Reversal of endothelial progenitor cell dysfunction in patients with type 2 diabetes using a conditioned medium of human embryonic stem cell-derived endothelial cells.

BACKGROUND: The potential clinical application of bone marrow or peripheral blood-derived progenitor cells for cardiovascular regeneration in patients with diabetes mellitus (DM) is limited by their functional impairment. We sought to determine the mechanisms of impaired therapeutic efficacy of peripheral blood-derived progenitor cells in type 2 DM patients and evaluated the use of cell-free conditioned medium obtained from human embryonic stem cell-derived endothelial-like cells (ESC-ECs) to reverse their functional impairment. METHODS: The angiogenic potential of late outgrowth endothelial cells (OECs) and cytokine profile of the conditional medium of proangiogenic cells (PACs) derived from peripheral blood-mononuclear cells of healthy control and DM patients and ESC-ECs was compared by in vitro tube formation assay and a multiplex bead-based immunoassay kit, respectively. The in vivo angiogenic potential of ESC-ECs derived conditioned medium in rescuing the functional impairment of PB-PACs in DM patients was investigated using a hindlimb ischemia model. RESULTS: Human ESC-ECs had similar functional and phenotypic characteristics as OECs in healthy controls. Cytokine profiling showed that vascular endothelial growth factor, stromal cell-derived factor 1 and placental growth factor were down-regulated in PACs from DM patients. Tube formation assay that revealed functional impairment of OECs from DM patients could be rescued by ESC-ECs conditioned medium. Administration of ESC-ECs conditioned medium restored the therapeutic efficacy of PB-PACs from DM patients in a mouse model of hindlimb ischemia. CONCLUSIONS: Our results showed that peripheral blood-derived progenitor cells from DM patients have impaired function because of defective secretion of angiogenic cytokines, which could be restored by supplementation of ESC-ECs conditioned medium.

Expression of Lmna-R225X nonsense mutation results in dilated cardiomyopathy and conduction disorders (DCM-CD) in mice: Impact of exercise training.

AIMS: To recapitulate progressive human dilated cardiomyopathy (DCM) and heart block in the Lmna R225X mutant mice model and investigate the molecular basis of LMNA mutation induced cardiac conduction disorders (CD); To investigate the potential interventional impact of exercise endurance. METHODS AND RESULTS: A Lmna R225X knock-in mice model in either heterozygous or homozygous genotype was generated. Electrical remodeling was observed with higher occurrence of AV block from neonatal and aged mutant mice as measured by surface electrocardiogram and atrio-ventricular Wenckebach point detection. Histological and molecular profiles revealed an increase in apoptotic cells and activation of caspase-3 activities in heart tissue. Upon aging, extracellular cellular matrix (ECM) remodeling appeared with accumulation of collagen in Lmna R225X mutant hearts as visualized by Masson's trichrome stain. This could be explained by the upregulated ECM gene expression, such as Fibronectin: Fn1, collagen: Col12a1, intergrin: Itgb2 and 3, as detected by microarray gene chip. Also, endurance exercise for 3 month improved the ventricular ejection fraction, attenuated fibrosis and cardiomyocytes apoptosis in the aged mutant mice. CONCLUSIONS: The mechanism of LMNA nonsense mutation induced cardiac conduction defects through AV node fibrosis is due to upregulated ECM gene expression upon activation of cardiac apoptosis. Lmna R225X mutant mice hold the potential for serving as in vivo models to explore the mechanism and therapeutic methods for AV block or myopathy associated with the aging process.

TOP2A overexpression in hepatocellular carcinoma correlates with early age onset, shorter patients survival and chemoresistance.

Genomic gain represents an important mechanism in the activation of proto-oncogenes. In many instances, induced oncogenes hold clinical implications both as prognostic markers and targets for therapeutic design. In hepatocellular carcinoma (HCC), although chromosomal gains are common, information on underlying oncogenes induced remains minimal. Here, we examined 7 causal sites of HCC for overexpressed genes by array-based transcriptional mapping. In 22 HCC cell lines and early passages of cultures studied, clusters of up-regulated genes were indicated, where TOP2A expression ranked the highest. Distinct TOP2A transcriptions were confirmed in an independent series of HCC tumors relative to adjacent non-tumoral liver (p=0.0018). By tissue microarray analysis of 172 HCC, we found TOP2A expressions correlated with advance histological grading (p<0.001), microvascular invasion (p=0.004) and an early age onset of the malignancy (

Identification of PEG10 as a progression related biomarker for hepatocellular carcinoma.

Widespread DNA copy number alterations are well recognized in hepatocellular carcinoma (HCC), although the affected genes expression remained largely undefined. In this study, we performed genome-wide analysis on HCC to examine the relationship between gene copy number and corresponding transcriptional changes. To ensure analysis on a homogenous population of tumor cells, integrative analysis of array-based CGH and expression profilings was performed on 20 HCC cell lines using a 19,200-element cDNA microarray platform. Further validation studies were carried out on a large series of primary HCC tumors and paired adjacent non-malignant liver to ascertain finding. Correlative analyses highlighted 31 candidate genes that manifested both copy gains and gene up-regulations (R2>0.5; p<0.05). Of interest was over-expressed paternally expressed 10 (PEG10) resided within the chromosome region 7q21 that has been implicated in the progression of HCC. Quantitative PCR and qRT-PCR studies verified concurrent genomic gains and over-expression of PEG10 in HCC cell lines and primary tumors (34/40 cases; 85%). In addition, qRT-PCR demonstrated a significant progressive trend of increasing PEG10 expressions from the putative pre-malignant adjacent livers to early resectable HCC tumors, and to late inoperable HCCs (p=0.007). In summary, the present study demonstrated the usefulness of integrated genomic and expression profilings in identifying candidate genes within regions of genomic alteration. Our results also suggested that PEG10 may be a potential biomarker in the progressive development of HCC, and that genomic gain represents one of the major mechanisms in the induction of PEG10 over-expressions.

Clonal relationship of tumor nodules in hepatocellular carcinoma: a hierarchical clustering analysis of comparative genomic hybridization data.

Information on the clonal relationship between tumor nodules in patients diagnosed with hepatocellular carcinoma (HCC) holds prognostic significance in the prediction of recurrence and postoperative treatment. Here, we investigated the clonal relationships in 11 focal nodules from 5 patients with HCC by comparative genomic hybridization (CGH) and spectral karyotyping. CGH analysis indicated tumor nodules in 5 (100%) of 5 cases to share similar patterns of genomic imbalances, suggesting nodules to be clonally related. This clonal relationship was further substantiated in 2 cases, where spectral karyotyping analysis indicated identical structural rearrangements between focal tumors. Few studies have attempted to differentiate multicentric growth from intrahepatic dissemination in HCC using CGH analysis; however, data interpretation remained subjective. In this study, we have extended our data to include published CGH findings on 57 nodules derived from 19 HCC cases. Simulation of CGH findings by unsupervised 2-way hierarchical clustering indicated tumor nodules in 20 (83.3%) of 24 cases to cluster concordantly, signifying a high incidence of clonal similarity. In sum, bioinformatic analysis of genomic profiles may represent a reliable research tool in analyzing clonal relationships among tumor nodules.

Generation of induced pluripotent stem cell lines from 3 distinct laminopathies bearing heterogeneous mutations in lamin A/C.

The term laminopathies defines a group of genetic disorders caused by defects in the nuclear envelope, mostly the lamins. Lamins are the main constituents of the nuclear lamina, a filamentous meshwork associated with the inner nuclear membrane that provides mechanical stability and plays important roles in processes such as transcription, DNA replication and chromatin organization. More than 300 mutations inlamin A/C have been associated with diverse clinical phenotypes, understanding the molecular basis of these diseases may provide a rationale for treating them. Here we describe the generation of induced pluripotent stem cells (iPSCs) from a patient with inherited dilated cardiomiopathy and 2 patients with distinct accelerated forms of aging, atypical Werner syndrome and Hutchinson Gilford progeria, all of which are caused by mutations in lamin A/C. These cell lines were pluripotent and displayed normal nuclear membrane morphology compared to donor fibroblasts. Their differentiated progeny reproduced the disease phenotype, reinforcing the idea that they represent excellent tools for understanding the role of lamin A/C in normal physiology and the clinical diversity associated with these diseases.

Identification of PFTAIRE protein kinase 1, a novel cell division cycle-2 related gene, in the motile phenotype of hepatocellular carcinoma cells.

UNLABELLED: Metastasis is a major cause of cancer morbidity and mortality in individuals with hepatocellular carcinoma (HCC), yet little is known about the underlying molecular basis. Using genetic information derived from chromosome-based comparative genomic hybridization, we have reported previously on regional chromosome 7q21-q22 gains in close association with HCC progression. In this study, we undertook cDNA microarray-based comparative genomic hybridization, to examine the 7q21-q22 region for the involved gene(s) in HCC. High-resolution mapping analysis highlighted 7 candidates, namely PFTAIRE protein kinase 1 (PFTK1), ODAG, CDK6, CAS1, PEX1, SLC25A, and PEG10, within the region. Quantitative reverse transcription (RT)-PCR evaluation further indicated upregulation of a single candidate gene, PFTK1, that correlated significantly with both advanced metastatic HCCs (P = 0.032) and tumor microvascular invasion (P = 0.012). Given that little is known about the function(s) of PFTK1, which is a novel cell division cycle (Cdc)2-related gene, we examined its potential role in the motile phenotype of HCC cells by both ectopic expression and knockdown investigations. RNA-interference knockdown of PFTK1 in invasive Hep3B cells resulted in a significant reduction in cell invasion, chemotactic migration, and cell motility (P < 0.001). Conversely, ectopic expression of PFTK1 in noninvasive HKCI-C3 cells induced substantial cellular invasion and migration (P < or = 0.007). In neither cell line was there any effect on cell viability. Immunofluorescence showed marked filamentous actin polymerizations in PFTK1-expressing cells. CONCLUSION: In this study, we have thus provided preliminary evidence that overexpression of PFTK1 may confer a motile phenotype in malignant hepatocytes that accounts for the association of upregulation of this gene in metastatic HCC.

Novel identification of zyxin upregulations in the motile phenotype of hepatocellular carcinoma.

Genome-wide copy number aberrations are common in hepatocellular carcinoma, although the precise genetic events underlying disease progression remain poorly defined. Previous work from our group has indicated several regional chromosomal gains such as chromosome 7q34-q36 that are associated with advanced metastatic tumors. Although the distal chromosome 7q gains have also been implicated in the progression of other malignancies, information on underlying targeted genes is limited. In this study, we have examined the chromosome 7q34-q36 region for involved gene(s) (or genes of interest). An integrated array-based comparative genomic hybridization and transcriptional mapping analyses has enabled us to identify a single candidate, zyxin on chromosome 7q34-q36. This array-derived finding was supported by quantitative reverse transcription-polymerase chain reaction, which also indicated common upregulations of zyxin in hepatocellular carcinoma tumors compared to their corresponding nonmalignant liver tissue (17/52 cases; 33%). Although there was no correlation between zyxin expression and tumor stagings, there was a significant increase in messenger RNA levels in hepatocellular carcinoma cases that presented with multifocal disease (211.5 +/- 936.9-fold) compared to those with solitary lesions (3.5 +/- 6.3-fold). Moreover, recurrence after resection was common in cases that displayed zyxin overexpressions in the initial resected tumor (P = 0.05). Functional examination of zyxin by small interfering RNA-mediated knockdown in Hep3B cell line indicated a significant inhibition on cell migration through porous membrane (P = 0.002) and invasion through matrigel-coated membrane (P = 0.005). In summary, mapping of chromosome 7q34-q36 has led to the identification of frequent zyxin overexpressions in hepatocellular carcinoma, and a potential role for zyxin in conferring a motile phenotype.

Positional expression profiling indicates candidate genes in deletion hotspots of hepatocellular carcinoma.

Molecular characterizations of hepatocellular carcinoma have indicated frequent allelic losses on chromosomes 4q, 8p, 16q and 17p, where the minimal deleted regions have been further defined on 4q12-q23, 4q31-q35, 8p21-p22, 16q12.1-q23.1 and 17p13. Despite these regions are now well-recognized in early liver carcinogenesis, few underlying candidate genes have been identified. In an effort to define affected genes within common deleted loci of hepatocellular carcinoma, we conducted transcriptional mapping by high-resolution cDNA microarray analysis. In 20 hepatocellular carcinoma cell lines and 20 primary tumors studied, consistent downregulations of novel transcripts were highlighted throughout the entire genome and within sites of frequent losses. The array-derived candidates including fibrinogen gamma peptide (FGG, at 4q31.3), vitamin D binding protein (at 4q13.3), fibrinogen-like 1 (FGL1, at 8p22), metallothionein 1G (MT1G, at 16q12.2) and alpha-2-plasmin inhibitor (SERPINF2, at 17p13) were confirmed by quantitative reverse transcription-polymerase chain reaction, which also indicated a more profound downregulation of FGL1, MT1G and SERPINF2 relative to reported tumor-suppressor genes, such as DLC1 (8p22), E-cadherin (16q22.1) and TP53 (17p13.1). In primary hepatocellular carcinoma examined, a significant repression of MT1G by more than 100-fold was indicated in 63% of tumors compared to the adjacent nonmalignant liver (P = 0.0001). Significant downregulations of FGG, FGL1 and SERPINF2 were also suggested in 30, 23 and 33% of cases, respectively, compared to their nonmalignant counterparts (P < 0.016). In summary, transcriptional mapping by microarray indicated a number of previously undescribed downregulated genes in hepatocellular carcinoma, and highlighted potential candidates within common deleted regions.