BackgroundConnective tissue growth factor (CTGF) is widely thought to promote the development of fibrosis in collaboration with transforming growth factor (TGF)-β; however, most of the evidence for its involvement comes from correlative and culture-based studies. In this study, the importance of CTGF in tissue fibrosis was directly examined in three murine models of fibrotic disease: a novel model of multiorgan fibrosis induced by repeated intraperitoneal injections of CTGF and TGF-β2; the unilateral ureteral obstruction (UUO) renal fibrosis model; and an intratracheal bleomycin instillation model of pulmonary fibrosis.ResultsIntraperitoneal coadministration of CTGF and TGF-β2 elicited a profound fibrotic response that was inhibited by the human anti-CTGF antibody FG-3019, as indicated by the ability of FG-3019 to ameliorate the histologic signs of fibrosis and reduce the otherwise increased hydroxyproline:proline (Hyp:Pro) ratios by 25% in kidney (P < 0.05), 30% in liver (P < 0.01) and 63% in lung (P < 0.05). Moreover, administration of either cytokine alone failed to elicit a fibrotic response, thus demonstrating that CTGF is both necessary and sufficient to initiate fibrosis in the presence of TGF-β and vice versa. In keeping with this requirement for CTGF function in fibrosis, FG-3019 also reduced the renal Hyp:Pro response up to 20% after UUO (P < 0.05). In bleomycin-injured animals, a similar trend towards a FG-3019 treatment effect was observed (38% reduction in total lung Hyp, P = 0.056). Thus, FG-3019 antibody treatment consistently reduced excessive collagen deposition and the pathologic severity of fibrosis in all models.ConclusionCooperative interactions between CTGF and TGF-β signaling are required to elicit overt tissue fibrosis. This interdependence and the observed anti-fibrotic effects of FG-3019 indicate that anti-CTGF therapy may provide therapeutic benefit in different forms of fibroproliferative disease.
Pancreatic ductal adenocarcinoma (PDA) is characterized by abundant desmoplasia and poor tissue perfusion. These features are proposed to limit the access of therapies to neoplastic cells and blunt treatment efficacy. Indeed, several agents that target the PDA tumor microenvironment promote concomitant chemotherapy delivery and increased antineoplastic response in murine models of PDA. Prior studies could not determine whether chemotherapy delivery or microenvironment modulation per se were the dominant features in treatment response, and such information could guide the optimal translation of these preclinical findings to patients. To distinguish between these possibilities, we used a chemical inhibitor of cytidine deaminase to stabilize and thereby artificially elevate gemcitabine levels in murine PDA tumors without disrupting the tumor microenvironment. Additionally, we used the FG-3019 monoclonal antibody (mAb) that is directed against the pleiotropic matricellular signaling protein connective tissue growth factor (CTGF/CCN2). Inhibition of cytidine deaminase raised the levels of activated gemcitabine within PDA tumors without stimulating neoplastic cell killing or decreasing the growth of tumors, whereas FG-3019 increased PDA cell killing and led to a dramatic tumor response without altering gemcitabine delivery. The response to FG-3019 correlated with the decreased expression of a previously described promoter of PDA chemotherapy resistance, the X-linked inhibitor of apoptosis protein. Therefore, alterations in survival cues following targeting of tumor microenvironmental factors may play an important role in treatment responses in animal models, and by extension in PDA patients.
Hypoxia-inducible transcription factor (HIF) is regulated by two oxygen-dependent events that are catalyzed by the HIF prolyl 4-hydroxylases (HIF-P4Hs) and HIF asparaginyl hydroxylase (FIH). We have purified the three recombinant human HIF-P4Hs to near homogeneity and characterized their catalytic properties and inhibition and those of FIH. The specific activities of the HIF-P4Hs were at least 40-50 mol/mol/min, and they and FIH catalyzed an uncoupled decarboxylation of 2-oxoglutarate in the absence of any peptide substrate. The purified HIF-P4Hs showed considerable activities even without added Fe2+, their apparent Km values for iron being markedly lower than that of FIH. Desferrioxamine and several metals were effective inhibitors of FIH, but surprisingly, ineffective inhibitors of the HIF-P4Hs in vitro, especially of HIF-P4H-2. Desferrioxamine and cobalt were more effective in cultured insect cells synthesizing recombinant HIF-P4H-2, but complete inhibition was not achieved and most of the enzyme was inactivated irreversibly. Cobalt also rapidly inactivated HIF-P4Hs during storage at 4 degrees C. The well-known stabilization of HIF-alpha by cobalt and nickel is thus not due to a simple competitive inhibition of HIF-P4Hs. The effective inhibition of FIH by these metals and zinc probably leads to full transcriptional activity of HIF-alpha even in concentrations that produce no stabilization of HIF-alpha.
These results indicate that blocking CTGF attenuates radiation-induced pulmonary remodeling and can reverse the process after initiation. CTGF has a central role in radiation-induced fibrogenesis, and FG-3019 may benefit patients with radiation-induced pulmonary fibrosis or patients with other forms or origin of chronic fibrotic diseases.
Hypoxia-inducible factor (HIF) prolyl 4-hydroxylases are a family of iron-and 2-oxoglutarate-dependent dioxygenases that negatively regulate the stability of several proteins that have established roles in adaptation to hypoxic or oxidative stress. These proteins include the transcriptional activators HIF-1␣ and HIF-2␣. The ability of the inhibitors of HIF prolyl 4-hydroxylases to stabilize proteins involved in adaptation in neurons and to prevent neuronal injury remains unclear. We reported that structurally diverse low molecular weight or peptide inhibitors of the HIF prolyl 4-hydroxylases stabilize HIF-1␣ and up-regulate HIF-dependent target genes (e.g. enolase, p21 waf1/cip1 , vascular endothelial growth factor, or erythropoietin) in embryonic cortical neurons in vitro or in adult rat brains in vivo. We also showed that structurally diverse HIF prolyl 4-hydroxylase inhibitors prevent oxidative death in vitro and ischemic injury in vivo. Taken together these findings identified low molecular weight and peptide HIF prolyl 4-hydroxylase inhibitors as novel neurological therapeutics for stroke as well as other diseases associated with oxidative stress.Iron maintains a unique role in physiology via its ability to change readily its oxidation state in response to changes in its local environment. A general simplification of its primary function is that it mediates one-electron redox reactions. This chemical property of iron enables it to act as an essential component in several biological activities, including as a cofactor for enzymes such as tyrosine hydroxylase. Oxygen binding to biomolecules such as hemoglobin and myoglobin is also coordinated by iron. Indeed iron deficiency can lead to a host of disorders, including anemia and restless legs syndrome (1).Paradoxically, the biochemical properties that make iron beneficial in many biological processes appear to be a drawback when the balance between its accumulation/sequestration within cellular compartments and its release is disturbed in favor of iron accumulation (2). Indeed, iron overload is associated with several neurological conditions (3-5). For example, the iron content of nigral Lewy bodies is elevated in patients with Parkinson disease (6 -9). Alzheimer disease has also been found to be associated with an increase in the iron content of senile plaques (10 -15). Accumulation of mitochondrial iron has been shown to play a role in Friedrich ataxia (16,17). Similarly, changes in intracellular free iron levels have been observed in cerebral ischemia (18 -20). Direct evidence that disrupted iron homeostasis contributes to injury rather than simply being caused by it has been obtained by treatment with low molecular weight iron chelators or by overexpression of iron storage proteins. Small molecule iron chelators such as deferoxamine mesylate (DFO) 2 inhibit neuronal injury in rodent models of stroke (21), Parkinson disease (22), and multiple sclerosis (23). Moreover, DFO and some other metal chelators such as clioquinol have been shown to slow the progressi...
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