversely correlated to the availability of molecular oxygen. At least two oxygen-regulated isoforms of HIF␣ have been identified, HIF-1␣ and HIF-2␣, sharing a high degree of sequence homology and a similar domain structure (4). Regulation of HIF is primarily governed by oxygen-dependent hydroxylation of its HIF␣ subunits, which influences protein stability and transcriptional activity.Growth and behavior of tumor cells is strongly dependent on their microenvironment, where hypoxia is both a stress factor and an important signal (for a review, see Refs. 5 and 6). Dating back to 1927, Otto Warburg had already described that tumor cells have a much increased utilization of the glycolytic pathway (7). Since then, a number of studies have established that indeed HIF is necessary to activate glycolysis in tumor cells in order to maintain energy homeosta...

Prostaglandins (PG) have been described as mediators in spinal nociceptive processing after peripheral inflammation. Enzymes essential for PG biosynthesis, cyclooxygenase isozymes COX-1 and COX-2, have not yet been investigated in the spinal cord. In two studies on rats with adjuvant-induced peripheral inflammation levels of mRNA expression of both COX isoforms were analyzed in the lumbar section of the spinal curd using reverse transcription-polymerase chain reaction (RT-PCR) technique. We could show that mRNA of both COX isoforms is expressed constitutively in the spinal cord with COX-2 as the predominant isoform. Six hours after induction of peripheral inflammation, levels of COX-2 mRNA expression were raised significantly in respect to untreated control rats and returned to baseline within 3 days after induction of inflammation. COX-2 might therefore be regarded as the COX isozyme responsible for spinal PG release in nociceptive processing under a peripheral inflammatory stimulus.l~ey words: Cyclooxygenase-2; Inflammation; Spinal cord; R F-PCR densa of the kidney [8] the testis or the brain, where COX-2 is not only expressed constitutively but also considered to be the dominating COX isoform [9][10][11]. In the central nervous system (CNS) prostaglandins maintain important functions as neuroregulators [12]. It has been reported that PGs are released from the spinal cord by various processes as stimulation of afferent nerves [13], noxious thermal stimulation [14] and by increased potassium levels [15]. Prostaglandins are also known to be involved in transmission of nociceptive information in the spinal cord after peripheral inflammation [16]. Functional evidence thus indicates a role for cyclooxygenases in the spinal cord, expression of the enzyme itself, however, has not yet been investigated. We conducted two studies to determine the spinal presence and distribution of COX isozymes using the rat model of adjuvant-induced inflammation. Tissue samples of hindpaws and the lumbar section of the spinal cord were taken before and after induction of inflammation and examined for levels of COX mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR).

Background-Hypercholesterolemia, a risk factor for cardiovascular disease, is associated with inflammation and hypercoagulability. Both can be mediated by the CD40 system. This study investigated whether the CD40 system is upregulated in patients with moderate hypercholesterolemia and whether it is influenced by therapy with a hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitor. Methods and Results-Fifteen patients with moderate hypercholesterolemia and 15 healthy control subjects were investigated. CD154 and P-selectin were analyzed on platelets and CD40 was analyzed on monocytes before and under therapy with the statin cerivastatin by double-label flow cytometry. Blood concentrations of soluble CD154 and monocyte chemoattractant protein-1 (MCP-1) were evaluated. Our main findings were as follows. Patients with moderate hypercholesterolemia showed a significant increase of CD154 and P-selectin on platelets and CD40 on monocytes compared with healthy subjects. Soluble CD154 showed a nonsignificant trend for higher plasma levels in patients. A positive correlation was found for total or LDL cholesterol and CD154, but not for CD40 on monocytes. The latter was upregulated in vitro by C-reactive protein, which was found to be significantly elevated in patients with moderate hypercholesterolemia. CD154 on platelets proved to be biologically active because it enhanced the release of MCP-1, which was markedly elevated in an in vitro platelet-endothelial cell coculture model and in the serum of patients. Short-term therapy with a HMG-CoA reductase inhibitor significantly downregulated CD40 on monocytes and serum levels of MCP-1. Conclusion-Patients with moderate hypercholesterolemia show upregulation of the CD40 system, which may contribute to the known proinflammatory, proatherogenic, and prothrombotic milieu found in these patients. (Circulation. 2001; 104:2395-2400.)

Cells in various anatomical locations are constantly exposed to mechanical forces from shear, tensile and compressional forces. These forces are significantly exaggerated in a number of pathological conditions arising from various etiologies e.g., hypertension, obstruction and hemodynamic overload. Increasingly persuasive evidence suggests that altered mechanical signals induce local production of soluble factors that interfere with the physiologic properties of tissues and compromise normal functioning of organ systems. Two immediate early gene‐encoded members of the family of the Cyr61/CTGF/Nov proteins referred to as cysteine‐rich protein 61 (Cyr61/CCN1) and connective tissue growth factor (CTGF/CCN2), are highly expressed in several mechanical stress‐related pathologies, which result from either increased externally applied or internally generated forces by the actin cytoskeleton. Both Cyr61 and CTGF are structurally related but functionally distinct multimodular proteins that are expressed in many organs and tissues only during specific developmental or pathological events. In vitro assessment of their biological activities revealed that Cyr61 expression induces a genetic reprogramming of angiogenic, adhesive and structural proteins while CTGF promotes distinctively extracellular matrix accumulation (i.e., type I collagen) which is the principal hallmark of fibrotic diseases. At the molecular level, expression of the Cyr61 and CTGF genes is regulated by alteration of cytoskeletal actin dynamics orchestrated by various components of the signaling machinery, i.e., small Rho GTPases, mitogen‐activated protein kinases, and actin binding proteins. This review discusses the mechanical regulation of the Cyr61 and CTGF in various tissues and cell culture models with a special attention to the cytoskeletally based mechanisms involved in such regulation.

Connective tissue growth factor (CTGF, CCN2) is a secreted matricellular protein, the functions of which depend on the interactions with other molecules in the microcellular environment. As an example of context-dependent activity of CTGF, this review will outline different aspects of CTGF function in relation to angiogenesis. CTGF is barely expressed in normal adult tissue, but is strongly upregulated in fibrotic tissue and is also increased during development, in wound healing, or in certain types of cancer. Accordingly, gene expression of CTGF is tightly regulated. To highlight the complexity of the regulation of CTGF gene expression, we discuss here the mechanisms involved in CTGF regulation by TGFbeta in different cell types, and the mechanisms related to CTGF gene expression in cells exposed to mechanical forces. Finally, we will touch upon novel aspects of epigenetic regulation of CTGF gene expression. (c) 2009 International Union of Biochemistry and Molecular Biology, Inc.

Objective-Atherosclerotic blood vessels overexpress connective tissue growth factor (CTGF) mRNA, but the role of CTGF in atherosclerosis remains controversial. To assess the hypothesis that CTGF is involved in atherosclerotic plaque progression, we investigated CTGF protein expression and distribution in the different types of plaque morphology. Methods and Results-Serial cross-sections of 45 human carotid plaques were immunohistochemically analyzed for the presence of CTGF protein, neovascularization (von Willebrand factor), macrophages (CD68), and T cells (CD3). The lesions were categorized according to American Heart Association (AHA) classification as fibrous (type IV and V) or complicated plaques (type VI). The levels of CTGF were significantly higher in complicated compared with fibrous plaques (Pϭ0.002). CTGF accumulated particularly in the rupture-prone plaque shoulder and in the areas of neovascularization or infiltration with inflammatory cells. Macrophage-like cells stained positive for CTGF protein in plaques. Subsequent in vitro studies showed that although monocyte-derived macrophages do not produce CTGF on stimulation with transforming growth factor-␤, lipopolysaccharide, or thrombin, they take it up from culture medium. Furthermore, CTGF induces mononuclear cell chemotaxis in a dose-dependent manner. Key Words: connective tissue growth factor Ⅲ atherosclerosis Ⅲ plaque development Ⅲ chemotaxis A therosclerosis is a chronic multifactorial disease characterized by the accumulation of lipids, fibrous tissue, and inflammatory cells in the large arteries. Whereas the earliest type of lesion consists mainly of lipid-laden foam cells and some T cells, the feature of advanced lesions is the accumulation of lipid-rich necrotic debris, encapsulated by a fibrous cap consisting of extracellular matrix produced by smooth muscle cells (SMCs). In the process of plaque development, complex cellular interactions between cells of the vessel wall and the immune system result in thinning of the fibrous cap, growing lipid core, increased inflammatory activity, and neovascularization. These processes lead to plaque instability and may result in plaque rupture, which is a common pathogenetic feature in a majority of acute manifestations of atherosclerosis, such as acute coronary syndrome and stroke. 1 Connective tissue growth factor (CTGF), a potent angiogenic, chemotactic, and extracellular matrix-inducing growth factor, is produced by a wide variety of cells, including endothelial cells (ECs), SMCs, and fibroblasts. At low levels, CTGF supports wound healing by connective tissue formation after tissue injury and plays a role in angiogenesis and skeletal development. 2 However, overexpression of CTGF gene was implicated in progression of many chronic inflammatory-fibroproliferative disorders, such as glomerulosclerosis, pulmonary fibrosis, and cirrhosis. 3 As reported by Oemar et al, 4 CTGF mRNA is expressed at very high levels in atherosclerotic but not in normal human blood vessels. CTGF-producing cells in plaques...

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1 It was supposed that inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase (statins) might inhibit the expression of the ®brosis-related factor CTGF (connective tissue growth factor) by interfering with the isoprenylation of Rho proteins. 2 The human renal ®broblast cell line TK173 was used as an in vitro model system to study the statin-mediated modulation of the structure of the actin cytoskeleton and of the expression of CTGF mRNA. 3 Incubation of the cells with simvastatin or lovastatin time-dependently and reversibly changed cell morphology and the actin cytoskeleton with maximal e ects observed after about 18 h. 4 Within the same time period, statins reduced the basal expression of CTGF and interfered with CTGF induction by lysophosphatidic acid (LPA) or transforming growth factor beta. Simvastatin and lovastatin proved to be much more potent than pravastatin (IC 50 1 ± 3 mM compared to 500 mM). 5 The inhibition of CTGF expression was prevented when the cells were incubated with mevalonate or geranylgeranylpyrophosphate (GGPP) but not by farnesylpyrophosphate (FPP). Speci®c inhibition of geranylgeranyltransferase-I by GTI-286 inhibited LPA-mediated CTGF expression whereas an inhibitor of farnesyltransferases FTI-276 was ine ective. 6 Simvastatin reduced the binding of the small GTPase RhoA to cellular membranes. The e ect was prevented by mevalonate and GGPP, but not FPP. 7 These data are in agreement with the hypothesis that interference of statins with the expression of CTGF mRNA is primarily due to interference with the isoprenylation of RhoA, in line with previous studies, which have shown that RhoA is an essential mediator of CTGF induction. 8 The direct interference of statins with the synthesis of CTGF, a protein functionally related to the development of ®brosis, may thus be a novel mechanism underlying the bene®cial e ects of statins observed in renal diseases.