Thrombospondin-1 differentially regulates microRNAs in vascular smooth muscle cells.

Thrombospondin-1 (TSP-1) is an important regulator of vascular smooth muscle cell (VSMC) physiology and gene expression. MicroRNAs (microRNA), small molecules that regulate protein translation, have emerged as potent regulators of cell function. MicroRNAs have been shown to be involved in intimal hyperplasia, atherosclerosis, and upregulated in the vasculature in diabetes. The purpose of this study was to identify microRNAs regulated by TSP-1 in vascular smooth muscle cells (VSMCs). Human VSMCs were treated for 6 h with basal media or TSP-1 both supplemented with 0.2% FBS. Cells were then snap frozen and RNA extracted. An Affymetrix GeneChip microRNA array analysis was performed in triplicate on three separate collections. Confirmatory qrtPCR was performed. Data were analyzed by ANOVA or t test, with significance set at p < 0.05. MicroRNAs identified were subjected to KEGG pathway analysis using the DIANA tools miRPath online tool. TSP-1 upregulated 22 microRNAs and downregulated 18 microRNAs in VSMCs (p < 0.05). The most upregulated microRNA was miR-512-3p (45.12 fold). The microRNA most downregulated by TSP-1 was miR-25-5p, which was decreased by 9.61. Of note, five members of the mir-17-92 cluster were downregulated. KEGG analysis revealed that thirty-three cellular signaling pathways were impacted by these microRNAs and that nine pathways were relevant to vascular disease. MicroRNAs regulate protein expression at the level of translation and may represent a significant mechanism by which TSP-1 regulates VSMC function. Several of the microRNAs identified have a role in vascular function. The miR-17-92 cluster family, which was found to exhibit reduced expression in this study, is known to be involved in angiogenesis and vascular function. TSP-1 regulates multiple microRNAs in VSMCs adding a new layer of complexity to TSP-1 regulation of VSMC function.

Association between autism spectrum disorder in individuals with velocardiofacial (22q11.2 deletion) syndrome and PRODH and COMT genotypes.

Velocardiofacial (VCFS; 22q11.2 deletion) syndrome is a genetic disorder that results from a hemizygous deletion of the q11.2 region on chromosome 22, and is associated with greatly increased risk for psychiatric disorders, including autism spectrum disorder (ASD) and schizophrenia. There is emerging evidence for the involvement of catechol-O-methyltransferase (COMT) and proline dehydrogenase (oxidase) 1 (PRODH) in the psychiatric phenotype of individuals with VCFS. Here, we tested the hypothesis that PRODH and COMT are associated with ASD in youths with VCFS. We found that individuals with VCFS and the low-activity alleles of both PRODH and COMT (rs4819756A and rs4680A) were more likely to present with ASD as compared with individuals with VCFS and the high-activity alleles of these genes [P<0.05; odds ratio=6.0 (95% confidence interval=1.27-28.26; N=87)]. Our results suggest that PRODH and COMT may interact to contribute to the ASD phenotype in individuals with VCFS.

Thrombospondin-1: a proatherosclerotic protein augmented by hyperglycemia.

OBJECTIVE: Diabetes is associated with a more aggressive form of atherosclerosis. Thrombospondin-1 (TSP-1), an extracellular matrix protein, is an acute-phase reactant that induces vascular smooth muscle (VSMC) migration and proliferation in areas of vascular injury and is also up-regulated in VSMCs exposed to hyperglycemia. This study tested the hypothesis that hyperglycemia amplifies the expression of genes induced by TSP-1 in VSMCs. METHODS: Human aortic VSMCs were cultured in Dulbecco Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics. Cells were used between passages three and five. VSMCs were preincubated in DMEM containing 0.2% FBS with 5 mM glucose (normoglycemia), 25 mM glucose (hyperglycemia), 25 mM mannose (osmotic control), TSP-1 (20 microg/mL), 25 mM glucose + TSP-1 (20 microg/mL), or 25 mM mannose + TSP-1 (20 microg/mL). Total RNA was extracted. Microarray analysis was performed and analyzed by analysis of variance. P < .05 was considered significant. Quantitative real-time polymerase chain reaction (rtPCR) was used to confirm selected up-regulated genes. RESULTS: Microarray analysis revealed: (1) hyperglycemia altered 30 genes; (2) TSP-1 altered 212 genes, of which 8 were altered similarly to VSMCs exposed to 25 mM glucose; (3) TSP-1 up-regulated 10 genes associated with atherosclerosis and 4 others with diabetic vascular disease; (4) hyperglycemia combined with TSP-1 altered expression of 2822 genes. The three genes most up-regulated by TSP-1 in a normoglycemic environment were uridine 5'-diphosphoglucose (UDP-glucose) dehydrogenase (UGDH, 127%), transforming growth factor beta-2 (TGFbeta2, 116%), and hyaluronan synthase 2 (HAS2, 113%). Further, TSP-1 altered the expression of genes in 13 canonical pathways; however, when combined with hyperglycemia, 53 canonical pathways were affected. CONCLUSION: Quantitative rtPCR confirmed that genes in several of these pathways for TSP-1 and hyperglycemia combined with TSP-1 were up-regulated. These findings suggest that TSP-1 may be germane to the progression of atherosclerosis and may have a large effect with concurrent hyperglycemia.

Cyclosporine inhibition of angiogenesis involves the transcription factor HESR1.

PURPOSE: Angiogenesis is critical in normal development and in tumor growth. Experimentally, cyclosporine A (CyA) inhibits angiogenesis in an in vivo mouse model and an in vitro capillary tube model. The mechanisms behind its antiangiogenic effects are not well characterized. To determine which nuclear factor, if any, may be involved in the antiangiogenic effects of CyA, we performed a microarray analysis of human aortic endothelial cells (HAEC) subjected to CyA and another calcineurin inhibitor, FK 506. METHODS: HAEC were divided into four groups: (1) HAEC incubated with CyA 2 microg/mL; (2) HAEC incubated with CyA 10 microg/mL; (3) HAEC incubated with FK 506 1 microg/mLl for 24 h; and (4) HAEC as control. We used Affymetrix GeneChip U133-A for gene expression analysis and validated our results with quantitative reverse transcription-polymerase chain reaction. RESULTS: At a 2 microg/mL dose, CyA treated HAEC revealed a 44-fold increase in the expression of hairy enhancer of split-related protein 1 (HESR1) and 1.73-fold down-regulation of transcripts encoding for the vascular endothelial growth factor (VEGF) receptor (VEGFR2). At 10 microg/mL, the expression of the HESR1 transcript was 57-fold higher than control, and VEGFR2 exhibited a 1.93-fold down-regulation. Quantitative reverse transcription-polymerase chain reaction confirmed a significant (P < 0.0001) increase in expression of HESR1 in CyA treated cells. In contrast, the expression level of HESR1 was not affected by the FK 506 treatment. CONCLUSION: CyA demonstrate antiangiogenic activities linked to an overexpression of HESR1 transcription factor, and down-regulation of VEGFR2. Thus, use of high-dose CyA may provide a novel treatment in angiogenesis dependent disease.

Complete maternal uniparental isodisomy of chromosome 4 in a subject with major depressive disorder detected by high density SNP genotyping arrays.

Uniparental isodisomy (iUPD) is a rare genetic condition caused by non-disjunction during meiosis that ultimately leads to a duplication of either the maternal or paternal chromosome in the affected individual. Two types of disorders can result, those due to imprinted genes and those due to homozygosity of recessive disease-causing mutations. Here, we describe the third known case of complete chromosome 4 iUPD of maternal origin. This condition became apparent during whole genome linkage studies of psychiatric disorders in the Portuguese population. The proband is an adult female with normal fertility and no major medical complaints, but a history of major depressive disorder and multiple suicide attempts. The proband's siblings and parents had normal chromosome 4 genotypes and no history of mood disturbance. A brief review of other studies lends support for the possibility that genes on chromosome 4 might confer risk for mood disorders. We conclude that chromosome 4 maternal uniparental disomy (UPD) is a rare disorder that may present with a major depressive phenotype. The lack of a common disease phenotype between this and two other cases of chromosome 4 iUPD [Lindenbaum et al. [1991] Am J Med Genet 49(Suppl 285):1582; Spena et al. [2004] Eur J Hum Genet 12:891-898) would suggest that there is no vital maternal gene imprinting on chromosome 4. However, since there is no reported case of paternal chromosome 4 UPD, paternal gene imprinting on chromosome 4 cannot be excluded.

Gene expression profiles of intact and regenerating zebrafish retina.

PURPOSE: Investigate the molecular determinants of retinal regeneration in adult vertebrates by analyzing the gene expression of control and post-lesion retina of adult zebrafish, a system that regenerates following injury. METHODS: Gene expression of zebrafish retina and brain were determined with DNA microarray, RT-PCR, and real-time quantitative PCR analyses. Damaged retinas and their corresponding controls were analyzed 2-5 days post-lesion (acute injury condition) or 14 d post-lesion (cell regeneration condition). RESULTS: Expected similarities and differences in the gene expression profile of zebrafish retina and brain were observed, confirming the applicability of the gene expression techniques. Mechanical lesion of retina triggered significant, time-dependent changes in retinal gene expression. The induced transcriptional changes were consistent with cellular phenomena known to occur, in a time-dependent manner, subsequent to retinal lesion, including cell cycle progression, axonal regeneration, and regenerative cytogenesis. CONCLUSIONS: The results indicate that retinal regeneration in adult zebrafish involves a complex set of induced, targeted changes in gene transcription, and suggest that these molecular changes underlie the ability of the adult vertebrate retina to regenerate.

Gene expression analysis of peripheral blood leukocytes from discordant sib-pairs with schizophrenia and bipolar disorder reveals points of convergence between genetic and functional genomic approaches.

We performed global RNA transcript analysis and comprehensive gene group analysis of peripheral blood leukocyte (PBL) RNA from two groups of matched sib-pairs that were discordant for either schizophrenia (n = 33 sib-pairs) or bipolar disorder (n = 5 sib-pairs). The pairs chosen for these analyses were selected from families with known patterns of genetic linkage (5q for schizophrenia and 6q for bipolar disorder). At the single gene level, we obtained lists of the transcripts with the most significant changes in expression and from these lists determined those with the highest degree of predictive power for classifying subjects according to diagnosis in these samples. At the gene group level, we comprehensively analyzed pairwise expression changes of more than 4,000 functional groups and cytogenetic locations, and present a novel method of displaying these data that we term "cytogenomic" mapping. Verification of selected changes in expression was performed using quantitative real-time RT-PCR. Our results provide compelling evidence for the utility of analyzing PBL RNA for changes in expression in neuropsychiatric disorders.

Genetic linkage of bipolar disorder to chromosome 6q22 is a consistent finding in Portuguese subpopulations and may generalize to broader populations.

We recently reported genome-wide significant linkage to chromosome 6q for bipolar disorder, in a study of 25 Portuguese families, using the Human Mapping Assay Xba 131 (HMA10K). To explore the generalizability of this finding, we reanalyzed our SNP linkage data according to the families' geographic origin. Specifically, the 25 families included 20 families from the Portuguese island collection (PIC; 15 families from the Azores Islands and 5 from the Madeira Islands) and 5 families from continental Portugal. Non-parametric linkage analysis (NPL) was performed as previously described and indicated that each of these subpopulations showed evidence of linkage for the same region. To further address the potential generalizability of these findings to other populations, we have also examined allelic heterozygosity in our subpopulations and in three reference populations (Caucasian, East Asian, and African-American). This analysis indicated that the PIC population is highly correlated to the Caucasian reference population (R = 0.86) for all of chromosome 6. In contrast allelic heterozygosity was more weakly correlated between PIC and both East Asian (R = 0.37) and African-American (R = 0.32) reference populations. Taken together these observations suggest a shared genetic liability among Portuguese populations for bipolar disorder on chromosome 6q, and that the PIC population is likely representative of Caucasians in general.