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.
Phosphorylation-dependent modulation of the vanilloid receptor TRPV1 is one of the key mechanisms mediating the hyperalgesic effects of inflammatory mediators, such as prostaglandin E 2 (PGE 2 ). However, little is known about the molecular organization of the TRPV1 phosphorylationcomplexandspecificallyaboutscaffoldingproteinsthatpositiontheproteinkinaseA(PKA)holoenzymeproximaltoTRPV1foreffective and selective regulation of the receptor. Here, we demonstrate the critical role of the A-kinase anchoring protein AKAP150 in PKA-dependent modulation of TRPV1 function in adult mouse dorsal root ganglion (DRG) neurons. We found that AKAP150 is expressed in ϳ80% of TRPV1-positive DRG neurons and is coimmunoprecipitated with the capsaicin receptor. In functional studies, PKA stimulation with forskolin markedly reduced desensitization of TRPV1. This effect was blocked by the PKA selective inhibitors KT5720 [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9, 12-epoxy-1H-diindolo[1,2,3-fg:3Ј,2Ј,1Ј-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylicacid hexyl ester] and H89 (N-[2-( p-bromo-cinnamylamino)-ethyl]-5-isoquinoline-sulfon-amide 2HCl), as well as by the AKAP inhibitory peptide Ht31. Similarly, PGE 2 decreased TRPV1 desensitization in a manner sensitive to the PKA inhibitor KT5720. Both the forskolin and PGE 2 effects were strongly impaired in DRG neurons from knock-in mice that express a mutant AKAP150 lacking the PKA-binding domain (⌬36 mice). Protein kinase C-dependent sensitization of TRPV1 remained intact in ⌬36 mice. The PGE 2 /PKA signaling defect in DRG neurons from ⌬36 mice was rescued by overexpressing the full-length human ortholog of AKAP150 in these cells. In behavioral testing, PGE 2 -induced thermal hyperalgesia was significantly diminished in ⌬36 mice. Together, these data suggest that PKA anchoring by AKAP150 is essential for the enhancement of TRPV1 function by activation of the PGE 2 /PKA signaling pathway.
The SMAD-mediated induction of connective tissue growth factor (CTGF), a fibroproliferative cytokine, by transforming growth factor (TGF)beta is required for the development of sustained fibrosis in humans. Here, we show that in fibroblasts, activation of the Ras/MEK/ERK pathway is required for the SMAD-mediated induction of CTGF by TGFbeta2. We then show that activation of protein kinase A (PKA) in fibroblasts is able to block Ras/MEK/ERK signaling and abolish the fibrotic response. Previously, we found that prostacyclin agonists were able to prevent the induction of CTGF in fibroblasts, and in patients with the fibrotic disease scleroderma. Here, we confirm the in vitro and in vivo antifibrotic effects of prostacyclin derivatives and show that these effects are due to PKA-dependent inhibition of the Ras/MEK/ERK pathway. Ras/MEK/ERK does not directly affect SMAD signaling. The coordinate and varied biological responses to TGFbeta are in part due to the interactions of signaling pathways within target cells. Specific inhibition of fibroblast Ras/MEK/ERK signaling might prevent fibrosis while leaving other physiological effects of TGFbeta unaltered.
Purpose The 2012 Surviving Sepsis Campaign pediatric guidelines recommend stress dose hydrocortisone (SDH) in children experiencing catecholamine dependent septic shock with suspected or proven absolute adrenal insufficiency. We evaluated whether SDH therapy in children with catecholamine dependent septic shock correlated with random serum total cortisol (rSTC) levels and was associated with improved outcomes. Methods With IRB approval, we retrospectively enrolled consecutive children (1 month to 18 years old) admitted to the pediatric intensive care unit (PICU) from January 1, 2013 to December 31, 2013 with catecholamine dependent septic shock who had rSTC levels measured prior to potential SDH therapy. The cohort was dichotomized to rSTC < 18 mcg/dL and ≥ 18 mcg/dL. Associations of SDH with outcomes: PICU mortality, PICU and hospital length of stay (LOS), ventilator free days (VFD) and vasopressor free days (VPFD) were examined. Results Seventy children with catecholamine dependent septic shock and measured rSTC levels were eligible (16% PICU mortality). While 43% (30/70) had rSTC < 18 mcg/dL, 60% (42/70) received SDH. Children with rSTC < 18 mcg/dL had lower severity of illness and lower vasopressor-inotrope scores than those with rSTC ≥ 18 mcg/dL (all p < 0.05). Children with SDH had higher severity of illness and PICU mortality than those without SDH (all p<0.05). Mean rSTC levels were similar in children with and without SDH (21.1 vs. 18.7 mcg/dL, p=0.69). In children with rSTC < 18 mcg/dL, SDH was associated with greater PICU and hospital LOS, and fewer VFD (all p<0.05). In children with rSTC > 18 mcg/dL, SDH was associated with greater PICU mortality, and fewer VFD and VPFD (all p<0.05). Conclusions SDH therapy in children with catecholamine dependent septic shock correlated more with severity of illness than rSTC levels and was associated with worse outcomes, irrespective of rSTC levels.
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