Growth differentiation factor 11 is a circulating factor that reverses age-related cardiac hypertrophy.

The most common form of heart failure occurs with normal systolic function and often involves cardiac hypertrophy in the elderly. To clarify the biological mechanisms that drive cardiac hypertrophy in aging, we tested the influence of circulating factors using heterochronic parabiosis, a surgical technique in which joining of animals of different ages leads to a shared circulation. After 4 weeks of exposure to the circulation of young mice, cardiac hypertrophy in old mice dramatically regressed, accompanied by reduced cardiomyocyte size and molecular remodeling. Reversal of age-related hypertrophy was not attributable to hemodynamic or behavioral effects of parabiosis, implicating a blood-borne factor. Using modified aptamer-based proteomics, we identified the TGF-ß superfamily member GDF11 as a circulating factor in young mice that declines with age. Treatment of old mice to restore GDF11 to youthful levels recapitulated the effects of parabiosis and reversed age-related hypertrophy, revealing a therapeutic opportunity for cardiac aging.

Functional variations modulating PRKCA expression and alternative splicing predispose to multiple sclerosis.

The protein kinase C alpha (PRKCA) gene, encoding a Th17-cell-selective kinase, was repeatedly associated with multiple sclerosis (MS), but the underlying pathogenic mechanism remains unknown. We replicated the association in Italians (409 cases, 723 controls), identifying a protective signal in the PRKCA promoter (P = 0.033), and a risk haplotype in intron 3 (P = 7.7 × 10(-4); meta-analysis with previously published data: P = 4.01 × 10(-8)). Expression experiments demonstrated that the protective signal is associated with alleles conferring higher PRKCA expression levels, well fitting our observation that MS patients have significantly lower PRKCA mRNA levels in blood. The risk haplotype was shown to be driven by a GGTG ins/del polymorphism influencing the heterogeneous nuclear ribonucleoprotein H-dependent inclusion/skipping of a PRKCA alternative exon 3*. Indeed, exon 3* can be present in two different versions in PRKCA mRNAs (out-of-frame 61 bp or in-frame 66 bp long), and is preferentially included in transcripts generated through a premature polyadenylation event. The GGTG insertion downregulates 3* inclusion and shifts splicing towards the 66 bp isoform. Both events reduce the nonsense-mediated mRNA-decay-induced degradation of exon 3*-containing mRNAs. Since we demonstrated that the protein isoform produced through premature polyadenylation aberrantly localizes to the plasma membrane and/or in cytoplasmic clusters, dysregulated PRKCA 3* inclusion may represent an additional mechanism relevant to MS susceptibility.

Mutations in Hedgehog pathway genes in fetal rhabdomyomas.

Ligand-independent, constitutive activation of Hedgehog signalling in mice expressing a mutant, activated SmoM2 allele results in the development of multifocal, highly differentiated tumours that express myogenic markers (including desmin, actin, MyoD and myogenin). The histopathology of these tumours, commonly classified as rhabdomyosarcomas, more closely resembles human fetal rhabdomyoma (FRM), a benign tumour that can be difficult to distinguish from highly differentiated rhabdomyosarcomas. We evaluated the spectrum of Hedgehog (HH) pathway gene mutations in a cohort of human FRM tumours by targeted Illumina sequencing and fluorescence in situ hybridization testing for PTCH1. Our studies identified functionally relevant aberrations at the PTCH1 locus in three of five FRM tumours surveyed, including a PTCH1 frameshift mutation in one tumour and homozygous deletions of PTCH1 in two tumours. These data suggest that activated Hedgehog signalling contributes to the biology of human FRM.

Polymorphisms in the genes coding for iron binding and transporting proteins are associated with disability, severity, and early progression in multiple sclerosis.

BACKGROUND: Iron involvement/imbalance is strongly suspected in multiple sclerosis (MS) etiopathogenesis, but its role is quite debated. Iron deposits encircle the veins in brain MS lesions, increasing local metal concentrations in brain parenchyma as documented by magnetic resonance imaging and histochemical studies. Conversely, systemic iron overload is not always observed. We explored the role of common single nucleotide polymorphisms (SNPs) in the main iron homeostasis genes in MS patients. METHODS: By the pyrosequencing technique, we investigated 414 MS cases [Relapsing-remitting (RR), n=273; Progressive, n=141, of which: Secondary (SP), n=103 and Primary (PP), n=38], and 414 matched healthy controls. Five SNPs in 4 genes were assessed: hemochromatosis (HFE: C282Y, H63D), ferroportin (FPN1: -8CG), hepcidin (HEPC: -582AG), and transferrin (TF: P570S). RESULTS: The FPN1-8GG genotype was overrepresented in the whole MS population (OR=4.38; 95%CI, 1.89-10.1; P<0.0001) and a similar risk was found among patients with progressive forms. Conversely, the HEPC -582GG genotype was overrepresented only in progressive forms (OR=2.53; 95%CI, 1.34-4.78; P=0.006) so that SP and PP versus RR yielded significant outputs (P=0.009). For almost all SNPs, MS disability score (EDSS), severity score (MSSS), as well as progression index (PI) showed a significant increase when comparing homozygotes versus individuals carrying other genotypes: HEPC -582GG (EDSS, 4.24±2.87 vs 2.78±2.1; P=0.003; MSSS, 5.6±3.06 vs 3.79±2.6; P=0.001); FPN1-8GG (PI, 1.11±2.01 vs 0.6±1.31; P=0.01; MSSS, 5.08±2.98 vs 3.85±2.8; P=0.01); HFE 63DD (PI, 1.63±2.6 vs 0.6±0.86; P=0.009). Finally, HEPC -582G-carriers had a significantly higher chance to switch into the progressive form (HR=3.55; 1.83-6.84; log-rank P=0.00006). CONCLUSIONS: Polymorphisms in the genes coding for iron binding and transporting proteins, in the presence of local iron overload, might be responsible for suboptimal iron handling. This might account for the significant variability peculiar to MS phenotypes, particularly affecting MS risk and progression paving the way for personalized pharmacogenetic applications in the clinical practice.

Molecular characterization of three novel splicing mutations causing factor V deficiency and analysis of the F5 gene splicing pattern.

BACKGROUND: Factor V deficiency is a rare autosomal recessive hemorrhagic disorder, associated with bleeding manifestations of variable severity. In the present study, we investigated the molecular basis of factor V deficiency in three patients, and performed a comprehensive analysis of the factor V gene (F5) splicing pattern. DESIGN AND METHODS: Mutational screening was performed by DNA sequencing. Wild-type and mutant F5 mRNA were expressed by transient transfection in COS-1 cells, followed by reverse-transcriptase polymerase chain reaction and sequencing. Real-time reverse-transcriptase polymerase chain reaction was used to evaluate degradation of mRNA carrying premature termination codons. RESULTS: Mutational screening identified three hitherto unknown splicing mutations (IVS8+6T>C, IVS21+1G>A, and IVS24+1_+4delGTAG). Production of mutant transcripts in COS-1 cells demonstrated that both IVS21+1G>A and IVS24+1_+4delGTAG cause the activation of cryptic donor splice sites, whereas IVS8+6T>C causes exon-8 skipping (F5-Delta 8-mRNA). Interestingly, F5-Delta 8-mRNA was also detected in wild-type transfected samples, human liver, platelets, and HepG2 cells, demonstrating that F5 exon-8 skipping takes place physiologically. Since F5-Delta 8-mRNA bears a premature termination codons, we investigated whether this transcript is subjected to nonsense-mediated mRNA decay degradation. The results confirmed the involvement of nonsense-mediated mRNA decay in the degradation of F5 PTC(+) mRNA. Moreover, a comprehensive analysis of the F5 splicing pattern led to the identification of two in-frame splicing variants resulting from skipping of exons 3 and 5-6. CONCLUSIONS: The functional consequences of three splicing mutations leading to FV deficiency were elucidated. Furthermore, we report the identification of three alternatively spliced F5 transcripts.

Coagulation factor V gene analysis in five Indian patients: identification of three novel small deletions.

Congenital factor V (FV) deficiency is a rare coagulopathy associated with moderate to severe bleeding symptoms. A total of 34 mutations, all located in the FV gene (F5), have been described in patients with severe FV deficiency, only eight of them being of Asian descent. Sequencing of F5 in five unrelated Indian patients identified three novel small deletions in exon 13, all present in the homozygous state (g.50936-50937delAA or AG and g.51660delA, both occurring in two different patients, and g.52162delC). Besides widening the knowledge on the mutational spectrum of FV deficiency in Asian populations, these data will also be useful for purposes of prenatal diagnosis.

The Vitamin D Receptor Regulates Tissue Resident Macrophage Response to Injury.

Ligand-dependent actions of the vitamin D receptor (VDR) play a pleiotropic role in the regulation of innate and adaptive immunity. The liganded VDR is required for recruitment of macrophages during the inflammatory phase of cutaneous wound healing. Although the number of macrophages in the granulation tissue 2 days after wounding is markedly reduced in VDR knockout (KO) compared with wild-type mice, VDR ablation does not alter macrophage polarization. Parabiosis studies demonstrate that circulatory chimerism with wild-type mice is unable to rescue the macrophage defect in the wounds of VDR KO mice and reveal that wound macrophages are of local origin, regardless of VDR status. Wound cytokine analyses demonstrated a decrease in macrophage colony-stimulating factor (M-CSF) protein levels in VDR KO mice. Consistent with this, induction of M-CSF gene expression by TGFß and 1,25-dihydroxyvitamin D was impaired in dermal fibroblasts isolated from VDR KO mice. Because M-CSF is important for macrophage self-renewal, studies were performed to evaluate the response of tissue resident macrophages to this cytokine. A decrease in M-CSF induced proliferation and cyclin D1 expression was observed in peritoneal resident macrophages isolated from VDR KO mice, suggesting an intrinsic macrophage abnormality. Consistent with this, wound-healing assays in mice with macrophage-specific VDR ablation demonstrate that a normal wound microenvironment cannot compensate for the absence of the VDR in macrophages and thus confirm a critical role for the macrophage VDR in the inflammatory response to injury.

Loss of insulin signaling in vascular endothelial cells accelerates atherosclerosis in apolipoprotein E null mice.

To determine whether insulin action on endothelial cells promotes or protects against atherosclerosis, we generated apolipoprotein E null mice in which the insulin receptor gene was intact or conditionally deleted in vascular endothelial cells. Insulin sensitivity, glucose tolerance, plasma lipids, and blood pressure were not different between the two groups, but atherosclerotic lesion size was more than 2-fold higher in mice lacking endothelial insulin signaling. Endothelium-dependent vasodilation was impaired and endothelial cell VCAM-1 expression was increased in these animals. Adhesion of mononuclear cells to endothelium in vivo was increased 4-fold compared with controls but reduced to below control values by a VCAM-1-blocking antibody. These results provide definitive evidence that loss of insulin signaling in endothelium, in the absence of competing systemic risk factors, accelerates atherosclerosis. Therefore, improving insulin sensitivity in the endothelium of patients with insulin resistance or type 2 diabetes may prevent cardiovascular complications.