Modeling and rescue of the vascular phenotype of Williams-Beuren syndrome in patient induced pluripotent stem cells.

Elastin haploinsufficiency in Williams-Beuren syndrome (WBS) leads to increased vascular smooth muscle cell (SMC) proliferation and stenoses. Our objective was to generate a human induced pluripotent stem (hiPS) cell model for in vitro assessment of the WBS phenotype and to test the ability of candidate agents to rescue the phenotype. hiPS cells were reprogrammed from skin fibroblasts of a WBS patient with aortic and pulmonary stenosis and healthy control BJ fibroblasts using four-factor retrovirus reprogramming and were differentiated into SMCs. Differentiated SMCs were treated with synthetic elastin-binding protein ligand 2 (EBPL2) (20 µg/ml) or the antiproliferative drug rapamycin (100 nM) for 5 days. We generated four WBS induced pluripotent stem (iPS) cell lines that expressed pluripotency genes and differentiated into all three germ layers. Directed differentiation of BJ iPS cells yielded an 85%-92% pure SMC population that expressed differentiated SMC markers, were functionally contractile, and formed tube-like structures on three-dimensional gel assay. Unlike BJ iPS cells, WBS iPS cells generated immature SMCs that were highly proliferative, showed lower expression of differentiated SMC markers, reduced response to the vasoactive agonists, carbachol and endothelin-1, impaired vascular tube formation, and reduced calcium flux. EBPL2 partially rescued and rapamycin fully rescued the abnormal SMC phenotype by decreasing the smooth muscle proliferation rate and enhancing differentiation and tube formation. WBS iPS cell-derived SMCs demonstrate an immature proliferative phenotype with reduced functional and contractile properties, thereby recapitulating the human disease phenotype. The ability of rapamycin to rescue the phenotype provides an attractive therapeutic candidate for patients with WBS and vascular stenoses.

Simvastatin reverses cardiac hypertrophy caused by disruption of the bradykinin 2 receptor.

Bradykinin 2 receptor (B2R) deficiency predisposes to cardiac hypertrophy and hypertension. The pathways mediating these effects are not known. Two-month-old B2R knockout (KO) and wild-type (WT) mice were assigned to 4 treatment groups (n = 12-14/group): control (vehicle); nitro-L-arginine methyl ester (L-NAME) an NO synthase inhibitor; simvastatin (SIM), an NO synthase activator; and SIM+L-NAME. Serial echocardiography was performed and blood pressure (BP) at 6 weeks was recorded using a micromanometer. Myocardial eNOS and mitogen-activated protein kinase (MAPK, including ERK, p38, and JNK) protein expression were measured. Results showed that (i) B2RKO mice had significantly lower ejection fraction than did WT mice (61% +/- 1% vs. 73% +/- 1%), lower myocardial eNOS and phospho-eNOS, normal systolic BP, and higher LV mass, phospho-p38, and JNK; (ii) L-NAME increased systolic BP in KO mice (117 +/- 19 mm Hg) but not in WT mice and exacerbated LV hypertrophy and dysfunction; and (iii) in KO mice, SIM decreased hypertrophy, p38, and JNK, improved function, increased capillary eNOS and phospho-eNOS, and prevented L-NAME-induced LV hypertrophy without lowering BP. We conclude that disruption of the B2R causes maladaptive cardiac hypertrophy with myocardial eNOS downregulation and MAPK upregulation. SIM reverses these abnormalities and prevents the development of primary cardiac hypertrophy as well as hypertrophy secondary to L-NAME-induced hypertension.

Isolation of a bone marrow-derived stem cell line with high proliferation potential and its application for preventing acute fatal liver failure.

Transplantation of hepatocytes or hepatocyte-like cells of extrahepatic origin is a promising strategy for treatment of acute and chronic liver failure. We examined possible utility of hepatocyte-like cells induced from bone marrow cells for such a purpose. Clonal cell lines were established from the bone marrow of two different rat strains. One of these cell lines, rBM25/S3 cells, grew rapidly (doubling time, approximately 24 hours) without any appreciable changes in cell properties for at least 300 population doubling levels over a period of 300 days, keeping normal diploid karyotype. The cells expressed CD29, CD44, CD49b, CD90, vimentin, and fibronectin but not CD45, indicating that they are of mesenchymal cell origin. When plated on Matrigel with hepatocyte growth factor and fibroblast growth factor-4, the cells efficiently differentiated into hepatocyte-like cells that expressed albumin, cytochrome P450 (CYP) 1A1, CYP1A2, glucose 6-phosphatase, tryptophane-2,3-dioxygenase, tyrosine aminotransferase, hepatocyte nuclear factor (HNF)1 alpha, and HNF4alpha. Intrasplenic transplantation of the differentiated cells prevented fatal liver failure in 90%-hepatectomized rats. In conclusion, a clonal stem cell line derived from adult rat bone marrow could differentiate into hepatocyte-like cells, and transplantation of the differentiated cells could prevent fatal liver failure in 90%-hepatectomized rats. The present results indicate a promising strategy for treating human fatal liver diseases.

Fine deletion mapping of chromosome 2q21-37 shows three preferentially deleted regions in oral cancer.

We analysed the loss of heterozygosity (LOH) of long arm of chromosome 2 by using 16 polymorphic microsatellite markers in 39 matched oral normal and cancer tissues, and defined the deletional mapping of the region with putative tumor suppressor genes. LOH was detected at least one location in 33 of 39 (85%) tumor tissues. Frequent deletions were detected at the locations of microsatellite markers, D2S2304 (35%), D2S111 (40%), D2S155 (35%), D2S1327 (29%), D2S164 (29%), D2S125 (68%) and D2S140 (32%). Three preferentially deleted regions at 2q21-24, 2q33-35 and 2q37.3 were observed. Several candidate tumor suppressor genes in these regions such as LRP1B, CASP8, CASP10, BARD1, ILKAP, PPP1R7, and ING5, are located. Further molecular analysis of each gene should be performed to clarify their roles in oral carcinogenesis.

Epigenetic alterations of BRG1 leads to cancer development through its nuclear-cytoplasmic shuttling abnormalities.

SWI/SNF is a multiprotein chromatin remodeling complex important for gene regulation. BRG1 and its close relative BRM, have ATPase activity necessary for transcriptional regulation by conformational change of nucleosomes. Due to this role on gene expression, several members of SWI/SNF complex including BRG1 and BRM function as a tumor suppressor or negative regulator of cellular proliferation. On the other hand, the shuttling of proteins between nucleus and cytoplasm is strongly involved in the regulation of cell cycle and proliferation. Many of tumor suppressor gene (TSG)s including p53, BRCA1, ING1 play some of their functions through nucleocytoplasmic shuttling. Abnormalities related with this process abrogate the subcellular localization of the TSGs and lead to cancer development. We recently demonstrated BRG1 as a TSG in oral cancer. Our analysis also revealed an interesting finding that one of the splicing forms of BRG1 is selectively lost in cancer tissue as compared to normal counterparts. Our further analysis revealed a putative nuclear retention signal domain for this splicing form. In this article, we speculate the possible mechanism for the inactivation of BRG1 gene in oral cancer through an abnormality in its subcellular localization.