Kazuhiro Kobuke

Fibulin-5/DANCE is essential for elastogenesis in vivo.

The elastic fibre system has a principal role in the structure and function of various types of organs that require elasticity, such as large arteries, lung and skin. Although elastic fibres are known to be composed of microfibril proteins (for example, fibrillins and latent transforming growth factor (TGF)-β-binding proteins) and polymerized elastin, the mechanism of their assembly and development is not well understood. Here we report that fibulin-5 (also known as DANCE), a recently discovered integrin ligand, is an essential determinant of elastic fibre organization. fibulin-5-/- mice generated by gene targeting exhibit a severely disorganized elastic fibre system throughout the body. fibulin-5-/- mice survive to adulthood, but have a tortuous aorta with loss of compliance, severe emphysema, and loose skin (cutis laxa). These tissues contain fragmented elastin without an increase of elastase activity, indicating defective development of elastic fibres. Fibulin-5 interacts directly with elastic fibres in vitro, and serves as a ligand for cell surface integrins αvβ3, αvβ5 and α9β1 through its amino-terminal domain. Thus, fibulin-5 may provide anchorage of elastic fibres to cells, thereby acting to stabilize and organize elastic fibres in the skin, lung and vasculature.

DANCE, a novel secreted RGD protein expressed in developing, atherosclerotic, and balloon-injured arteries.

We have identified and characterized mouse, rat, and human cDNAs that encode a novel secreted protein of 448 amino acids named DANCE (developmental arteries andneural crest epidermal growth factor (EGF)-like). DANCE contains six calcium-binding EGF-like domains, one of which includes an RGD motif. Overexpression studies of recombinant DANCE protein document that DANCE is a secreted 66-kDa protein. DANCE and recently described protein S1–5 comprise a new EGF-like protein family. The human DANCE gene was mapped at chromosome 14q32.1. DANCE mRNA is mainly expressed in heart, ovary, and colon in adult human tissues. Expression profile analysis byin situ hybridization revealed prominent DANCE expression in developing arteries. DANCE is also expressed in neural crest cell derivatives, endocardial cushion tissue, and several other mesenchymal tissues. In adult vessels, DANCE expression is largely diminished but is reinduced in balloon-injured vessels and atherosclerotic lesions, notably in intimal vascular smooth muscle cells and endothelial cells that lose their ability to proliferate in late stage of injury. DANCE protein was shown to promote adhesion of endothelial cells through interaction of integrins and the RGD motif of DANCE. DANCE is thus a novel vascular ligand for integrin receptors and may play a role in vascular development and remodeling.

Metastasis-associated protein, S100A4 mediates cardiac fibrosis potentially through the modulation of p53 in cardiac fibroblasts

Metastasis-associated protein, S100A4 is suggested as a marker for fibrosis in several organs. It also modulates DNA binding of p53 and affects its function. However, the functional role of S100A4 in the myocardium has remained unclear. Therefore, we investigated the role of S100A4 and its relationship with p53 in cardiac fibrosis. In Dahl-rat hypertensive heart disease model, S100A4 was upregulated in the hypertrophic myocardium and further activated during transition to heart failure (HF). It was expressed in various cells including fibroblasts. In in vitro cardiac fibroblasts, the knockdown of S100A4 significantly suppressed both cell proliferation and collagen expressions. S100A4 co-localized and interacted with p53 in the nucleus. S100A4 knockdown increased the expression of p53-downstream genes, p21 and mdm2, and concomitant knockdown of p53 recovered cell proliferation and collagen expression. Transverse aortic constriction (TAC) was performed in S100A4 knockout (KO) mice, which showed a similar baseline-phenotype to wild type (WT) mice. Although there was no difference in hypertrophic response, KO mice showed reduced interstitial fibrosis, decreased myofibroblasts, and suppressed expressions of collagens and profibrotic cytokines in the left ventricle. Also, DNA microarray analysis showed that S100A4 knockout in vivo had a significant impact on expressions of p53-associated genes. These findings suggest that S100A4 modulates p53 function in fibroblasts and thereby mediates myocardial interstitial fibrosis through two distinct mechanisms; cell proliferation and collagen expression. Blockade of S100A4 may have therapeutic potential in cardiac hypertrophy and HF by attenuating cardiac fibrosis.

A common disease-associated missense mutation in alpha-sarcoglycan fails to cause muscular dystrophy in mice

Limb-girdle muscular dystrophy type 2D (LGMD2D) is caused by autosomal recessive mutations in the alpha-sarcoglycan gene. An R77C substitution is the most prevalent cause of the disease, leading to disruption of the sarcoglycan-sarcospan complex. To model this common mutation, we generated knock-in mice with an H77C substitution in alpha-sarcoglycan. The floxed neomycin (Neo)-cassette retained at the targeted H77C alpha-sarcoglycan locus caused a loss of alpha-sarcoglycan expression, resulting in muscular dystrophy in homozygotes, whereas Cre-mediated deletion of the floxed Neo-cassette led to recovered H77C alpha-sarcoglycan expression. Contrary to expectations, mice homozygous for the H77C-encoding allele expressed both this mutant alpha-sarcoglycan and the other components of the sarcoglycan-sarcospan complex in striated muscle, and did not develop muscular dystrophy. Accordingly, conditional rescued expression of the H77C protein in striated muscle of the alpha-sarcoglycan-deficient mice prevented the disease. Adding to the case that the behavior of mutant alpha-sarcoglycan is different between humans and mice, mutant human R77C alpha-sarcoglycan restored the expression of the sarcoglycan-sarcospan complex when introduced by adenoviral vector into the skeletal muscle of previously created alpha-sarcoglycan null mice. These findings indicate that the alpha-sarcoglycan with the most frequent missense mutation in LGMD2D is correctly processed, is transported to the sarcolemma, and is fully functional in mouse muscle. Our study presents an unexpected difference in the behavior of a missense-mutated protein in mice versus human patients, and emphasizes the need to understand species-specific protein quality control systems.

Role of Cytokines in Autoimmune Myocarditis and Cardiomyopathy

Cellular as well as humoral autoimmune responses are critically associated with the pathogenesis and progression of myocarditis and cardiomyopathy. Cytokines appear to play critical roles in accentuating or regulating autoimmune mechanisms in these disorders. However, depending on the triggers of autoimmune responses against the heart, such as viral or parasitic infections and experimental immunization with cardiac myosin, the effect of each cytokine on autoimmune myocardial disease may vary. Cytokines may represent new therapeutic targets in the treatment and prevention of autoimmunity-mediated myocarditis and cardiomyopathy, though the etiology and variability in the type of autoimmune responses should be taken into account in the development of cytokine/anti-cytokine treatment of these disorders.

Quantitative myocardial perfusion analysis using multi-row detector CT in acute myocardial infarction

Objective To assess the feasibility of quantitative myocardial perfusion imaging (MPI) in acute myocardial infarction (AMI), using multi-row detector CT (MDCT) with a model-based deconvolution method. Design, setting, patients and interventions Fifteen normal subjects with normal coronary arteries and 26 patients with AMI after reperfusion therapy underwent MPI with MDCT. Perfusion parameters: tissue blood flow (TBF), tissue blood volume (TBV) and mean transit time (MTT) were obtained and compared with clinical parameters, angiography and single-photon emission CT (SPECT) data. Furthermore, the MPI data were compared with data from myocardial magnetic resonance (MR) in 10 subjects. Results The TBF and TBV of infarcted myocardium were significantly lower than those of non-infarcted areas (TBF, 51.96±19.42 vs 108.84±13.29 ml/100 g/min, p<0.01; TBV, 4.47±2.23 vs 9.79±2.58 ml/100 g, p<0.01). The MTT of infarcted areas did not differ from that of non-infarcted areas. The defect areas on TBV colour maps were significantly associated with peak creatine kinase level, QRS score and SPECT defect score. The ratio of TBF or TBV in the epicardial to endocardial side was significantly higher in infarct myocardium with good collateral circulation than in myocardium with poor/no collateral circulation (p<0.01 for both). The TBF measurements with CT- and MR-MPI were in good agreement by linear regression analysis (R=0.55, p<0.01). Conclusions This study demonstrated that MDCT perfusion imaging with deconvolution analysis could quantitatively detect myocardial perfusion abnormalities in patients with AMI and may provide the basis for the non-invasive and quantitative assessment of myocardial infarction.

OC29 is preferentially expressed in the presumptive sensory organ region of the otocyst

The mammalian inner ear derives from the otocyst. Molecular mechanisms underlying inner ear development are largely unknown. We have isolated a secreted molecule, OC29, from a rat otocyst cDNA library by the signal sequence trap method. OC29 was revealed to be a rat homologue of human WFIKKN. OC29 is preferentially expressed in the developing inner ear and the dorsal neural tube. In the inner ear, the expression of OC29 is first detectable at embryonic day 11.5 (E11.5), broadly in the dorsolateral region of the otocyst, which gives rise to the vestibular organ. At E12.5, the expression of OC29 becomes restricted to the presumptive sensory region, mainly to the BMP4‐positive presumptive cristae, and expression becomes reduced at later stages. These results suggest that OC29 may have a role in the early development of the inner ear sensory organ, particularly in the formation of the cristae of the semicircular canals. Developmental Dynamics 231:766–774, 2004.

Collagen VI deficiency reduces muscle pathology, but does not improve muscle function, in the γ-sarcoglycan-null mouse

Muscular dystrophy is characterized by progressive skeletal muscle weakness and dystrophic muscle exhibits degeneration and regeneration of muscle cells, inflammation and fibrosis. Skeletal muscle fibrosis is an excessive deposition of components of the extracellular matrix including an accumulation of Collagen VI. We hypothesized that a reduction of Collagen VI in a muscular dystrophy model that presents with fibrosis would result in reduced muscle pathology and improved muscle function. To test this hypothesis, we crossed γ-sarcoglycan-null mice, a model of limb-girdle muscular dystrophy type 2C, with a Col6a2-deficient mouse model. We found that the resulting γ-sarcoglycan-null/Col6a2Δex5 mice indeed exhibit reduced muscle pathology compared with γ-sarcoglycan-null mice. Specifically, fewer muscle fibers are degenerating, fiber size varies less, Evans blue dye uptake is reduced and serum creatine kinase levels are lower. Surprisingly, in spite of this reduction in muscle pathology, muscle function is not significantly improved. In fact, grip strength and maximum isometric tetanic force are even lower in γ-sarcoglycan-null/Col6a2Δex5 mice than in γ-sarcoglycan-null mice. In conclusion, our results reveal that Collagen VI-mediated fibrosis contributes to skeletal muscle pathology in γ-sarcoglycan-null mice. Importantly, however, our data also demonstrate that a reduction in skeletal muscle pathology does not necessarily lead to an improvement of skeletal muscle function, and this should be considered in future translational studies.

Intravenous amiodarone homogeneously prolongs ventricular repolarization in patients with life-threatening ventricular tachyarrhythmia

BACKGROUND The most critical adverse effects of class III drugs are marked QT prolongation and torsade de pointes. Even though intravenous amiodarone (iv-Amio) is a representative class III drug, it peculiarly inhibits both clinical ventricular tachycardia/fibrillation (VT/VF) and proarrhythmic effects. To test the hypothesis that iv-Amio homogeneously prolongs repolarization, we evaluated electrocardiographic changes before and during short-term amiodarone therapy, focusing closely on the ventricular dispersion of repolarization. METHODS Twenty-seven consecutive patients treated with iv-Amio for VT/VF as a first-line antiarrhythmic therapy were enrolled in this study. Twelve-lead electrocardiography was recorded before and during amiodarone therapy to evaluate the following electrocardiographic intervals: R-R, QRS, QT, QRS to T-peak (QTp), and T-peak to T-end (Tp-e; as an index of dispersion of repolarization). Repolarization indices were corrected to the heart rate by Bazetts method (QTc, c-QTp, c-Tp-e). RESULTS Amiodarone suppressed VT/VF in 19/27 (70%) patients without conferring any proarrhythmic effect. The QTc, c-QTp, and R-R interval were significantly prolonged during amiodarone (476±45ms vs 511±45ms, p<0.05; 338±40ms vs 364±35ms, p<0.05; 762±272ms vs 870±189ms, p<0.05; respectively), whereas the c-Tp-e and QRS durations did not change significantly (139±33ms vs 145±41ms, p=0.25; 96±20ms vs 97±21ms, p=0.33; respectively). CONCLUSIONS Iv-Amio homogeneously prolongs repolarization and properly inhibits original VT/VF recurrence without inducing torsade de pointes.

Difference in statin effects on neointimal coverage after implantation of drug-eluting stents.

ObjectiveThis study was carried out to examine the difference in effects between rosuvastatin and pravastatin on neointimal formation after the placement of a drug-eluting stent (DES). Materials and methodsForty patients who underwent placement of a DES in our hospital were prospectively randomized to receive rosuvastatin (n=20) or pravastatin (n=20), and analyzed by optical coherence tomography at the chronic stage. The main outcome measure was comparison of neointimal coverage analyzed at a strut level. ResultsA significant reduction in total cholesterol, low-density lipoprotein, and white blood cell count was observed during the study in the rosuvastatin group (total cholesterol, from 4.82±0.90 to 4.43±0.77 mmol/l, P=0.038; low-density lipoprotein, from 2.85±0.76 to 2.34±0.57 mmol/l, P=0.006; white blood cell count, from 5810±1399 to 5355±1257/µl, P=0.048), but not in the pravastatin group. Although not statistically significant, C-reactive protein was lower in the rosuvastatin than in the pravastatin group at the chronic stage (1.14±1.21 vs. 7.67±13.67 mg/l, P=0.051). Malapposed and uncovered struts were significantly less frequent in the rosuvastatin group than in the pravastatin group (malapposed, 0.06 vs. 0.60%, P<0.001; uncovered, 6.49 vs. 11.29%, P<0.001). The difference in uncovered struts was maintained even when stent types were analyzed separately (everolimus-eluting stent, 4.81 vs. 6.21%, P=0.007; sirolimus-eluting stent, 14.40 vs. 20.86%, P<0.001). Comparison of neointimal thickness between the rosuvastatin and the pravastatin groups showed inconsistent results depending on the stent types analyzed. ConclusionCompared with pravastatin, the use of rosuvastatin resulted in lower frequency of uncovered and malapposed struts after the placement of a DES, which might be mediated through improved inflammatory and lipid profiles.