Towards An Understanding Of The Etiology Of Abdominal Aortic Aneurysms: Identification Of Genes Implicated In Aaa Risk And Development

Abdominal aortic aneurysm (AAA) is a common disease for which mechanisms of formation are still not well understood. Despite a strong genetic component to AAA risk, specific risk alleles are still largely unidentified. AAA is also a localized disease with a majority occurring in the infrarenal abdominal aorta and is six times more common than aneurysms of the thoracic aorta. To determine whether risk alleles are present in functional positional candidate genes. we: 1. performed a genetic association study using DNA from AAA cases and controls in ten candidate genes and 2. performed exon sequencing on three genes with evidence of nominal association. To identify genes with regional expression in the aorta of potential relevance to AAA: 1. Illumina microarrays were used to perform a gene expression profiling of entire baboon aortas and 2. protein expression of one candidate gene, HOXA4, was examined in further detail. Single nucleotide polymorphisms in three positional candidate genes, CEBPG, PEPD and CD22, that had both functions relevant to AAA and expression in AAA tissue exhibited nominal association and further studied by DNA sequencing of exons. Sequence variants were identified in PEPD and CD22, but not CEPBG, but no sequence variants had predicted functional changes. Analysis of the gene expression profiles from whole length baboon aortas identified 26 genes that exhibited lengthwise expression changes along aorta, including three genes from the homeobox (HOX) family of transcription factors. Comparison of HOX gene transcripts between AAA and control abdominal aortic tissue identified ten HOX genes that were significantly downregulated in AAA tissue. The most significantly downregulated HOX gene, HOXA4, also exhibited spatial expression changes and was studied in human aortic tissue. IImmunohistochemical staining of sections of human aorta using an antibody directed against HOXA4 showed expression in all three histological layers of the aorta, primarily by endothelial cells and smooth muscle cells. Immunohistochemical analysis and immunofluorescent staining of cultured cells in vitro demonstrated protein expression localizing to the nucleus and perinuclear regions of endothelial and smooth muscle cells Quantitative analysis of HOXA4 protein by western blot in 24 paired human thoracic and abdominal aortic samples showed that, consistent with the microarray results, HOXA4 had significantly lower expression (p 〈 0.0001) in the abdominal aorta. A negative correlation was also shown between HOXA4 was demonstrated between HOXA4 protein levels and increasing in both thoracic and abdominal aorta. In conclusion, several novel genes with potential roles in AAA etiology and pathogenesis have been identified using two different methods. Two plausible functional candidate genes from a region with evidence of genetic linkage, CD22 and PEPD, were shown to have evidence of nominal association, suggesting that variation in or around these genes may contribute to the risk of developing an AAA. HOXA4 is novel candidate gene with differential expression by aortic location and age. Lower levels of HOXA4 in the abdominal aorta and further decreases in expression with increasing age suggest that HOXA4 may have a protective function with respect to AAA

Immunohistochemical Analysis of the Natural Killer Cell Cytotoxicity Pathway in Human Abdominal Aortic Aneurysms

Our previous analysis using genome-wide microarray expression data revealed extreme overrepresentation of immune related genes belonging the Natural Killer (NK) Cell Mediated Cytotoxicity pathway (hsa04650) in human abdominal aortic aneurysm (AAA). We followed up the microarray studies by immunohistochemical analyses using antibodies against nine members of the NK pathway (VAV1, VAV3, PLCG1, PLCG2, HCST, TYROBP, PTK2B, TNFA, and GZMB) and aortic tissue samples from AAA repair operations (n = 6) and control aortae (n = 8) from age-, sex- and ethnicity-matched donors from autopsies. The results confirmed the microarray results. Two different members of the NK pathway, HCST and GRZB, which act at different steps in the NK-pathway, were actively transcribed and translated into proteins in the same cells in the AAA tissue demonstrated by double staining. Furthermore, double staining with antibodies against CD68 or CD8 together with HCST, TYROBP, PTK2B or PLCG2 revealed that CD68 and CD8 positive cells expressed proteins of the NK-pathway but were not the only inflammatory cells involved in the NK-pathway in the AAA tissue. The results provide strong evidence that the NK Cell Mediated Cytotoxicity Pathway is activated in human AAA and valuable insight for future studies to dissect the pathogenesis of human AAA

Regional expression of HOXA4 along the aorta and its potential role in human abdominal aortic aneurysms

<p>Abstract</p> <p>Background</p> <p>The infrarenal abdominal aorta exhibits increased disease susceptibility relative to other aortic regions. Allograft studies exchanging thoracic and abdominal segments showed that regional susceptibility is maintained regardless of location, suggesting substantial roles for embryological origin, tissue composition and site-specific gene expression.</p> <p>Results</p> <p>We analyzed gene expression with microarrays in baboon aortas, and found that members of the HOX gene family exhibited spatial expression differences. <it>HOXA4 </it>was chosen for further study, since it had decreased expression in the abdominal compared to the thoracic aorta. Western blot analysis from 24 human aortas demonstrated significantly higher HOXA4 protein levels in thoracic compared to abdominal tissues (<it>P </it>< 0.001). Immunohistochemical staining for HOXA4 showed nuclear and perinuclear staining in endothelial and smooth muscle cells in aorta. The <it>HOXA4 </it>transcript levels were significantly decreased in human abdominal aortic aneurysms (AAAs) compared to age-matched non-aneurysmal controls (<it>P </it>< 0.00004). Cultured human aortic endothelial and smooth muscle cells stimulated with INF-γ (an important inflammatory cytokine in AAA pathogenesis) showed decreased levels of HOXA4 protein (<it>P </it>< 0.0007).</p> <p>Conclusions</p> <p>Our results demonstrated spatial variation in expression of HOXA4 in human aortas that persisted into adulthood and that downregulation of <it>HOXA4 </it>expression was associated with AAAs, an important aortic disease of the ageing population.</p

Analysis of positional candidate genes in the AAA1 susceptibility locus for abdominal aortic aneurysms on chromosome 19

ABSTRACT: BACKGROUND: Abdominal aortic aneurysm (AAA) is a complex disorder with multiple genetic risk factors. Using affected relative pair linkage analysis, we previously identified an AAA susceptibility locus on chromosome 19q13. This locus has been designated as the AAA1 susceptibility locus in the Online Mendelian Inheritance in Man (OMIM) database. METHODS: Nine candidate genes were selected from the AAA1 locus based on their function, as well as mRNA expression levels in the aorta. A sample of 394 cases and 419 controls was genotyped for 41 SNPs located in or around the selected nine candidate genes using the Illumina GoldenGate platform. Single marker and haplotype analyses were performed. Three genes (CEBPG, PEPD and CD22) were selected for DNA sequencing based on the association study results, and exonic regions were analyzed. Immunohistochemical staining of aortic tissue sections from AAA and control individuals was carried out for the CD22 and PEPD proteins with specific antibodies. RESULTS: Several SNPs were nominally associated with AAA (p < 0.05). The SNPs with most significant p-values were located near the CCAAT enhancer binding protein (CEBPG), peptidase D (PEPD), and CD22. Haplotype analysis found a nominally associated 5-SNP haplotype in the CEBPG/PEPD locus, as well as a nominally associated 2-SNP haplotype in the CD22 locus. DNA sequencing of the coding regions revealed no variation in CEBPG. Seven sequence variants were identified in PEPD, including three not present in the NCBI SNP (dbSNP) database. Sequencing of all 14 exons of CD22 identified 20 sequence variants, five of which were in the coding region and six were in the 3'-untranslated region. Five variants were not present in dbSNP. Immunohistochemical staining for CD22 revealed protein expression in lymphocytes present in the aneurysmal aortic wall only and no detectable expression in control aorta. PEPD protein was expressed in fibroblasts and myofibroblasts in the media-adventitia border in both aneurysmal and non-aneurysmal tissue samples. CONCLUSIONS: Association testing of the functional positional candidate genes on the AAA1 locus on chromosome 19q13 demonstrated nominal association in three genes. PEPD and CD22 were considered the most promising candidate genes for altering AAA risk, based on gene function, association evidence, gene expression, and protein expression

Transcriptional (ChIP-Chip) Analysis of ELF1, ETS2, RUNX1 and STAT5 in Human Abdominal Aortic Aneurysm

We investigated transcriptional control of gene expression in human abdominal aortic aneurysm (AAA). We previously identified 3274 differentially expressed genes in human AAA tissue compared to non-aneurysmal controls. Four expressed transcription factors (ELF1, ETS2, STAT5 and RUNX1) were selected for genome-wide chromatin immunoprecipitation. Transcription factor binding was enriched in 4760 distinct genes (FDR &lt; 0.05), of which 713 were differentially expressed in AAA. Functional classification using Gene Ontology (GO), KEGG, and Network Analysis revealed enrichment in several biological processes including “leukocyte migration” (FDR = 3.09 × 10−05) and “intracellular protein kinase cascade” (FDR = 6.48 × 10−05). In the control aorta, the most significant GO categories differed from those in the AAA samples and included “cytoskeleton organization” (FDR = 1.24 × 10−06) and “small GTPase mediated signal transduction” (FDR = 1.24 × 10−06). Genes up-regulated in AAA tissue showed a highly significant enrichment for GO categories “leukocyte migration” (FDR = 1.62 × 10−11), “activation of immune response” (FDR = 8.44 × 10−11), “T cell activation” (FDR = 4.14 × 10−10) and “regulation of lymphocyte activation” (FDR = 2.45 × 10−09), whereas the down-regulated genes were enriched in GO categories “cytoskeleton organization” (FDR = 7.84 × 10−05), “muscle cell development” (FDR = 1.00 × 10−04), and “organ morphogenesis” (FDR = 3.00 × 10−04). Quantitative PCR assays confirmed a sub-set of the transcription factor binding sites including those in MTMR11, DUSP10, ITGAM, MARCH1, HDAC8, MMP14, MAGI1, THBD and SPOCK1

Peripheral Blood Mitochondrial DNA Damage as a Potential Noninvasive Biomarker of Diabetic Retinopathy

Citation: Mishra M, Lillvis J, Seyoum B, Kowluru RA. Peripheral blood mitochondrial DNA damage as a potential noninvasive biomarker of diabetic retinopathy. Invest Ophthalmol Vis Sci. 2016;57:4035-4044. DOI:10.1167/iovs.16-19073 PURPOSE. In the development of diabetic retinopathy, retinal mitochondria become dysfunctional, and mitochondrial DNA (mtDNA) is damaged. Because retinopathy is a progressive disease, and circulating glucose levels are high in diabetes, our aim was to investigate if peripheral blood mtDNA damage can serve as a potential biomarker of diabetic retinopathy. METHODS. Peripheral blood mtDNA damage was investigated by extended-length PCR in rats and mice, diabetic for 10 to 12 months (streptozotocin-induced, type 1 model), and in 12-and 40-week-old Zucker diabetic fatty rats (ZDF, type 2). Mitochondrial copy number (in gDNA) and transcription (in cDNA) were quantified by qPCR. Similar parameters were measured in blood from diabetic patients with/without retinopathy. RESULTS. Peripheral blood from diabetic rodents had significantly increased mtDNA damage and decreased copy numbers and transcription. Lipoic acid administration in diabetic rats, or Sod2 overexpression or MMP-9 knockdown in mice, the therapies that prevent diabetic retinopathy, also ameliorated blood mtDNA damage and restored copy numbers and transcription. Although blood from 40-week-old ZDF rats had significant mtDNA damage, 12-week-old rats had normal mtDNA. Diabetic patients with retinopathy had increased blood mtDNA damage, and decreased transcription and copy numbers compared with diabetic patients without retinopathy and nondiabetic individuals. CONCLUSIONS. Type 1 diabetic rodents with oxidative stress modulated by pharmacologic/ genetic means, and type 2 animal model and patients with/without diabetic retinopathy, demonstrate a strong relation between peripheral blood mtDNA damage and diabetic retinopathy, and suggest the possibility of use of peripheral blood mtDNA as a noninvasive biomarker of diabetic retinopathy

AMD Genomics: Non-Coding RNAs as Biomarkers and Therapeutic Targets

Age-related macular degeneration (AMD) is a progressive neurodegenerative disease that is the world&rsquo;s leading cause of blindness in the aging population. Although the clinical stages and forms of AMD have been elucidated, more specific prognostic tools are required to determine when patients with early and intermediate AMD will progress into the advanced stages of AMD. Another challenge in the field has been the appropriate development of therapies for intermediate AMD and advanced atrophic AMD. After numerous negative clinical trials, an anti-C5 agent and anti-C3 agent have recently shown promising results in phase 3 clinical trials, in terms of slowing the growth of geographic atrophy, an advanced form of AMD. Interestingly, both drugs appear to be associated with an increased incidence of wet AMD, another advanced form of the disease, and will require frequent intravitreal injections. Certainly, there remains a need for other therapeutic agents with the potential to prevent progression to advanced stages of the disease. Investigation of the role and clinical utility of non-coding RNAs (ncRNAs) is a major advancement in biology that has only been minimally applied to AMD. In the following review, we discuss the clinical relevance of ncRNAs in AMD as both biomarkers and therapeutic targets

Threshold-Based Ordering of Sequential Actions during Drosophila Courtship.

Goal-directed animal behaviors are typically composed of sequences of motor actions whose order and timing are critical for a successful outcome. Although numerous theoretical models for sequential action generation have been proposed, few have been supported by the identification of control neurons sufficient to elicit a sequence. Here, we identify a pair of descending neurons that coordinate a stereotyped sequence of engagement actions during Drosophila melanogaster male courtship behavior. These actions are initiated sequentially but persist cumulatively, a feature not explained by existing models of sequential behaviors. We find evidence consistent with a ramp-to-threshold mechanism, in which increasing neuronal activity elicits each action independently at successively higher activity thresholds

Threshold-based ordering of sequential actions during Drosophila courtship

Goal-directed animal behaviors are typically composed of sequences of motor actions whose order and timing are critical for a successful outcome. Although numerous theoretical models for sequential action generation have been proposed, few have been supported by the identification of control neurons sufficient to elicit a sequence. Here, we identify a pair of descending neurons that coordinate a stereotyped sequence of engagement actions during Drosophila melanogaster male courtship behavior. These actions are initiated sequentially but persist cumulatively, a feature not explained by existing models of sequential behaviors. We find evidence consistent with a ramp-to-threshold mechanism, in which increasing neuronal activity elicits each action independently at successively higher activity thresholds.McKellar et al. show that Drosophila courtship includes a stereotyped behavioral motif consisting of several overlapping actions. These multiple actions are controlled by a single pair of sexually dimorphic descending neurons. These neurons elicit each successive action at progressively higher threshold spike counts