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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.
PMCID: PMC3254126  PMID: 22168796
2.  HOXA10 is expressed in response to sex steroids at the time of implantation in the human endometrium. 
Journal of Clinical Investigation  1998;101(7):1379-1384.
Hox genes are well-known transcriptional regulators that play an essential role in directing embryonic development. Mice that are homozygous for a targeted disruption of the Hoxa10 gene exhibit uterine factor infertility. We have recently demonstrated that HOXA10 is expressed in the adult human uterus. To examine expression of HOXA10 during the menstrual cycle, Northern blot analysis and in situ hybridization were performed. Expression of HOXA10 dramatically increased during the midsecretory phase of the menstrual cycle, corresponding to the time of implantation and increase in circulating progesterone. Expression of HOXA10 in cultured endometrial cells was stimulated by estrogen or progesterone. Stimulation of HOXA10 by progesterone was concentration-dependent within the physiologic range, and the effect of estrogen was inhibited by cycloheximide. These results identify sex steroids as novel regulators of HOX gene expression. HOXA10 may have an important function in regulating endometrial development during the menstrual cycle and in establishing conditions necessary for implantation in the human.
PMCID: PMC508715  PMID: 9525980
3.  HOXA1 is overexpressed in oral squamous cell carcinomas and its expression is correlated with poor prognosis 
BMC Cancer  2012;12:146.
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.
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.
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).
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.
PMCID: PMC3351375  PMID: 22498108
Oral cancer; HOXA1; Cellular proliferation; Prognosis
4.  Pharmacologically-induced thoracic and abdominal aortic aneurysms in mice 
Hypertension  2010;55(5):1267-1274.
Aortic aneurysms are common among the elderly population. Large majority of aortic aneurysms are located at two distinct aneurysm-prone regions—the abdominal aorta and thoracic aorta involving the ascending aorta. In this study, we combined two factors that are associated with human aortic aneurysms—hypertension and degeneration of elastic lamina—to induce an aortic aneurysm in mice. Roles of hemodynamic conditions in the formation of aortic aneurysms were assessed using (1) two different methods for inducing hypertension, and (2) anti-hypertensive agents.
In nine-week-old C57BL/6J male mice, hypertension was induced by angiotensin-II or deoxycorticosterone acetate (DOCA)-salt hypertension; degeneration of elastic lamina was induced by infusion of beta-aminopropionitrile, a lysyl oxidase inhibitor. Irrespective of the methods for inducing hypertension, mice developed thoracic and abdominal aortic aneurysms (38-50% and 30-49 %, respectively). Aneurysms were found at the two aneurysm-prone regions with site-specific morphological and histological characteristics. Treatment with anti-hypertensive agent, amlodipine, normalized blood pressure and dramatically reduced aneurysm formation in the mice that received angiotensin-II and beta-aminopropionitrile. However, a treatment with captopril, angiotensin converting enzyme inhibitor, did not affect blood pressure or the incidence of aortic aneurysms in the mice that received deoxycorticosterone acetate-salt and beta-aminopropionitrile.
In summary, we have shown that a combination of hypertension and pharmacologically-induced degeneration of elastic laminas can induce both thoracic and abdominal aortic aneurysms with site-specific characteristics. The aneurysm formation in this model was dependent on hypertension, but not on direct effects of angiotensin-II to the vascular wall.
PMCID: PMC2859958  PMID: 20212272
aorta; aneurysm; hypertension; angiotensin-II; lysyl oxidase; hemodynamics; remodeling
5.  Epigenetic Regulation of Early Osteogenesis and Mineralized Tissue Formation by a HOXA10-PBX1-Associated Complex 
Cells, Tissues, Organs  2011;194(2-4):146-150.
Homeodomain-containing (HOX) factors such as the abdominal class homeodomain protein HOXA10 and the TALE-family protein PBX1 form coregulatory complexes and are potent transcriptional and epigenetic regulators of tissue morphogenesis. We have identified that HOXA10 and PBX1 are expressed in osteoprogenitors; however, their role in osteogenesis has not been established. To determine the mechanism of HOXA10-PBX-mediated regulation of osteoblast commitment and the related gene expression, PBX1 or HOX10 were depleted (shRNA or genetic deletion, respectively) or exogenously expressed in C3H10T1/2, bone marrow stromal progenitors, and MC3T3-E1 (preosteoblast) cells. Overexpression of HOXA10 increased the expression of osteoblast-related genes, osteoblast differentiation and mineralization; expression of PBX1 impaired osteogenic commitment of pluripotent cells and the differentiation of osteoblasts. In contrast, the targeted depletion of PBX1 by shRNA increased the expression of bone marker genes (osterix, alkaline phosphatase, BSP, and osteocalcin). Chromatin-associated PBX1 and HOXA10 were present at osteoblast-related gene promoters preceding gene expression, but PBX1 was absent from promoters during the transcription of bone-related genes, including osterix (Osx). Further, PBX1 complexes were associated with histone deacetylases normally linked with chromatin inactivation. Loss of PBX1 but not of HOXA10 from the Osx promoter was associated with increases in the recruitment of histone acetylases (p300), as well as decreased H3K9 methylation, reflecting transcriptional activation. We propose PBX1 plays a central role in attenuating the activity of HOXA10 as an activator of osteoblast-related genes and functions to establish the proper timing of gene expression during osteogenesis, resulting in proper matrix maturation and mineral deposition in differentiated osteoblasts.
PMCID: PMC3178072  PMID: 21597276
Chromatin remodeling during osteogenesis; Osterix histone acetylation; Pbx inhibition of osteoblastogenesis
6.  Identification of novel Hoxa1 downstream targets regulating hindbrain, neural crest and inner ear development 
Developmental biology  2011;357(2):295-304.
Hoxgenes play a crucial role during embryonic patterning and organogenesis. Of the 39 Hox genes, Hoxa1 is the first to be expressed during embryogenesis and the only anterior Hox gene linked to a human syndrome. Hoxa1 is necessary for proper development of the brainstem, inner ear and heart in humans and mice; however, almost nothing is known about the molecular downstream targets through which it exerts its function. To gain insight into the transcriptional network regulated by this protein, we performed microarray analysis on tissue microdissected from the prospective rhombomere 3–5 region of Hoxa1 null and wild type embryos. Due to the very early and transient expression of this gene, dissections were performed on early somite stage embryos during an eight-hour time window of development. Our array yielded a list of around 300 genes differentially expressed between the two samples. Many of the identified genes play a role in a specific developmental or cellular process. Some of the validated targets regulate early neural crest induction and specification. Interestingly, three of these genes, Zic1, Hnf1b and Foxd3, were down-regulated in the posterior hindbrain, where cardiac neural crest cells arise, which pattern the outflow tract of the heart. Other targets are necessary for early inner ear development, e.g. Pax8 and Fgfr3 or are expressed in specific hindbrain neurons regulating respiration, e.g. Lhx5. These findings allow us to propose a model where Hoxa1 acts in a genetic cascade upstream of genes controlling specific aspects of embryonic development, thereby providing insight into possible mechanisms underlying the human HoxA1-syndrome.
PMCID: PMC3176680  PMID: 21784065
Hoxa1; microarray; neural crest; inner ear; rhombomere
7.  Regional Heterogeneity within the Aorta: Relevance to Aneurysm Disease 
Vascular remodeling within the aorta results in a loss of structural integrity with consequent aneurysm formation. This degradation is more common in the abdominal aorta, but also occurs above the diaphragm in the thoracic aorta. Conventionally, the aorta has been considered a large vascular conduit with uniform cellular and extracellular structure and function. Evidence is accumulating, however, to suggest that variations exist between the thoracic and abdominal aorta, thereby demonstrating regional heterogeneity. Further pathophysiologic studies of aortic dilation in each of these regions have identified disparities in atherosclerotic plaque deposition, vessel mechanics, protease profiles, and cell signaling pathways. Improved understanding of this spatial heterogeneity may promote evolution in the management of aneurysm disease through computational models of aortic wall stress, imaging of proteolytic activity, targeted pharmacologic treatment, and the application of region-specific gene therapy.
PMCID: PMC2679174  PMID: 19026791
thoracic aorta; abdominal aorta; aortic aneurysm; matrix metalloproteinase
8.  Mechanical Properties of Suprarenal and Infrarenal Abdominal Aorta: Implications for Mouse Models of Aneurysms 
Medical engineering & physics  2011;33(10):1262-1269.
Multiple mouse models have been developed to increase our understanding of the natural history of abdominal aortic aneurysms. An advantage of such models is that one can quantify the time course of changes in geometry, histology, cell biology, and mechanics as a lesion develops. One of the most commonly used mouse models yields lesions in the suprarenal abdominal aorta whereas most other models target the infrarenal abdominal aorta, consistent with the clinical observation that nearly all abdominal aneurysms in humans occur in the infrarenal aorta. Understanding reasons for similarities and differences between diverse mouse models and human lesions may provide increased insight that would not be possible studying a single situation alone. Toward this end, however, we must first compare directly the native structure and properties of these two portions of the abdominal aorta in the mouse. In this paper, we present the first biaxial mechanical data and nonlinear constitutive descriptors for the suprarenal and infrarenal aorta in mice, which reveals only subtle mechanical differences despite marked morphological and histological differences. Such data promise to increase our ability to understand and model the natural history of these deadly lesions.
PMCID: PMC3235688  PMID: 21742539
AAA; stress; strain; stiffness; murine models
9.  Improved Surgical Technique for the Establishment of a Murine Model of Aortic Transplantation 
Microsurgery  1998;18(6):368-371.
Aortic allotransplantation is a reliable procedure to study the evolvement of chronic rejection in mice. The progressive nature of this process in mice is characterized by diffuse and concentric myointimal proliferation which is inevitably associated with variable degrees of luminal constriction. These vascular changes are comparable to those that are witnessed in organ allografts undergoing chronic rejection in humans, underscoring its utility as a model of choice for the study of the development of this lesion. Whilst improved surgical technique has resulted in markedly enhanced graft survival, the results are far from being acceptable. Realizing this limitation, we embarked on developing a modified technique for aortic transplantation which would allow for improved graft survival in mice. A bypass conduit was created by end-to-side anastomosis of a segment of the donor's thoracic aorta into the infrarenal portion of the recipient's abdominal aorta. Using this technique, the graft survival was >98% with evidence in allotransplanted aorta of morphological changes pathognomonic of chronic rejection. On the contrary, no histopathological anomalies were discerned in aortic grafts transplanted across syngeneic animals. This modified surgical approach ameliorates the unacceptably high graft loss associated with earlier techniques, further extending the utility of this model as a tool to study the molecular and cellular mechanisms rudiment to the evolvement of chronic rejection.
PMCID: PMC3005259  PMID: 9846999
10.  AKT2 Confers Protection Against Aortic Aneurysms and Dissections 
Circulation research  2012;112(4):618-632.
Aortic aneurysm and dissection (AAD) are major diseases of the adult aorta caused by progressive medial degeneration of the aortic wall. Although the overproduction of destructive factors promotes tissue damage and disease progression, the role of protective pathways is unknown.
In this study, we examined the role of AKT2 in protecting the aorta from developing AAD.
Methods and Results
AKT2 and phospho-AKT levels were significantly downregulated in human thoracic AAD tissues, especially within the degenerative medial layer. Akt2-deficient mice showed abnormal elastic fibers and reduced medial thickness in the aortic wall. When challenged with angiotensin II (AngII), these mice developed aortic aneurysm, dissection, and rupture with features similar to those in humans, in both thoracic and abdominal segments. Aortas from Akt2-deficient mice displayed profound tissue destruction, apoptotic cell death, and inflammatory cell infiltration that were not observed in aortas from wild-type mice. Additionally, AngII-infused Akt2-deficient mice showed significantly elevated expression of matrix metalloproteinase (MMP)-9 and reduced expression of tissue inhibitor of metalloproteinase (TIMP)-1. In cultured human aortic vascular smooth muscle cells, AKT2 inhibited the expression of MMP-9 and stimulated the expression of TIMP-1 by preventing the binding of transcription factor forkhead box protein O1 (FOXO1) to the MMP-9 and TIMP-1 promoters.
Impaired AKT2 signaling may contribute to increased susceptibility to the development of AAD. Our findings provide evidence of a mechanism that underlies the protective effects of AKT2 on the aortic wall and that may serve as a therapeutic target in the prevention of AAD.
PMCID: PMC3586338  PMID: 23250987
Aortic aneurysm and dissection; AKT; FOXO1; MMP-9; TIMP-1
11.  Obesity Promotes Inflammation in Periaortic Adipose Tissue and Angiotensin II-Induced Abdominal Aortic Aneurysm Formation 
Obesity promotes macrophage infiltration into adipose tissue and is associated with increases in several cardiovascular diseases. Infusion of angiotensin II (AngII) to mice induces formation of abdominal aortic aneurysms (AAAs) with profound medial and adventitial macrophage infiltration. We sought to determine if obesity promotes macrophage infiltration and proinflammatory cytokines in periaortic adipose tissue surrounding abdominal aortas and increases AngII-induced AAAs.
Methods and Results
Hypertrophied white adipocytes surrounded abdominal aortas while brown adipocytes surrounded thoracic aortas of obese mice. mRNA abundance of macrophage proinflammatory chemokines and their receptors were elevated with obesity to a greater extent in abdominal compared to thoracic periaortic adipose tissue. Periaortic adipose tissue explants surrounding abdominal aortas of obese mice released greater concentrations of MCP-1 and promoted more macrophage migration than explants from thoracic aortas. Male C57BL/6 mice were fed a high fat (HF) diet for 1, 2, or 4 months and then infused with AngII (1,000 ng/kg/min) for 28 days. AAA incidence increased progressively with the duration of HF feeding (18%, 36% and 60%, respectively). Similarly, AngII-infused ob/ob mice exhibited increased AAAs compared to lean controls (76% compared to 32%, respectively, P<0.05). Infusion of AngII to obese mice promoted further macrophage infiltration into periaortic and visceral adipose tissue, and obese mice exhibiting AAAs had greater macrophage content in visceral adipose tissue than mice not developing AAAs.
Increased macrophage accumulation in periaortic adipose tissue surrounding abdominal aortas of AngII-infused obese mice is associated with enhanced AAA formation.
PMCID: PMC2753598  PMID: 19608970
obesity; angiotensin II; abdominal aortic aneurysm
12.  Homeobox A7 increases cell proliferation by up-regulation of epidermal growth factor receptor expression in human granulosa cells 
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.
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.
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.
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.
PMCID: PMC2904782  PMID: 20540809
13.  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.
PMCID: PMC207712  PMID: 2001989
14.  Androgen Increases AT1a Receptor Expression in Abdominal Aortas to Promote Angiotensin II-Induced AAAs in Apolipoprotein E Deficient Mice 
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).
Androgen promotes AT1aR mRNA abundance in abdominal aortas associated with increased AngII-induced AAAs.
PMCID: PMC2757112  PMID: 18451329
Angiotensin; Aneurysms; Androgen; Atherosclerosis; Sex Hormones
15.  Novel pathways in the pathobiology of human abdominal aortic aneurysms 
Abdominal aortic aneurysm (AAA), a dilatation of the infrarenal aorta, typically affects males > 65 years. The pathobiological mechanisms of human AAA are poorly understood. The goal of this study was to identify novel pathways involved in the development of AAAs.
A custom-designed “AAA-chip” was used to assay 43 of the differentially expressed genes identified in a previously published microarray study between AAA (n = 15) and control (n = 15) infrarenal abdominal aorta. Protein analyses were performed on selected genes.
Altogether 38 of the 43 genes on the “AAA-chip” showed significantly different expression. Novel validated genes in AAA pathobiology included ADCY7, ARL4C, BLNK, FOSB, GATM, LYZ, MFGE8, PRUNE2, PTPRC, SMTN, TMODI and TPM2. These genes represent a wide range of biological functions, such as calcium signaling, development and differentiation, as well as cell adhesion not previously implicated in AAA pathobiology. Protein analyses for GATM, CD4, CXCR4, BLNK, PLEK, LYZ, FOSB, DUSP6, ITGA5 and PTPRC confirmed the mRNA findings.
The results provide new directions for future research into AAA pathogenesis to study the role of novel genes confirmed here. New treatments and diagnostic tools for AAA could potentially be identified by studying these novel pathways.
PMCID: PMC3782105  PMID: 22797469
gene expression; vascular biology; aorta; abdominal aortic aneurysm
16.  Cdx Protein Interaction with Hoxa5 Regulatory Sequences Contributes to Hoxa5 Regional Expression along the Axial Skeleton 
Molecular and Cellular Biology  2005;25(4):1389-1401.
Hox gene functions are intimately linked to correct developmental expression of the genes. The identification of cis-acting regulatory sequences and their associated trans-acting factors constitutes a key step in deciphering the mechanisms underlying the correct positioning of the functional domain of Hox genes along the anterior-posterior axis. We have identified DNA elements driving Hoxa5 regionalized expression in mice, using the 2.1-kb mesodermal enhancer (MES) localized in Hoxa5 3′ flanking sequences as a starting point. The MES sequence comprises regulatory elements targeting Hoxa5 expression in the limbs, the urogenital and gastrointestinal tracts, and the cervical-upper thoracic region of the prevertebral column. A 164-bp DNA fragment within the MES caudally restricts Hoxa5 expression at the level of prevertebra 10, corresponding to the posterior limit of its functional domain. Cdx proteins directly bind to this element in vitro via two conserved sites. Preventing Cdx binding by mutating the sites causes caudal expansion of the transgene expression domain. Of all three murine Cdx proteins that bind this element in vitro, Cdx4 has emerged as a potential regional posterior repressor of Hoxa5 expression. The restrictive control provided by Cdx interactions with Hoxa5 regulatory sequences may be one of the critical events in cervicothoracic axial specification.
PMCID: PMC548006  PMID: 15684390
17.  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.
PMCID: PMC3976385  PMID: 24705708
18.  Decreased Collagen and Increased Matrix Metalloproteinase-13 in Experimental Abdominal Aortic Aneurysms in Males Compared with Females 
Surgery  2009;147(2):258-267.
This study examined gender differences in collagen regulation during rodent experimental abdominal aortic aneurysm (AAA) formation.
Infrarenal aortas of male and female rats were perfused with elastase or saline (control). Aortic diameters were measured at baseline (day 0), and on post-operative days 7 and 14. TGF-β1, collagen subtypes I and III, and MMP-13 (collagenase) expression and/or protein levels from aortic tissue were determined by RT-PCR and Western Blotting. Aortic tissue was stained for total collagen, neutrophils, and macrophages using immunohistochemistry on days 4 and 7.
Seven and 14 days following perfusion, aortic diameter increased significantly in elastase-perfused males compared with females (P<0.001 for each). Four and 7 days post-perfusion, significantly more neutrophils and macrophages were present in elastase-perfused males compared with females. Seven days post-perfusion, protein levels of TGF-β1 were lower in males compared with females (P=0.04). Type I collagen levels also decreased on days 7 (P<0.001) and 14 (P=0.002), and type III collagen levels decreased on days 7 (P<0.001) and 14 (P<0.001), in males compared with females. By Masson's Trichrome, less adventitial collagen was present in the elastase-perfused males compared with females. MMP-13 expression (P<0.001) and protein levels (P=0.006) in elastase-perfused males were higher than females on day 14.
This study documents a decrease in types I and III collagen with a concurrent increase in MMP-13 following elastase perfusion in males compared with females. These data suggest that alterations in extracellular matrix collagen turnover may be responsible for altered AAA formation between genders.
PMCID: PMC3017342  PMID: 19767051
Aorta; aneurysm; collagen; estrogen; MMP-13; gender differences
19.  Juxtarenal Aortic Aneurysm after Successful Repair of Ruptured Infrarenal Aneurysm 
Texas Heart Institute Journal  1989;16(2):113-116.
Eight months after successful repair of a ruptured infrarenal aneurysm, a 50-year-old hypertensive male patient was found to have a 2nd aneurysm restricted to the juxtarenal segment of the aorta, and not in continuity with the infrarenal lesion. While there have been reports both of true aneurysms arising independently in the abdominal (infrarenal) and thoracic (suprarenal) aortic segments, and of false aneurysms arising secondary to abdominal aortic repair (at the suture line or as dilatations of residual aneurysmal tissue), we believe our case unusual in that the new aneurysm was a discrete and “true” thoracoabdominal dilatation arising just above the renal arteries and terminating just below the superior mesenteric artery. This strictly juxtarenal dilatation conforms better to a descriptive pattern for atherosclerotic occlusive disease than it does to the standard scheme for classifying thoracoabdominal aortic aneurysms. (Texas Heart Institute Journal 1989;16:113-6)
PMCID: PMC324861  PMID: 15227224
Aorta, abdominal; aorta, thoracic; aortic aneurysm; aortic rupture/aorta, abdominal; arteriosclerosis; nomenclature, diagnostic
20.  Molecular Mechanisms of Treatment Resistance in Endometriosis: The Role of Progesterone–Hox Gene Interactions 
HOX genes, encoding homeodomain transcription factors, are dynamically expressed in endometrium, where they are necessary for endometrial growth, differentiation, and implantation. In human endometrium, the expression of HOXA10 and HOXA11 is driven by sex steroids, with peak expression occurring at time of implantation in response to rising progesterone levels. However, the maximal HOXA10 and HOXA11 expression fails to occur in women with endometriosis. In endometriosis, altered progesterone receptor expression or diminished activity may lead to attenuated or dysregulated progesterone response and decreased expression of progesterone-responsive genes including HOX genes in the eutopic endometrium. In turn, other mediators of endometrial receptivity that are regulated by HOX genes, such as pinopodes, αvβ3 integrin, and IGFBP-1, are downregulated in endometriosis. HOXA10 hypermethylation has recently been demonstrated to silence HOXA10 gene expression and account for decreased HOXA10 in the endometrium of women with endometriosis. Silencing of progesterone target genes by methylation is an epigenetic mechanism that mediates progesterone resistance. The relatively permanent nature of methylation may explain the widespread failure of treatments for endometriosis-related infertility.
PMCID: PMC3107856  PMID: 20104430
HOX genes; implantation; endometrium; endometriosis
21.  Positive Transcriptional Feedback Controls Hydrogenase Expression in Alcaligenes eutrophus H16 
Journal of Bacteriology  1999;181(18):5684-5692.
The protein HoxA is the central regulator of the Alcaligenes eutrophus H16 hox regulon, which encodes two hydrogenases, a nickel permease and several accessory proteins required for hydrogenase biosynthesis. Expression of the regulatory gene hoxA was analyzed. Screening of an 8-kb region upstream of hoxA with a promoter probe vector localized four promoter activities. One of these was found in the region immediately 5′ of hoxA; the others were correlated with the nickel metabolism genes hypA1, hypB1, and hypX. All four activities were independent of HoxA and of the minor transcription factor ς54. Translational fusions revealed that hoxA is expressed constitutively at low levels. In contrast to these findings, immunoblotting studies revealed a clear fluctuation in the HoxA pool in response to conditions which induce the hox regulon. Quantitative transcript assays indicated elevated levels of hyp mRNA under hydrogenase-derepressing conditions. Using interposon mutagenesis, we showed that the activity of a remote promoter is required for hydrogenase expression and autotrophic growth. Site-directed mutagenesis revealed that PMBH, which directs transcription of the structural genes of the membrane-bound hydrogenase, contributes to the expression of hoxA under hydrogenase-derepressing conditions. Thus, expression of the hox regulon is governed by a positive feedback loop mediating amplification of the regulator HoxA. These results imply the existence of an unusually large (ca. 17,000-nucleotide) transcript.
PMCID: PMC94088  PMID: 10482509
22.  MicroRNA expression signature in human abdominal aortic aneurysms 
BMC Medical Genomics  2012;5:25.
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.
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®.
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.
Our genome-wide approach revealed several differentially expressed miRNAs in human AAA tissue suggesting that miRNAs play a role in AAA pathogenesis.
PMCID: PMC3507654  PMID: 22704053
Apoptosis; Microarray analysis; Vascular biology; miRNA-mRNA analysis; Network analysis
23.  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.
PMCID: PMC1900059  PMID: 17452460
24.  HoxA-11 and FOXO1A Cooperate to Regulate Decidual Prolactin Expression: Towards Inferring the Core Transcriptional Regulators of Decidual Genes 
PLoS ONE  2009;4(9):e6845.
During the menstrual cycle, the ovarian steroid hormones estrogen and progesterone control a dramatic transcriptional reprogramming of endometrial stromal cells (ESCs) leading to a receptive state for blastocyst implantation and the establishment of pregnancy. A key marker gene of this decidualization process is the prolactin gene. Several transcriptional regulators have been identified that are essential for decidualization of ESCs, including the Hox genes HoxA-10 and HoxA-11, and the forkhead box gene FOXO1A. While previous studies have identified downstream target genes for HoxA-10 and FOXO1A, the role of HoxA-11 in decidualization has not been investigated. Here, we show that HoxA-11 is required for prolactin expression in decidualized ESC. While HoxA-11 alone is a repressor on the decidual prolactin promoter, it turns into an activator when combined with FOXO1A. Conversely, HoxA-10, which has been previously shown to associate with FOXO1A to upregulate decidual IGFBP-1 expression, is unable to upregulate PRL expression when co-expressed with FOXO1A. By co-immunoprecipitation and chromatin immunoprecipitation, we demonstrate physical association of HoxA-11 and FOXO1A, and binding of both factors to an enhancer region (−395 to −148 relative to the PRL transcriptional start site) of the decidual prolactin promoter. Because FOXO1A is induced upon decidualization, it serves to assemble a decidual-specific transcriptional complex including HoxA-11. These data highlight cooperativity between numerous transcription factors to upregulate PRL in differentiating ESC, and suggest that this core set of transcription factors physically and functionally interact to drive the expression of a gene battery upregulated in differentiated ESC. In addition, the functional non-equivalence of HoxA-11 and HoxA-10 with respect to PRL regulation suggests that these transcription factors regulate distinct sets of target genes during decidualization.
PMCID: PMC2731163  PMID: 19727442
25.  Differential Expression of Sphingosine-1-Phosphate Receptors in Abdominal Aortic Aneurysms 
Mediators of Inflammation  2012;2012:643609.
Objective. Inflammation plays a key role in the pathophysiology of abdominal aortic aneurysms (AAAs). Newly discovered Sphingosine-1-Phosphate Receptors (S1P receptors) are critical in modulating inflammatory response via prostaglandin production. The aim of the current study was to investigate the expression of different S1P receptors in AAAs and compared with normal aortas at the protein level. Materials and Methods. Aortic specimens were harvested during aortic reconstructive surgery for the AAA group or during organ transplant for the control group. The protein expression of S1P1, 2 and 3 in AAAs and normal aortas was assessed by Western blotting and immunohistochemical analysis. Results. There were 40 AAAs and 20 control aortas collected for the receptor analysis. For Western blot analysis, S1P1 expression was not detected in either group; S1P2 protein was constitutively detected in both types of aortas but its expression level was significantly decreased by 73% (P < 0.05) in AAAs compared with the control group. In contrast, strong S1P3 expression was detected in AAAs aortas but not in normal aortas. Immumohistochemical staining showed similar results, except a weak S1P3 signal was detectable in normal aortas. Conclusions. Western blot and staining results consistently showed the down-regulation of the S1P2 protein with simultaneous up-regulation of the S1P3 protein in AAAs. Since those newly discovered receptors play an important role in the inflammatory cascade, the modulating of S1P signaling, particularly via S1P2 and S1P3, could represent novel therapeutic targets in future AAA treatments.
PMCID: PMC3323867  PMID: 22547907

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