Loeys-Dietz syndrome (LDS) is a connective tissue disorder that is characterized by a high risk for aneurysmand dissection throughout the arterial tree and phenotypically resembles Marfan syndrome. LDS is caused by heterozygous missense mutations in either TGF-β receptor gene (TGFBR1 or TGFBR2), which are predicted to result in diminished TGF-β signaling; however, aortic surgical samples from patients show evidence of paradoxically increased TGF-β signaling. We generated 2 knockin mouse strains with LDS mutations in either Tgfbr1 or Tgfbr2 and a transgenic mouse overexpressing mutant Tgfbr2. Knockin and transgenic mice, but not haploinsufficient animals, recapitulated the LDS phenotype. While heterozygous mutant cells had diminished signaling in response to exogenous TGF-β in vitro, they maintained normal levels of Smad2 phosphorylation under steady-state culture conditions, suggesting a chronic compensation. Analysis of TGF-β signaling in the aortic wall in vivo revealed progressive upregulation of Smad2 phosphorylation and TGF-β target gene output, which paralleled worsening of aneurysm pathology and coincided with upregulation of TGF-β1 ligand expression. Importantly, suppression of Smad2 phosphorylation and TGF-β1 expression correlated with the therapeutic efficacy of the angiotensin II type 1 receptor antagonist losartan. Together, these data suggest that increased TGF-β signaling contributes to postnatal aneurysm progression in LDS.

Background-Marfan syndrome (MFS) is caused by mutations in the fibrillin-1 gene and dysregulation of transforming growth factor-␤ (TGF-␤). Recent evidence suggests that losartan, an angiotensin II type 1 blocker that blunts TGF-␤ activation, may be an effective treatment for MFS. We hypothesized that dysregulation of TGF-␤ might be mirrored in circulating TGF-␤ concentrations. Methods and Results-Serum obtained from MFS mutant mice (Fbn1C1039G/ϩ ) treated with losartan was analyzed for circulating TGF-␤1 concentrations and compared with those from placebo-treated and wild-type mice. Aortic root size was measured by echocardiography. Data were validated in patients with MFS and healthy individuals. In mice, circulating total TGF-␤1 concentrations increased with age and were elevated in older untreated Fbn1 C1039G/ϩ mice compared with wild-type mice (Pϭ0.01; nϭ16; meanϮSEM, 115Ϯ8 ng/mL versus nϭ17; meanϮSEM, 92Ϯ4 ng/mL). Losartan-treated Fbn1 C1039G/ϩ mice had lower total TGF-␤1 concentrations compared with age-matched Fbn1

We sought to identify fibroblast growth factor receptor 2 (FGFR2) kinase domain mutations that confer resistance to the pan-FGFR inhibitor, dovitinib, and explore the mechanism of action of the drug-resistant mutations. We cultured BaF3 cells overexpressing FGFR2 in high concentrations of dovitinib and identified 14 dovitinib-resistant mutations, including the N550K mutation observed in 25% of FGFR2(mutant) endometrial cancers (ECs). Structural and biochemical in vitro kinase analyses, together with BaF3 proliferation assays, showed that the resistance mutations elevate the intrinsic kinase activity of FGFR2. BaF3 lines were used to assess the ability of each mutation to confer cross-resistance to PD173074 and ponatinib. Unlike PD173074, ponatinib effectively inhibited all the dovitinib-resistant FGFR2 mutants except the V565I gatekeeper mutation, suggesting ponatinib but not dovitinib targets the active conformation of FGFR2 kinase. EC cell lines expressing wild-type FGFR2 were relatively resistant to all inhibitors, whereas EC cell lines expressing mutated FGFR2 showed differential sensitivity. Within the FGFR2(mutant) cell lines, three of seven showed marked resistance to PD173074 and relative resistance to dovitinib and ponatinib. This suggests that alternative mechanisms distinct from kinase domain mutations are responsible for intrinsic resistance in these three EC lines. Finally, overexpression of FGFR2(N550K) in JHUEM-2 cells (FGFR2(C383R)) conferred resistance (about five-fold) to PD173074, providing independent data that FGFR2(N550K) can be associated with drug resistance. Biochemical in vitro kinase analyses also show that ponatinib is more effective than dovitinib at inhibiting FGFR2(N550K). We propose that tumors harboring mutationally activated FGFRs should be treated with FGFR inhibitors that specifically bind the active kinase.

Excessive transforming growth factor-␤ (TGF-␤) signaling characterizes the progression of aortic aneurysm in mouse models of Marfan syndrome, a systemic disorder of the connective tissue that is caused by mutations in the gene encoding the extracellular matrix protein fibrillin-1. Fibrillin-1 mutations are believed to promote abnormal Smad2/3 signaling by impairing the sequestration of latent TGF-␤ complexes into the extracellular matrix. Here we report that promiscuous Smad2/3 signaling is the cell-autonomous phenotype of primary cultures of vascular smooth muscle cells (VSMC) explanted from the thoracic aortas of Fbn1 mutant mice with either neonatal onset or progressively severe aortic aneurysm. This cellular phenotype was characterized in VSMC isolated from Fbn1-null (mgN/mgN) mice, which recapitulate the most severe form of Marfan syndrome. We found that loss of fibrillin-1 deposition promotes the production of intracellular reactive oxygen species and abnormal accumulation of phosphorylated TGF-␤-activated kinase 1 and p38 MAPK, in addition to increasing the levels of endogenous phospho-Smad2. We showed that improper Smad2/3 signaling in Fbn1-null VSMC is in part stimulated by phospho-p38 MAPK, which is in turn activated in response to signals other than those mediated by the kinase activity of the ALK5 receptor. Consistent with these cell culture data, in vivo analyses documented that phospho-p38 MAPK accumulates earlier than phospho-Smad2 in the aortic wall of mgN/mgN mice and that systemic inhibition of phospho-p38 MAPK activity lowers the levels of phospho-Smad2 in this tissue. Collectively, these findings indicate that improper activation of p38 MAPK is a precursor of constitutive Smad2/3 signaling in the aortic wall of a mouse model of neonatal lethal Marfan syndrome.

An increase in left ventricular collagen (cardiac fibrosis) is a detrimental process that adversely affects heart function. Strong evidence implicates the infiltration of inflammatory cells as a critical part of the process resulting in cardiac fibrosis. Inflammatory cells are capable of releasing arachidonic acid, which may be further metabolized by cyclooxygenase, lipoxygenase, and cytochrome P450 monooxygenase enzymes to biologically active products, including PGs, leukotrienes, epoxyeicosatrienoic acids, and hydroxyeicosatetraenoic acids. Some of these products have profibrotic properties and may represent a pathway by which inflammatory cells initiate and mediate the development of cardiac fibrosis. In this study, we critically review the current literature on the potential link between this pathway and cardiac fibrosis.

Cyclooxygenase and lipoxygenase metabolism of arachidonic acid produces compounds important in cardiovascular control. Further, arachidonic acid can be metabolised by cytochrome p450 to produce epoxyeicosatrienoic acids (EETs). These derivatives are inactivated by soluble epoxide hydrolase (sEH). The potential role of these EETs in hypertension and cardiac remodelling has been determined using the selective sEH inhibitor, N-adamantyl-N'-dodecylurea (ADU), in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Experiments were performed on male Wistar rats following uninephrectomy alone (UNX rats) or uninephrectomy with administration of DOCA (25 mg every fourth day subcutaneously) and 1% NaCl in drinking water (DOCA-salt rats). ADU (10 mg/kg/d subcutaneously) was administered for 2 wk starting 2 wk after surgery. Cardiovascular structure and function were determined using organ wet weights, histological analysis of collagen and inflammation, isolated heart and thoracic aortic ring preparations, and electrophysiological measurements. DOCA-salt hypertensive rats developed hypertension, hypertrophy, perivascular and interstitial fibrosis, endothelial dysfunction, and prolongation of the cardiac action potential duration within 4 wk. Administration of ADU prevented the further increase in systolic blood pressure and left-ventricular wet weight and normalized endothelial function. ADU treatment did not change inflammatory cell infiltration, collagen deposition, or cardiac action potential duration. EETs may be involved in the development of hypertension and endothelial dysfunction in DOCA-salt rats, but not in excessive collagen deposition or electrophysiological abnormalities.

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The dystrophin-deficient (mdx) mouse remains the most commonly used model for Duchenne muscular dystrophy (DMD). Mdx mice show a predominantly covert cardiomyopathy, the hallmark of which is fibrosis. We compared mdx and normal mice at six ages (3, 6, 9, 12, 15, and 18 months) using in vivo assessment of cardiac function, selective collagen staining, and measures of TGF-β mRNA, Evans blue dye infiltration, macrophage infiltration, and aortic wall thickness. Clear temporal progression was demonstrated, including early fragility of cardiomyocyte membranes, which has an unrelated impact on cardiac function but is associated with macrophage infiltration and fibrosis. Aortic wall thickness is less in older mdx mice. Mdx mice display impaired responses to inotropic challenge from a young age; this is indicative of altered adrenoreceptor function. We draw attention to the paradox of ongoing fibrosis in mdx hearts without a strong molecular signature (in the form of TGF-β mRNA expression).