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1.  HOXA4 protein levels and localization in the aorta and in human abdominal aortic aneurysms 
BMC Physiology  2011;11:18.
This report presents evidence for the specificities of select commercially available HOXA4 antibodies in regards to concerns about the specificity of the HOXA4 antibody used by Lillvis et al. (Regional expression of HOXA4 along the aorta and its potential role in human abdominal aortic aneurysms. BMC Physiol 2011, 11:9). Using an antibody characterized extensively by us, Lillvis et al. report detecting HOXA4 at a size of 33 kDa despite our previous reports that HOXA4 is detected at ~37-39 kDa and that the ~30-33 kDa band is non-specific. Using small interfering RNA targeting HOXA4, forced expression of full-length HOXA4 and HOXA4-positive and -negative ovarian cancer cell lines, we confirm our previous findings that the ~30-33 kDa band is non-specific and that HOXA4 is detected at ~37-39 kDa. Moreover, we demonstrate that HOXA4 small interfering RNA reduces the ~37-39 kDa HOXA4 band, but not the ~30-33 kDa non-specific band, in a human acute monocytic leukemia cell line used by Lillvis et al. Western blot analysis performed with two additional commercially available HOXA4 antibodies also detected HOXA4 at ~37-39 kDa. Lastly, immunofluorescent staining of a HOXA4-negative ovarian cancer cell line with the antibody used by Lillvis et al. yields strong perinuclear staining, similar to that observed by Lillvis et al., which cannot be attributed to HOXA4. Our results highlight and briefly discuss the importance of careful antibody validation and selection for use in various applications.
doi:10.1186/1472-6793-11-18
PMCID: PMC3254126  PMID: 22168796
2.  Regulation of CDX4 gene transcription by HoxA9, HoxA10, the Mll-Ell oncogene and Shp2 during leukemogenesis 
Oncogenesis  2014;3(12):e135-.
Cdx and Hox proteins are homeodomain transcription factors that regulate hematopoiesis. Transcription of the HOX and CDX genes decreases during normal myelopoiesis, but is aberrantly sustained in leukemias with translocation or partial tandem duplication of the MLL1 gene. Cdx4 activates transcription of the HOXA9 and HOXA10 genes, and HoxA10 activates CDX4 transcription. The events that break this feedback loop, permitting a decreased Cdx4 expression during normal myelopoiesis, were previously undefined. In the current study, we find that HoxA9 represses CDX4 transcription in differentiating myeloid cells, antagonizing activation by HoxA10. We determine that tyrosine phosphorylation of HoxA10 impairs transcriptional activation of CDX4, but tyrosine phosphorylation of HoxA9 facilitates repression of this gene. As HoxA9 and HoxA10 are phosphorylated during myelopoiesis, this provides a mechanism for differentiation stage-specific Cdx4 expression. HoxA9 and HoxA10 are increased in cells expressing Mll-Ell, a leukemia-associated MLL1 fusion protein. We find that Mll-Ell induces a HoxA10-dependent increase in Cdx4 expression in myeloid progenitor cells. However, Cdx4 decreases in a HoxA9-dependent manner on exposure of Mll-Ell-expressing cells to differentiating cytokines. Leukemia-associated, constitutively active mutants of Shp2 block cytokine-induced tyrosine phosphorylation of HoxA9 and HoxA10. In comparison with myeloid progenitor cells that are expressing Mll-Ell alone, we find increased CDX4 transcription and Cdx4 expression in cells co-expressing Mll-Ell plus constitutively active Shp2. Increased Cdx4 expression is sustained on exposure of these cells to differentiating cytokines. Our results identify a mechanism for increased and sustained CDX4 transcription in leukemias co-overexpressing HoxA9 and HoxA10 in combination with constitutive activation of Shp2. This is clinically relevant, because MLL1 translocations and constitutive Shp2 activation co-exist in human myeloid leukemias.
doi:10.1038/oncsis.2014.49
PMCID: PMC4275563  PMID: 25531430
3.  Vascular smooth muscle cell PPARγ deletion promotes abdominal aortic aneurysms 
Objective
Peroxisome proliferator-activated receptor-gamma (PPARγ) is known to play an important role in the vasculature. However, the role of PPARγ in abdominal aortic aneurysms (AAA) is not well understood. We hypothesized that PPARγ in smooth muscle cells attenuates the development of AAA. In this study, we also investigated PPARγ-mediated signaling pathways that may prevent the development of AAA.
Methods and Results
In the present study, we determined whether periaortic application of CaCl2 renders vascular smooth muscle cell-selective PPARγ knockout (SMPG KO) mice more susceptible to destruction of normal aortic wall architecture. There is evidence of increased vessel dilatation in the abdominal aorta six weeks after 0.25 M periaortic CaCl2 application in SMPG KO mice compared to littermate controls (1.4±0.3 mm, n=8 versus 1.1±0.2 mm, n=7; P = .000119). Furthermore, results from SMPG KO mice indicate medial layer elastin degradation was greater six weeks after abluminal application of CaCl2 to the abdominal aorta (P <.01). Next, we found that activated cathepsin S, a potent elastin-degrading enzyme, is increased in SMPG KO mice when compared to wild-type controls. To further identify a role of PPARγ signaling in reducing the development of AAA, we demonstrated that adenoviral-mediated PPARγ overexpression in cultured rat aortic smooth muscle cells decreases (P = .022) the mRNA levels of cathepsin S. In addition, a chromatin immunoprecipitation (ChIP) assay detected PPARγ bound to a peroxisome proliferator-activated receptor response element (PPRE) −141 to −159 bp upstream of the cathepsin S gene sequence in mouse aortic smooth muscle cells. Also, adenoviral-mediated PPARγ overexpression and knockdown in cultured rat aortic smooth muscle cells decreases (P = .013) and increases (P = .018) expression of activated cathepsin S. Finally, immunohistochemistry demonstrated a greater inflammatory infiltrate in SMPG KO mouse aortas, as evidenced by elevations in F4/80 and tumor necrosis factor-alpha expression.
Conclusion
In this study, we identify PPARγ as an important contributor in attenuating the development of aortic aneurysms by demonstrating that loss of PPARγ in vascular smooth muscle cells promotes aortic dilatation and elastin degradation. Thus, PPARγ activation may be potentially promising medical therapy in reducing the risk of AAA progression and rupture.
doi:10.1016/j.jvs.2010.05.089
PMCID: PMC2949502  PMID: 20630681
4.  MicroRNA expression signature in human abdominal aortic aneurysms 
BMC Medical Genomics  2012;5:25.
Background
Abdominal aortic aneurysm (AAA) is a dilatation of the aorta affecting most frequently elderly men. Histologically AAAs are characterized by inflammation, vascular smooth muscle cell apoptosis, and extracellular matrix degradation. The mechanisms of AAA formation, progression, and rupture are currently poorly understood. A previous mRNA expression study revealed a large number of differentially expressed genes between AAA and non-aneurysmal control aortas. MicroRNAs (miRNAs), small non-coding RNAs that are post-transcriptional regulators of gene expression, could provide a mechanism for the differential expression of genes in AAA.
Methods
To determine differences in miRNA levels between AAA (n = 5) and control (n = 5) infrarenal aortic tissues, a microarray study was carried out. Results were adjusted using Benjamini-Hochberg correction (adjusted p < 0.05). Real-time quantitative RT-PCR (qRT-PCR) assays with an independent set of 36 AAA and seven control tissues were used for validation. Potential gene targets were retrieved from miRNA target prediction databases Pictar, TargetScan, and MiRTarget2. Networks from the target gene set were generated and examined using the network analysis programs, CytoScape® and Ingenuity Pathway Core Analysis®.
Results
A microarray study identified eight miRNAs with significantly different expression levels between AAA and controls (adjusted p < 0.05). Real-time qRT-PCR assays validated the findings for five of the eight miRNAs. A total of 222 predicted miRNA target genes known to be differentially expressed in AAA based on a prior mRNA microarray study were identified. Bioinformatic analyses revealed that several target genes are involved in apoptosis and activation of T cells.
Conclusions
Our genome-wide approach revealed several differentially expressed miRNAs in human AAA tissue suggesting that miRNAs play a role in AAA pathogenesis.
doi:10.1186/1755-8794-5-25
PMCID: PMC3507654  PMID: 22704053
Apoptosis; Microarray analysis; Vascular biology; miRNA-mRNA analysis; Network analysis
5.  Elastogenic Inductability of Smooth Muscle Cells from a Rat Model of Late Stage Abdominal Aortic Aneurysms 
Tissue Engineering. Part A  2011;17(13-14):1699-1711.
Although abdominal aortic aneurysms (AAA) can be potentially stabilized by inhibiting inflammatory cell recruitment and their release of proteolytic enzymes, active AAA regression is not possible without regeneration of new elastic matrix structures. Unfortunately, postneonatal vascular smooth muscle cells (SMCs), healthy, and likely more so, diseased cells, poorly synthesize or remodel elastic fibers, impeding any effort directed at regenerative AAA treatment. Previously, we determined the eleastogenic benefits of oligomers (HA-o; 4–6 mers) of the glycosaminoglycan, hyaluronan (HA) and transforming growth factor-β1 (TGF-β1) to healthy SMCs. Since AAAs are often diagnosed only late in development when matrix disruption is severe, we now determine if elastogenic upregulation of SMCs from late-stage AAAs (>100% diameter increase) is possible. AAAs were induced by perfusion of rat infrarenal aortae with porcine pancreatic elastase. Elastic matrix degradation, vessel expansion (∼120%), inflammatory cell infiltration, and enhanced activity of matrix-metalloproteases (MMPs) 2 and 9 resulted, paralleling human AAAs. Aneurysmal SMCs (EaRASMCs) maintained a diseased phenotype in 2D cell culture and exhibited patterns of gene expression different from healthy rat aortic SMCs (RASMCs). Relative to passage-matched healthy RASMCs, unstimulated EaRASMCs produced far less tropoelastin and matrix elastin. Exogenous TGF-β and HA-o (termed “factors”) significantly decreased EaRASMC proliferation and enhanced tropoelastin synthesis, though only at the highest provided dose combination (20 mg/mL of HA-o, 10 ng/mL of TGF-β); despite such enhancement, tropoelastin amounts were only ∼40% of amounts synthesized by healthy RASMC cultures. Differently, elastic matrix synthesis was enhanced beyond amounts synthesized by healthy RASMCs (112%), even at lower doses of factors (2 mg/mL of HA-o and 5 ng/mL of TGF-β). The factors also enhanced elastic fiber deposition over untreated EaRASMC cultures and restored several genes whose expression was altered in EaRASMC cultures back to levels expressed by healthy RASMCs. However, the activity of MMPs 2 and 9 generated by EaRASMC cultures was unaffected by the factors/factor dose. The study confirms that SMCs from advanced AAAs can be elastogenically induced, although much higher doses of elastogenic factors are required for induction relative to healthy SMCs. Also, the factors do not appear to inhibit MMP activity, vital to preserve existing elastic matrix structures that serve as nucleation sites for new elastic fiber deposition. Thus, to enhance net accumulation of newly regenerated elastic matrix, toward possibly regressing AAAs, codelivery of MMP inhibitors may be necessitated.
doi:10.1089/ten.tea.2010.0526
PMCID: PMC3118732  PMID: 21341992
6.  Analysis of In Situ and Ex Vivo Vascular Endothelial Growth Factor Receptor Expression During Experimental Aortic Aneurysm Progression 
Objective
Mural inflammation and neovascularization are characteristic pathologic features of abdominal aortic aneurysm (AAA) disease. Vascular endothelial growth factor receptor (VEGFR) expression may also mediate AAA growth and rupture. We examined VEGFR expression as a function of AAA disease progression in the Apolipoprotein E- deficient (Apo E−/−) murine AAA model.
Methods
Apo E−/− mice maintained on a high fat diet underwent continuous infusion with angiotensin II at 1000 ng/kg/min (Ang II) or vehicle (Control) via subcutaneous osmotic pump. Serial transabdominal ultrasound measurements of abdominal aortic diameter were recorded (n = 16 mice, 3 – 4 time points/ mouse) for up to 28 days. Near-infrared receptor fluorescent (NIRF) imaging was performed on Ang II mice (n = 9) and Controls (n=5) with scVEGF/Cy, a single-chain VEGF homo-dimer labeled with Cy5.5 fluorescent tracer (7–18 µg/mouse IV). NIRF with inactivated single chain VEGF/Cy tracer (scVEGF/In, 18 µg/mouse IV) was performed on two additional Ang II mice to control for non-receptor mediated tracer binding and uptake. Following image acquisition and sacrifice, aortae were harvested for analysis. An additional AAA mouse cohort received either an oral angiogenesis inhibitor or suitable negative or positive controls to clarify the significance of angiogenesis in experimental aneurysm progression.
Results
Aneurysms developed in the suprarenal aortic segment of all Ang II mice. Significantly greater fluorescent signal was obtained from aneurysmal aorta as compared to remote, uninvolved aortic segments in Ang II scVEGF/Cy mice or AAA in scVEGF/In mice or suprarenal aortic segments in Control mice. Signal intensity increased in a diameter-dependent fashion in aneurysmal segments. Immunostaining confirmed mural VEGFR-2 expression in medial smooth muscle cells. Treatment with an angiogenesis inhibitor attenuated AAA formation while decreasing mural macrophage infiltration and CD-31+ cell density.
Conclusion
Mural VEGFR expression, as determined by scVEGF/Cy fluorescent imaging and VEGFR-2 immunostaining, increases in experimental AAAs in a diameter-dependent fashion. Angiogenesis inhibition limits AAA progression. Clinical VEGFR expression imaging strategies, if feasible, may improve real-time monitoring of AAA disease progression and response to suppressive strategies.
doi:10.1161/ATVBAHA.109.187757
PMCID: PMC2752726  PMID: 19574559
Vascular endothelial growth factor; abdominal aortic aneurysms; imaging; neovascularization; angiogenesis inhibition
7.  Transient Exposure of Neonatal Female Mice to Testosterone Abrogates the Sexual Dimorphism of Abdominal Aortic Aneurysms 
Circulation research  2012;110(11):e73-e85.
Rationale
Abdominal aortic aneurysms (AAAs) exhibit marked sexual dimorphism with higher prevalences in men. Similarly, AAAs induced by angiotensin II (AngII) infusion into mice exhibit a higher prevalence in males. Testosterone promotes AAA pathology in adult male mice through regulation of angiotensin type 1A receptors (AT1aR) in abdominal aortas. However, mechanisms for sexual dimorphism of regional aortic angiotensin receptor expression and AAA formation are unknown.
Objective
To define the role of developmental testosterone exposures in sexual dimorphism of AAAs, we determined if exposure of neonatal female mice to testosterone confers adult susceptibility to AngII-induced AAAs.
Methods and Results
One day old female hypercholesterolemic mice were administered a single dose of either vehicle or testosterone. Neonatal testosterone administration increased abdominal aortic AT1aR mRNA abundance and promoted a striking increase in AngII-induced AAAs in adult females exhibiting low serum testosterone concentrations. AngII-induced atherosclerosis and ascending aortic aneurysms were also increased by testosterone administration to neonatal females. In contrast, neonatal testosterone administration in males had no effect on AngII-induced vascular pathologies. Deficiency of AT1aR in smooth muscle cells (SMCs) reduced effects of neonatal testosterone to promote AAAs in adult females, but did not alter atherosclerosis or ascending aortic aneurysms. Testosterone increased AT1aR mRNA abundance and hydrogen peroxide generation in cultured abdominal aortic SMCs. Increased AT1aR mRNA abundance was maintained during progressive passaging of female SMCs.
Conclusions
These data reveal an unrecognized role of transient sex hormone exposures during neonatal development as long-lasting mediators of regional aortic AT1aR expression and sexual dimorphism of AAAs.
doi:10.1161/CIRCRESAHA.111.253880
PMCID: PMC3518797  PMID: 22539767
aneurysm; sexual dimorphism; testosterone; vascular smooth muscle; angiotensin
8.  HOXA1 is overexpressed in oral squamous cell carcinomas and its expression is correlated with poor prognosis 
BMC Cancer  2012;12:146.
Background
HOX genes encode homeodomain-containing transcription factors involved in the regulation of cellular proliferation and differentiation during embryogenesis. However, members of this family demonstrated oncogenic properties in some malignancies. The present study investigated whether genes of the HOXA cluster play a role in oral cancer.
Methods
In order to identify differentially expressed HOXA genes, duplex RT-PCR in oral samples from healthy mucosa and squamous cell carcinoma was used. The effects of HOXA1 on proliferation, apoptosis, adhesion, invasion, epithelial-mesenchymal transition (EMT) and anchorage-independent growth were assessed in cells with up- and down-regulation of HOXA1. Immunohistochemical analysis using a tissue microarray (TMA) containing 127 oral squamous cell carcinomas (OSCC) was performed to determine the prognostic role of HOXA1 expression.
Results
We showed that transcripts of HOXA genes are more abundant in OSCC than in healthy oral mucosa. In particular, HOXA1, which has been described as one of the HOX members that plays an important role in tumorigenesis, was significantly more expressed in OSCCs compared to healthy oral mucosas. Further analysis demonstrated that overexpression of HOXA1 in HaCAT human epithelial cells promotes proliferation, whereas downregulation of HOXA1 in human OSCC cells (SCC9 cells) decreases it. Enforced HOXA1 expression in HaCAT cells was not capable of modulating other events related to tumorigenesis, including apoptosis, adhesion, invasion, EMT and anchorage-independent growth. A high number of HOXA1-positive cells was significantly associated with T stage, N stage, tumor differentiation and proliferative potential of the tumors, and was predictive of poor survival. In multivariate analysis, HOXA1 was an independent prognostic factor for OSCC patients (HR: 2.68; 95% CI: 1.59-2.97; p = 0.026).
Conclusion
Our findings indicate that HOXA1 may contribute to oral carcinogenesis by increasing tumor cell proliferation, and suggest that HOXA1 expression might be helpful as a prognostic marker for patients with OSCC.
doi:10.1186/1471-2407-12-146
PMCID: PMC3351375  PMID: 22498108
Oral cancer; HOXA1; Cellular proliferation; Prognosis
9.  Homeobox A7 increases cell proliferation by up-regulation of epidermal growth factor receptor expression in human granulosa cells 
Background
Homeobox (HOX) genes encode transcription factors, which regulate cell proliferation, differentiation, adhesion, and migration. The deregulation of HOX genes is frequently associated with human reproductive system disorders. However, knowledge regarding the role of HOX genes in human granulosa cells is limited.
Methods
To determine the role of HOXA7 in the regulation and associated mechanisms of cell proliferation in human granulosa cells, HOXA7 and epidermal growth factor receptor (EGFR) expressions were examined in primary granulosa cells (hGCs), an immortalized human granulosa cell line, SVOG, and a granulosa tumor cell line, KGN, by real-time PCR and Western blotting. To manipulate the expression of HOXA7, the HOXA7 specific siRNA was used to knockdown HOXA7 in KGN. Conversely, HOXA7 was overexpressed in SVOG by transfection with the pcDNA3.1-HOAX7 vector. Cell proliferation was measured by the MTT assay.
Results
Our results show that HOXA7 and EGFR were overexpressed in KGN cells compared to hGCs and SVOG cells. Knockdown of HOXA7 in KGN cells significantly decreased cell proliferation and EGFR expression. Overexpression of HOXA7 in SVOG cells significantly promoted cell growth and EGFR expression. Moreover, the EGF-induced KGN proliferation was abrogated, and the activation of downstream signaling was diminished when HOXA7 was knocked down. Overexpression of HOXA7 in SVOG cells had an opposite effect.
Conclusions
Our present study reveals a novel mechanistic role for HOXA7 in modulating granulosa cell proliferation via the regulation of EGFR. This finding contributes to the knowledge of the pro-proliferation effect of HOXA7 in granulosa cell growth and differentiation.
doi:10.1186/1477-7827-8-61
PMCID: PMC2904782  PMID: 20540809
10.  Overexpression of Catalase in Vascular Smooth Muscle Cells Prevents the Formation of Abdominal Aortic Aneurysms 
Arteriosclerosis, thrombosis, and vascular biology  2013;33(10):10.1161/ATVBAHA.113.302175.
Objective
Elevated levels of oxidative stress have been reported in abdominal aortic aneurysms (AAA), but which reactive oxygen species (ROS) promotes the development of AAA remains unclear. Here we investigate the effect of the hydrogen peroxide (H2O2) degrading enzyme catalase on the formation of AAA.
Approach and Results
AAA were induced with the application of calcium chloride (CaCl2) on mouse infrarenal aortas. The administration of PEG-catalase, but not saline, attenuated the loss of tunica media and protected against AAA formation (0.91±0.1 mm vs. 0.76±0.09 mm). Similarly, in a transgenic mouse model, catalase over-expression in the vascular smooth muscle cells (VSMC) preserved the thickness of tunica media and inhibited aortic dilatation by 50% (0.85±0.14 mm vs. 0.57±0.08 mm). Further studies showed that injury with CaCl2 decreased catalase expression and activity in the aortic wall. Pharmacologic administration or genetic over-expression of catalase restored catalase activity and subsequently decreased matrix metalloproteinase activity. In addition, a profound reduction in inflammatory markers and VSMC apoptosis was evident in aortas of catalase over-expressing mice. Interestingly, as opposed to infusion of PEG-catalase, chronic over-expression of catalase in VSMC did not alter the total aortic H2O2 levels.
Conclusions
The data suggest that a reduction in aortic wall catalase activity can predispose to AAA formation. Restoration of catalase activity in the vascular wall enhances aortic VSMC survival and prevents AAA formation primarily through modulation of matrix metalloproteinase activity.
doi:10.1161/ATVBAHA.113.302175
PMCID: PMC3880803  PMID: 23950141
AAA; catalase; hydrogen peroxide; VSMC; MMP
11.  Peptide inhibitor of CXCL4-CCL5 heterodimer formation, MKEY, inhibits aortic aneurysm initiation and progression in mice 
Objective
Macrophages are critical contributors in abdominal aortic aneurysm (AAA) disease. We examined the ability of MKEY, a peptide inhibitor of CXCL4-CCL5 interaction, to influence AAA progression in murine models.
Methods and Results
AAAs were created in 10-week-old male C57BL/6 mice by transient infrarenal aortic porcine pancreatic elastase (PPE) infusion. Mice were treated with MKEY via intravenous injection either 1) before PPE infusion, or 2) after aneurysm initiation. Immunostaining demonstrated CCL5 and CCR5 expression on aneurysmal aortae and mural monocytes/macrophages, respectively. MKEY treatment partially inhibited transmural AAA migration of adaptively transferred leukocytes in recipient mice. While all vehicle-pretreated mice developed AAA, aneurysms formed in only 60% (3/5) and 14% (1/7) of mice pretreated with MKEY at 10 and 20 mg /kg, respectively. MKEY pretreatment reduced aortic diameter enlargement, preserved medial elastin fibers and smooth muscle cells, and attenuated mural macrophage infiltration, angiogenesis, and aortic MMP2 & 9 expression following PPE infusion. MKEY initiated after PPE infusion also stabilized and/or reduced enlargement of existing AAAs. Finally, MKEY treatment was effective in limiting AAA formation following angiotensin II infusion in apolipoprotein E deficient mice.
Conclusion
MKEY suppresses AAA formation and progression in two complementary experimental models. Peptide inhibition of CXCL4-CCL5 interactions may represent a viable translational strategy to limit progression of human AAA disease.
doi:10.1161/ATVBAHA.112.300329
PMCID: PMC4158029  PMID: 23288157
abdominal aortic aneurysm; chemokines; CCL5; mice
12.  Global analysis of genes regulated by HOXA10 in decidualization reveals a role in cell proliferation 
Molecular human reproduction  2008;14(6):357-366.
Homeobox (HOX) A10 is essential for fertility as demonstrated in transgenic mice, specifically affecting implantation and decidualization. Its role in human decidualization, however, remains unknown. In this study, we used gene silencing followed by microarray analysis to decipher the role of HOXA10 during decidualization of human endometrial stromal cells (HESCs). HOXA10 was knocked down using siRNA oligonucleotide transfection and cells were treated with estradiol, medroxyprogesterone acetate and dibutyryl cAMP (H + cAMP) to induce decidualization. Genes significantly regulated were identified using the Affymetrix microarray chip. With this method, 2361 transcripts were significantly altered by 1.5-fold or higher (P < 0.05) with H + cAMP treatment only. Of these genes, 258 were significantly up-regulated by HOXA10 knockdown and 236 transcripts were significantly down-regulated by more than 1.5-fold, totaling 494 genes that were regulated by HOXA10 during decidualization. Data analysis using the Ingenuity System revealed that many of the genes regulated by HOXA10 knockdown during H + cAMP treatment were associated with cell cycle. Real-time PCR was used to confirm that HOXA10 knockdown decreased expression of the cell cycle genes CDC2 and CCNB2. In addition, a higher percentage of cells were arrested in the G2/M phase. Next, we observed that cell proliferation as measured by BrdU incorporation was decreased upon HOXA10 knockdown and H + cAMP treatment. Apoptosis, on the other hand, as measured by Annexin V staining was not influenced by siHOXA10 in decidualizing cells. Together, these data demonstrate that during decidualization of HESC, HOXA10 is actively involved in promoting cell proliferation through the regulation of hundreds of genes.
doi:10.1093/molehr/gan023
PMCID: PMC2731304  PMID: 18456676
decidualization; HOXA10; endometrium; gene silencing; microarray analysis
13.  HOXA9 Participates in the Transcriptional Activation of E-Selectin in Endothelial Cells▿  
Molecular and Cellular Biology  2007;27(12):4207-4216.
The homeobox gene HOXA9 has recently been shown to be an important regulator of endothelial cell (EC) differentiation and activation in addition to its role in embryonic development and hematopoiesis. In this report, we have determined that the EC-leukocyte adhesion molecule E-selectin is a key target for HOXA9. The depletion of HOXA9 protein in ECs resulted in a significant and specific decrease in tumor necrosis factor alpha (TNF-α)-induced E-selectin gene expression. In addition, HOXA9 specifically activated the E-selectin gene promoter in ECs. Progressive deletional analyses together with site-specific mutagenesis of the E-selectin promoter indicated that the Abd-B-like HOX DNA-binding motif, CAATTTTATTAA, located in the proximal region spanning bp −210 to −221 upstream of the transcription start site was crucial for the promoter induction by HOXA9. Both HOXA9 in EC nuclear extract and recombinant HOXA9 protein bound to this sequence in vitro. Moreover, we showed that HOXA9 binds temporally, in a TNF-α-dependent manner, to the region containing this Abd-B-like element in vivo. We have thus identified a novel and functionally critical cis-regulatory element for TNF-α-mediated transient expression of the E-selectin gene. Further, we provide evidence that HOXA9 acts as an obligate proinflammatory factor by mediating cytokine induction of E-selectin.
doi:10.1128/MCB.00052-07
PMCID: PMC1900059  PMID: 17452460
14.  Analysis of positional candidate genes in the AAA1 susceptibility locus for abdominal aortic aneurysms on chromosome 19 
BMC Medical Genetics  2011;12:14.
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.
doi:10.1186/1471-2350-12-14
PMCID: PMC3037298  PMID: 21247474
15.  Ultrasound Screening for Abdominal Aortic Aneurysm 
Executive Summary
Objective
The aim of this review was to assess the effectiveness of ultrasound screening for asymptomatic abdominal aortic aneurysm (AAA).
Clinical Need
Abdominal aortic aneurysm is a localized abnormal dilatation of the aorta greater than 3 cm. In community surveys, the prevalence of AAA is reported to be between 2% and 5.4%. Abdominal aortic aneurysms are found in 4% to 8% of older men and in 0.5% to 1.5% of women aged 65 years and older. Abdominal aortic aneurysms are largely asymptomatic. If left untreated, the continuing extension and thinning of the vessel wall may eventually result in rupture of the AAA. Often rupture may occur without warning, causing acute pain. Rupture is always life threatening and requires emergency surgical repair of the ruptured aorta. The risk of death from ruptured AAA is 80% to 90%. Over one-half of all deaths attributed to a ruptured aneurysm take place before the patient reaches hospital. In comparison, the rate of death in people undergoing elective surgery is 5% to 7%; however, symptoms of AAA rarely occur before rupture. Given that ultrasound can reliably visualize the aorta in 99% of the population, and its sensitivity and specificity for diagnosing AAA approaches 100%, screening for aneurysms is worth considering as it may reduce the incidence of ruptured aneurysms and hence reduce unnecessary deaths caused by AAA-attributable mortality.
Review Strategy
The Medical Advisory Secretariat used its standard search strategy to retrieve international health technology assessments and English-language journal articles from selected databases to determine the effectiveness of ultrasound screening for abdominal aortic aneurysms. Case reports, letters, editorials, nonsystematic reviews, non-human studies, and comments were excluded.
Questions asked:
Is population-based AAA screening effective in improving health outcomes in asymptomatic populations?
Is AAA screening acceptable to the population? Does this affect the effectiveness the screening program?
How often should population-based screening occur?
What are appropriate treatment options after screening based on the size of aneurysms?
Are there differences between universal and targeted screening strategies?
What are the harms of screening?
Summary of Findings
Population-based ultrasound screening is effective in men aged 65 to 74 years, particularly in those with a history of smoking. Screening reduces the incidence of AAA ruptures, and decreases rates of emergency surgical repair for AAA and AAA-attributable mortality.
Acceptance rates decline with increasing age and are lower for women. Low acceptance rates may affect the effectiveness of a screening program.
A one-time screen is sufficient for a population-based screening program with regard to initial negative scans and development of large AAAs.
There is no difference between early elective surgical repair and surveillance for small aneurysms (4.0–5.4 cm). Repeated surveillance of small aneurysms is recommended.
Targeted screening based on history of smoking has been found to detect 89% of prevalent AAAs and increase the efficiency of screening programs from statistical modeling data.
Women have not been studied for AAA screening programs. There is evidence suggesting that screening women for AAA should be considered with respect to mortality and case fatality rates in Ontario. It is important that further evaluation of AAAs in women occur.
There is a small risk of physical harm from screening. Less than 1% of aneurysms will not be visualized on initial screen and a re-screen may be necessary; elective surgical repair is associated with a 6% operative morality rate and about 3% of small aneurysms may rupture during surveillance. These risks should be communicated through informed consent prior to screening.
There is little evidence of severe psychological harms associated with screening.
Conclusions
Based on this review, the Medical Advisory Secretariat concluded that there is sufficient evidence to determine that AAA screening using ultrasound is effective and reduces negative health outcomes associated with the condition.
Moreover, screening for AAA is cost-effective, comparing favorably for the cost of per life year gained for screening programs for cervical cancer, hypertension, and breast cancer that are in practice in Ontario, with a high degree of compliance, and can be undertaken with a minimal effort at fewer than 10 minutes to screen each patient.
Overall, the clinical utility of an invitation to use ultrasound screening to identify AAA in men aged 65 to 74 is effective at reducing AAA-attributable mortality. The benefit of screening women is not yet established. However, Ontario data indicate several areas of concern including population prevalence, detection of AAA in women, and case management of AAA in women in terms of age cutoffs for screening and natural history of disease associated with age of rupture.
PMCID: PMC3379169  PMID: 23074490
16.  Endovascular Repair of Abdominal Aortic Aneurysm 
EXECUTIVE SUMMARY
The Medical Advisory Secretariat conducted a systematic review of the evidence on the effectiveness and cost-effectiveness of endovascular repair of abdominal aortic aneurysm in comparison to open surgical repair. An abdominal aortic aneurysm [AAA] is the enlargement and weakening of the aorta (major blood artery) that may rupture and result in stroke and death. Endovascular abdominal aortic aneurysm repair [EVAR] is a procedure for repairing abdominal aortic aneurysms from within the blood vessel without open surgery. In this procedure, an aneurysm is excluded from blood circulation by an endograft (a device) delivered to the site of the aneurysm via a catheter inserted into an artery in the groin. The Medical Advisory Secretariat conducted a review of the evidence on the effectiveness and cost-effectiveness of this technology. The review included 44 eligible articles out of 489 citations identified through a systematic literature search. Most of the research evidence is based on non-randomized comparative studies and case series. In the short-term, EVAR appears to be safe and comparable to open surgical repair in terms of survival. It is associated with less severe hemodynamic changes, less blood transfusion and shorter stay in the intensive care and hospital. However, there is concern about a high incidence of endoleak, requiring secondary interventions, and in some cases, conversion to open surgical repair. Current evidence does not support the use of EVAR in all patients. EVAR might benefit individuals who are not fit for surgical repair of abdominal aortic aneurysm and whose risk of rupture of the aneurysm outweighs the risk of death from EVAR. The long-term effectiveness and cost-effectiveness of EVAR cannot be determined at this time. Further evaluation of this technology is required.
OBJECTIVE
The objective of this health technology policy assessment was to determine the effectiveness and cost-effectiveness of endovascular repair of abdominal aortic aneurysms (EVAR) in comparison to open surgical repair (OSR).
BACKGROUND
Clinical Need
An abdominal aortic aneurysm (AAA) is a localized, abnormal dilatation of the aorta greater than 3 cm or 50% of the aortic diameter at the diaphragm. (1) A true AAA involves all 3 layers of the vessel wall. If left untreated, the continuing extension and thinning of the vessel wall may eventually result in rupture of the AAA. The risk of death from ruptured AAA is 80% to 90%. (61) Heller et al. (44) analyzed information from a national hospital database in the United States. They found no significant change in the incidence rate of elective AAA repair or ruptured AAA presented to the nation’s hospitals. The investigators concluded that technologic and treatment advances over the past 19 years have not affected the outcomes of patients with AAAs, and the ability to identify and to treat patients with AAAs has not improved.
Classification of Abdominal Aortic Aneurysms
At least 90% of the AAAs are affected by atherosclerosis, and most of these aneurysms are below the level of the renal arteries.(1)
An abdominal aortic aneurysm may be symptomatic or asymptomatic. An AAA may be classified according to their sizes:(7)
Small aneurysms: less than 5 cm in diameter.
Medium aneurysms: 5-7cm.
Large aneurysms: more than 7 cm in diameter.
Small aneurysms account for approximately 50% of all clinically recognized aneurysms.(7)
Aortic aneurysms may be classified according to their gross appearance as follows (1):
Fusiform aneurysms affect the entire circumference of a vessel, resulting in a diffusely dilated lesion
Saccular aneurysms involve only a portion of the circumference, resulting in an outpouching (protrusion) in the vessel wall.
Prevalence of Abdominal Aortic Aneurysms
In community surveys, the prevalence of AAA is reported to be between 1% and 5.4%. (61) The prevalence is related to age and vascular risk factors. It is more common in men and in those with a positive family history.
In Canada, Abdominal aortic aneurysms are the 10th leading cause of death in men 65 years of age or older. (60) Naylor (60) reported that the rate of AAA repair in Ontario has increased from 38 per 100,000 population in 1981/1982 to 54 per 100,000 population in 1991/1992. For the period of 1989/90 to 1991/92, the rate of AAA repair in Ontarians age 45 years and over was 53 per 100,000. (60) In the United States, about 200,000 new cases are diagnosed each year, and 50,000 to 60,000 surgical AAA repairs are performed. (2) Ruptured AAAs are responsible for about 15,000 deaths in the United States annually. One in 10 men older than 80 years has some aneurysmal change in his aorta. (2)
Symptoms of Abdominal Aortic Aneurysms
AAAs usually do not produce symptoms. However, as they expand, they may become painful. Compression or erosion of adjacent tissue by aneurysms also may cause symptoms. The formation of mural thrombi, a type of blood clots, within the aneurysm may predispose people to peripheral embolization, where blood vessels become blocked. Occasionally, an aneurysm may leak into the vessel wall and the periadventitial area, causing pain and local tenderness. More often, acute rupture occurs without any prior warning, causing acute pain and hypotension. This complication is always life-threatening and requires an emergency operation.
Diagnosis of Abdominal Aortic Aneurysms
An AAA is usually detected on routine examination as a palpable, pulsatile, and non-tender mass. (1)
Abdominal radiography may show the calcified outline of the aneurysms; however, about 25% of aneurysms are not calcified and cannot be visualized by plain x-ray. (1) An abdominal ultrasound provides more accurate detection, can delineate the traverse and longitudinal dimensions of the aneurysm, and is useful for serial documentation of aneurysm size. Computed tomography and magnetic resonance have also been used for follow-up of aortic aneurysms. These technologies, particularly contrast-enhanced computer tomography, provide higher resolution than ultrasound.
Abdominal aortography remains the gold standard to evaluate patients with aneurysms for surgery. This technique helps document the extent of the aneurysms, especially their upper and lower limits. It also helps show the extent of associated athereosclerotic vascular disease. However, the procedure carries a small risk of complications, such as bleeding, allergic reactions, and atheroembolism. (1)
Prognosis of Abdominal Aortic Aneurysms
The risk of rupture of an untreated AAA is a continuous function of aneurysm size as represented by the maximal diameter of the AAA. The annual rupture rate is near zero for aneurysms less than 4 cm in diameter. The risk is about 1% per year for aneurysms 4 to 4.9 cm, 11% per year for aneurysms 5 to 5.9 cm, and 25% per year or more for aneurysms greater than 6 cm. (7)
The 1-year mortality rate of patients with AAAs who do not undergo surgical treatment is about 25% if the aneurysms are 4 to 6 cm in diameter. This increases to 50% for aneurysms exceeding 6 cm. Other major causes of mortality for people with AAAs include coronary heart disease and stroke.
Treatment of Abdominal Aortic Aneurysms
Treatment of an aneurysm is indicated under any one of the following conditions:
The AAA is greater than 6 cm in diameter.
The patient is symptomatic.
The AAA is rapidly expanding irrespective of the absolute diameter.
Open surgical repair of AAA is still the gold standard. It is a major operation involving the excision of dilated area and placement of a sutured woven graft. The surgery may be performed under emergent situation following the rupture of an AAA, or it may be performed electively.
Elective OSR is generally considered appropriate for healthy patients with aneurysms 5 to 6 cm in diameter. (7) Coronary artery disease is the major underlying illness contributing to morbidity and mortality in OSR. Other medical comorbidities, such as chronic renal failure, chronic lung disease, and liver cirrhosis with portal hypertension, may double or triple the usual risk of OSR.
Serial noninvasive follow-up of small aneurysms (less than 5 cm) is an alternative to immediate surgery.
Endovascular repair of AAA is the third treatment option and is the topic of this review.
PMCID: PMC3387737  PMID: 23074438
17.  Clustering of Tissue-Specific Sub-TADs Accompanies the Regulation of HoxA Genes in Developing Limbs 
PLoS Genetics  2013;9(12):e1004018.
HoxA genes exhibit central roles during development and causal mutations have been found in several human syndromes including limb malformation. Despite their importance, information on how these genes are regulated is lacking. Here, we report on the first identification of bona fide transcriptional enhancers controlling HoxA genes in developing limbs and show that these enhancers are grouped into distinct topological domains at the sub-megabase scale (sub-TADs). We provide evidence that target genes and regulatory elements physically interact with each other through contacts between sub-TADs rather than by the formation of discreet “DNA loops”. Interestingly, there is no obvious relationship between the functional domains of the enhancers within the limb and how they are partitioned among the topological domains, suggesting that sub-TAD formation does not rely on enhancer activity. Moreover, we show that suppressing the transcriptional activity of enhancers does not abrogate their contacts with HoxA genes. Based on these data, we propose a model whereby chromatin architecture defines the functional landscapes of enhancers. From an evolutionary standpoint, our data points to the convergent evolution of HoxA and HoxD regulation in the fin-to-limb transition, one of the major morphological innovations in vertebrates.
Author Summary
Hox genes encode transcription factors with crucial roles during development. These genes are grouped in four different clusters names HoxA, B, C, and D. Mutations in genes of the HoxA and D clusters have been found in several human syndromes, affecting in some cases limb development. Despite their essential role and contrary to the genes of the HoxD cluster, little is known about how the HoxA genes are regulated. Here, we identified a large set of regulatory elements controlling HoxA genes during limb development. By studying spatial chromatin organization at the HoxA region, we found that the regulatory elements are spatially clustered regardless of their activity. Clustering of enhancers define tissue-specific chromatin domains that interact specifically with each other and with active genes in the limb. Our findings give support to the emerging concept that chromatin architecture defines the functional properties of genomes. Additionally, our study suggests a common constraint of the chromatin topology in the evolution of HoxA and HoxD regulation in the emergence of the hand/foot, which is one of the major morphological innovations in vertebrates.
doi:10.1371/journal.pgen.1004018
PMCID: PMC3873244  PMID: 24385922
18.  The Truncated Hoxa1 Protein Interacts with Hoxa1 and Pbx1 in Stem Cells 
Journal of cellular biochemistry  2009;106(3):427-443.
Hox genes contain a homeobox encoding a 60-amino acid DNA binding sequence. The Hoxa-1 gene (Hox1.6, ERA1) encodes two alternatively spliced mRNAs that encode distinct proteins, one with the homeodomain (Hoxa1-993), and another protein lacking this domain (Hoxa1-399). The functions of Hoxa1-399 are unknown. We detected Hoxa1-993 and Hoxa1-399 by immunoprecipitation using Hoxa1 antibodies. To assess whether Hoxa1-399 functions in cellular differentiation we analyzed Hoxb1, a Hoxa1 target gene. Hoxa1-993 and its cofactor, Pbx1, bind to the Hoxb1 SOct-R3 promoter to transcriptionally activate a luciferase reporter. Results from F9 stem cells that stably express ectopic Hoxa1-399 (the F9-399 line) show that Hoxa1-399 reduces this transcriptional activation. Gel shift assays demonstrate that Hoxa1-399 reduces Hoxa1-993/Pbx1 binding to the Hoxb1 SOct-R3 region. GST-pull down experiments suggest that Hoxa1-399, Hoxa1-993, and Pbx1 form a trimer. However, the F9-399 line exhibits no differences in RA-induced proliferation arrest or endogenous Hoxb1, Pbx1, Hoxa5, Cyp26a1, GATA4, or Meis mRNA levels when compared to F9 wild type.
doi:10.1002/jcb.22023
PMCID: PMC3923656  PMID: 19115252
Homeobox; Transcription Factor; Splice Variant; cell differentiation; F9 cells; retinoids; Hoxb1; Pbx; teratocarcinoma; stem cell; vitamin A
19.  Cyclooxygenase-2 Inhibition Attenuates Abdominal Aortic Aneurysm Progression in Hyperlipidemic Mice 
PLoS ONE  2012;7(11):e44369.
Abdominal aortic aneurysms (AAAs) are a chronic inflammatory disease that increase the risk of life-threatening aortic rupture. In humans, AAAs have been characterized by increased expression of cyclooxygenase-2 and the inactivation of COX-2 prior to disease initiation reduces AAA incidence in a mouse model of the disease. The current study examined the effectiveness of selective cyclooxygenase-2 (COX-2) inhibition on reducing AAA progression when administered after the initiation of AAA formation. AAAs were induced in hyperlipidemic apolipoprotein E-deficient mice by chronic angiotensin II (AngII) infusion and the effect of treatment with the COX-2 inhibitor celecoxib was examined when initiated at different stages of the disease. Celecoxib treatment that was started 1 week after initiating AngII infusion reduced AAA incidence by 61% and significantly decreased AAA severity. Mice treated with celecoxib also showed significantly reduced aortic rupture and mortality. Treatment with celecoxib that was started at a late stage of AAA development also significantly reduced AAA incidence and severity. Celecoxib treatment significantly increased smooth muscle alpha-actin expression in the abdominal aorta and did not reduce expression of markers of macrophage-dependent inflammation. These findings indicate that COX-2 inhibitor treatment initiated after formation of AngII-induced AAAs effectively reduces progression of the disease in hyperlipidemic mice.
doi:10.1371/journal.pone.0044369
PMCID: PMC3507882  PMID: 23209546
20.  YY1 Acts as a Transcriptional Activator of Hoxa5 Gene Expression in Mouse Organogenesis 
PLoS ONE  2014;9(4):e93989.
The Hox gene family encodes homeodomain-containing transcriptional regulators that confer positional information to axial and paraxial tissues in the developing embryo. The dynamic Hox gene expression pattern requires mechanisms that differentially control Hox transcription in a precise spatio-temporal fashion. This implies an integrated regulation of neighbouring Hox genes achieved through the sharing and the selective use of defined enhancer sequences. The Hoxa5 gene plays a crucial role in lung and gut organogenesis. To position Hoxa5 in the regulatory hierarchy that drives organ morphogenesis, we searched for cis-acting regulatory sequences and associated trans-acting factors required for Hoxa5 expression in the developing lung and gut. Using mouse transgenesis, we identified two DNA regions included in a 1.5-kb XbaI-XbaI fragment located in the Hoxa4-Hoxa5 intergenic domain and known to control Hoxa4 organ expression. The multifunctional YY1 transcription factor binds the two regulatory sequences in vitro and in vivo. Moreover, the mesenchymal deletion of the Yy1 gene function in mice results in a Hoxa5-like lung phenotype with decreased Hoxa5 and Hoxa4 gene expression. Thus, YY1 acts as a positive regulator of Hoxa5 expression in the developing lung and gut. Our data also support a role for YY1 in the coordinated expression of Hox genes for correct organogenesis.
doi:10.1371/journal.pone.0093989
PMCID: PMC3976385  PMID: 24705708
21.  Smooth muscle phenotypic modulation is an early event in aortic aneurysms 
Objectives
Vascular smooth muscle cells can undergo profound changes in phenotype, defined by coordinated repression of smooth muscle cell marker genes and production of matrix metalloproteinases in response to injury. However, little is known of the role of smooth muscle cells in aortic aneurysms. We hypothesized that smooth muscle cells undergo phenotypic modulation early in the development of aortic aneurysms.
Methods
Abdominal aortas from C57B6 mice (n = 79) were perfused with elastase or saline (control) and harvested at 1, 3, 7, or 14 days. Aortas were analyzed by means of quantitative polymerase chain reaction and immunohistochemistry for smooth muscle cell marker genes, including SM22A, smooth muscle α-actin, and matrix metalloproteinases 2 and 9. In complimentary experiments human aneurysms (n = 10) and control aorta (n = 10) were harvested at the time of surgical intervention and analyzed.
Results
By 14 days, aortic diameter was larger after elastase perfusion compared with control diameter (100% ± 9.6% vs 59.5% ± 18.9%, P = .0002). At 7 days, elastase-perfused mice had a 78% and 85% reduction in SM22α and smooth muscle α-actin expression, respectively, compared with that seen in control animals well before aneurysms were present, and these values remained repressed at 14 days. Immunohistochemistry confirmed less SM22α and smooth muscle α-actin in experimental aneurysms at 14 days in concert with increased matrix metalloproteinase 2 and 9 expression at 7 and 14 days. Similarly, human aneurysms had less SM22α and smooth muscle α-actin and increased matrix metalloproteinase 2 and 9 staining, compared with control values, as determined by means of quantitative polymerase chain reaction.
Conclusions
Aneurysms demonstrate smooth muscle cell phenotypic modulation characterized by downregulation of smooth muscle cell marker genes and upregulation of matrix metalloproteinases. These events in experimental models occur before aneurysm formation. Targeting smooth muscle cells to a reparative phenotype might provide a novel therapy in the treatment of aortic aneurysms.
doi:10.1016/j.jtcvs.2009.07.075
PMCID: PMC2956879  PMID: 19931668
22.  Androgen Increases AT1a Receptor Expression in Abdominal Aortas to Promote Angiotensin II-Induced AAAs in Apolipoprotein E Deficient Mice 
Objective
Castration of male apolipoprotein E deficient (apoE-/-) mice reduces angiotensin II (AngII)-induced abdominal aorta aneurysms (AAAs) to that of female mice. The purpose of this study was to determine whether this reduction is due to androgen-mediated regulation of aortic AngII type 1A receptors (AT1aR).
Methods and Results
AT1aR mRNA abundance in the AAA-prone region of abdominal aortas was 8-fold greater compared to thoracic aortas of male, but not female mice. AT1aR mRNA abundance decreased after castration in abdominal, but not thoracic aortas of male mice. Dihydrotestosterone (DHT, 0.16 mg/day) administration to castrated male mice restored AT1aR mRNA abundance in abdominal aortas, but had no effect in thoracic aortas. DHT also increased AT1aR mRNA abundance in abdominal aortas from female mice. Castrated male or female apoE-/- mice were administered DHT during infusion of saline or AngII (1,000 ng/kg/min for 28 days). DHT administration did not alter serum cholesterol concentrations, lipoprotein distributions, or atherosclerotic lesion areas in either male or female mice. However, administration of DHT increased AAA incidence in male (27% placebo vs. vs. 75% DHT) and female mice (28% placebo vs. 64% DHT).
Conclusions
Androgen promotes AT1aR mRNA abundance in abdominal aortas associated with increased AngII-induced AAAs.
doi:10.1161/ATVBAHA.107.160382
PMCID: PMC2757112  PMID: 18451329
Angiotensin; Aneurysms; Androgen; Atherosclerosis; Sex Hormones
23.  Bone marrow–derived MCP1 required for experimental aortic aneurysm formation and smooth muscle phenotypic modulation 
Objectives
This study tested the hypothesis that monocyte chemotactic protein 1 (MCP1) is required for abdominal aortic aneurysm (AAA) and smooth muscle phenotypic modulation in a mouse elastase perfusion model.
Methods
Infrarenal aortas of C57BL/6 (wild type [WT]) and MCP1 knockout (KO) mice were analyzed at 14 days after perfusion. Key cellular sources of MCP1 were identified using bone marrow transplantation. Cultured aortic smooth muscle cells (SMCs) were treated with MCP1 to assess its potential to directly regulate SMC contractile protein expression and matrix metalloproteinases (MMPs).
Results
Elastase perfused WT aortas had a mean dilation of 102% (n = 9) versus 53.7% for MCP1KO aortas (n = 9, P < .0001) and 56.3% for WT saline-perfused controls (n = 8). Cells positive for MMP9 and Mac-2 were nearly absent in the KO aortas. Complimentarily, the media of the KO vessels had abundant differentiated smooth muscle and intact elastic fibers and markedly less MMP2. Experiments in cultured SMCs showed MCP1 can directly repress smooth muscle markers and induce MMP2 and MMP9. Bone marrow transplantation studies showed that KO of MCP1 in bone marrow–derived cells protects from AAA formation. Moreover, KO in the bone was significantly more protective than global KO, suggesting an unexpected benefit to selectively depleting MCP1 in bone marrow–derived cells.
Conclusions
These results have shown that MCP1 derived from bone marrow cells is required for experimental AAA formation and that retention of nonbone marrow MCP1 limits AAA compared with global depletion. This protein contributes to macrophage infiltration into the AAA and can act directly on SMCs to reduce contractile proteins and induce MMPs.
doi:10.1016/j.jtcvs.2011.07.053
PMCID: PMC3627218  PMID: 21996300
24.  Three trans-acting regulatory functions control hydrogenase synthesis in Alcaligenes eutrophus. 
Journal of Bacteriology  1991;173(6):1845-1854.
Random Tn5 mutagenesis of the regulatory region of megaplasmid pHG1 of Alcaligenes eutrophus led to the identification of three distinct loci designated hoxA, hoxD, and hoxE. Sequencing of the hoxA locus revealed an open reading frame which could code for a polypeptide of 482 amino acids with a molecular mass of 53.5 kDa. A protein of comparable apparent molecular mass was detected in heterologous expression studies with a plasmid-borne copy of the hoxA gene. Amino acid alignments revealed striking homologies between HoxA and the transcriptional activators NifA and NtrC of Klebsiella pneumoniae and HydG of Escherichia coli. HoxA- mutants of A. eutrophus lacked both NAD-reducing soluble hydrogenase and membrane-bound hydrogenase. In HoxA- mutants, the synthesis of beta-galactosidase from a hoxS'-'lacZ operon fusion was drastically reduced, indicating that HoxA is essential for the transcription of hydrogenase genes. Mutants defective in hoxD and hoxE also lacked the catalytic activities of the two hydrogenases; however, in contrast to HoxA- mutants, they contained immunologically detectable NAD-reducing soluble hydrogenase and membrane-bound hydrogenase proteins, although at a reduced level. The low hydrogenase content in the HoxD- and HoxE- mutants correlated with a decrease in beta-galactosidase synthesized under the direction of a hoxS'-'lacZ operon fusion. Thus, hoxD and hoxE apparently intervene both in the regulation of hydrogenase synthesis and in subsequent steps leading to the formation of catalytically active enzymes.
Images
PMCID: PMC207712  PMID: 2001989
25.  Endothelin Type A Receptor (ETA) Expression Is Regulated by HOXA10 in Human Endometrial Stromal Cells 
Endothelin type A receptor (ETA) is a member of the superfamily of G protein-coupled receptors. Our laboratory conducted a microarray screen that identified ETA as target of HOXA10 transcriptional control in endometrium. Here, we confirm HOXA10-regulated ETA expression in endometrium. Endometrial biopsies were obtained from fertile reproductive-age individuals, and first trimester decidual samples were obtained at the time of elective termination. Immunohistochemistry (IHC) was used to identify ETA protein in endometrium as well as first trimester decidua. ETA was expressed in endometrial stromal cells throughout the menstrual cycle. ETA was also highly expressed in first trimester decidual cells. The regulatory relationship between HOXA10 and ETA was established by transient transfection analysis. The human endometrial stromal cell line (HESC) and the human endometrial epithelial cell line (Ishikawa) were transfected with pcDNA/HOXA10, HOXA10 small interfering RNA (siRNA), or respective controls. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed to determine expression levels of HOXA10 and ETA in each group. ETA gene expression increased 9-fold (P < .05) after pcDNA/HOXA10 transfection of HESC. ETA was not regulated by HOXA10 in Ishikawa cells. We conclude that ETA is expressed in normal endometrium and decidua. Expression of this receptor is regulated by an essential mediator of endometrial receptivity, HOXA10. ETA may enhance the proliferative potential of endometrial cells in a manner similar to that seen in vascular smooth muscle cells. ETA likely acts as a molecular mechanism by which HOXA10 promotes stromal cell growth and prostaglandin production in both the implantation window and decidua.
doi:10.1177/1933719110361961
PMCID: PMC3107845  PMID: 20371740
ETA; HOXA10; endometrium; decidua

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