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.
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.
Oral cancer; HOXA1; Cellular proliferation; Prognosis
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.
Apoptosis; Microarray analysis; Vascular biology; miRNA-mRNA analysis; Network analysis
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.
thoracic aorta; abdominal aorta; aortic aneurysm; matrix metalloproteinase
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.
Chromatin remodeling during osteogenesis; Osterix histone acetylation; Pbx inhibition of osteoblastogenesis
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.
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.
aorta; aneurysm; hypertension; angiotensin-II; lysyl oxidase; hemodynamics; remodeling
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)
Aorta, abdominal; aorta, thoracic; aortic aneurysm; aortic rupture/aorta, abdominal; arteriosclerosis; nomenclature, diagnostic
This study sought to characterize the inflammatory infiltrate in ascending thoracic aortic aneurysm (TAAs) in patients with Marfan syndrome (MFS), familial TAA (FTAA), and non-familial TAA cases.
TAAs are associated with a pathologic lesion termed medial degeneration, which was described as a noninflammtory lesion. TAAs are a complication of MFS and also can be inherited in an autosomal dominant manner of FTAA.
Full aortic segments were collected from patients undergoing elective repair with MFS (n=5), FTAA (n=6) and TAAs (n=9), along with control aortas (n=5). Immunohistochemistry staining was performed using antibodies directed against markers of lymphocytes and macrophages. Real-time PCR analysis was performed to quantify the expression level of T cell receptor β chain variable region gene.
Immunohistochemisty of TAA aortas demonstrated that the media and adventitia from MFS, FTAA and sporadic cases had increased numbers of T lymphocytes and macrophages when compared with control aortas. The number of T cells and macrophages in the aortic media of the aneurysm correlated inversely with the patient’s age at the time of prophylactic surgical repair of the aorta. Surprisingly, T cell receptor profiling indicated a similar clonal nature of the T cells in the aortic wall in a majority of aneurysms, whether the patient had MFS, FTAA or sporadic disease.
These results indicate that infiltration of inflammatory cells contributes to the pathogenesis of TAAs. Superantigen-driven stimulation of T lymphocytes in the aortic tissues of the TAA patients may contribute to the initial immune response.
This study sought to investigate the infiltration of T-lymphocytes and macrophage in the aortas of patients with MFS, FTAA and sporadic TAAs. The results indicate that infiltration of inflammatory cells contributes to the pathogenesis of TAAs and superantigen-driven stimulation of T-lymphocytes may contribute to the initial immune response.
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.
obesity; angiotensin II; abdominal aortic aneurysm
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.
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).
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.
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.
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.
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.
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.
aneurysm; sexual dimorphism; testosterone; vascular smooth muscle; angiotensin
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.
Aorta; aneurysm; collagen; estrogen; MMP-13; gender differences
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.
Aortic pathology progression and/or procedure related complications following endovascular repair should always be considered mostly in older patients. We herein describe a hybrid procedure for treatment of rapidly expanding thoracoabdominal aneurysm following endovascular treatment of a descending thoracic aortic aneurysm in an older patient.
A 82-year-old man at 18 months after endovascular surgery for a contained rupture of descending thoracic aortic aneurysm revealed a type IV thoracoabdominal aneurysm with significant increase of the aortic diameters at superior mesenteric and renal artery levels. A hybrid approach consisting of preventive visceral vessel revascularization and endovascular repair of entire abdominal aorta was performed. Under general anaesthesia and by xyphopubic laparotomy, the infrarenal aneurysmatic aorta and common iliac arteries were replaced by a bifurcated woven prosthetic graf. From each of the prosthetic branches two reverse 14x7 mm bifurcated PTFE prosthetic grafts were anastomized to both renal arteries and to the celiac axis and superior mesenteric artery, respectively. Vessel ischemia was restricted to the time required for anastomosis. Three 10 cm Gore endovascular stent-grafts for a total length of 15 cm, were used. The overlapping of the stent-grafts was carried out from the bottom upwards, starting from the aorto-iliac prosthetic body up to the healthy segment of thoracic aorta, 40 mm from the previous stent-grafts.
The patient was discharged on the 9th postoperative day.
This technique offers the advantage of a less invasive treatment, reducing the risk of paraplegia, visceral ischaemia and pulmonary complications, mostly in older patients.
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.
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.
Hoxa1; microarray; neural crest; inner ear; rhombomere
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.
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.
A case of thoracic-abdominal dissection after open surgical exclusion of an infrarenal aortic aneurysm is presented.
A 62-year-old woman was diagnosed with an infrarenal abdominal aortic aneurysm with a rapid increase in maximal diameter. She underwent surgery for aneurysm exclusion by an end-to-end aortoaortic bypass with Dacron collagen (Intervascular; WL Gore & Associates Inc, USA). After 15 days, she was admitted to the emergency department with intense epigastric and lumbar pain. Computed tomography angiography with contrast revealed an aortic dissection with origin in the proximal bypass anastomosis and cranial extension to the thoracic aorta. The true lumen at the level of the eighth thoracic vertebra was practically collapsed by the false lumen. The celiac trunk, and the mesenteric and renal arteries were perfused by the true lumen. After the acute phase of the aortic dissection, surgical repair was planned. Two paths of false lumen were found – one at the thoracic aorta and the second in the proximal bypass anastomosis. Surgical repair comprised two approaches. First, a Valiant Thoracic stent graft (Medtronic Inc, UK) was implanted distal from the left subclavian artery, expanding the collapsed true lumen and covering the false and dissected lumen. Second, an infrarenal Endurant abdominal stent graft (Medtronic Inc) was implanted. This second device was complemented with an aortic infrarenal extension using a Talent abdominal stent graft (Medtronic Inc) in the infrarenal aortic neck to achieve a hermetic seal. The postoperative clinical course was uneventful, and her symptoms were completely resolved in six months.
Arteritis must be taken into account in young patients with high inflammatory markers. Covered stents and endoprosthetic devices seem to be effective methods to seal the dissected lumen.
Aortic dissection; Arteritis
In the surgical treatment of abdominal aortic aneurysm, the single proximal cross-clamp can be placed at 3 alternative aortic levels: infrarenal, hiatal, and thoracic. We performed this retrospective study to evaluate the advantages and disadvantages of the 3 main aortic clamping locations. Eighty patients presented at our institution with abdominal aortic aneurysms from March 1993 through May 1998. Fifty of these patients had intact aneurysms and underwent elective surgery, and 30 had ruptured aneurysms that necessitated emergency surgery. Proximal aortic clamping was applied at the infrarenal level in 24 patients (22 from the intact aneurysm group, 2 from the ruptured group), at the hiatal level in 34 patients (22 intact, 12 ruptured), and at the thoracic level (descending aorta) via a limited left lateral thoracotomy in 22 patients (6 intact, 16 ruptured). Early mortality rates (within 30 days) were 4% (2 of 50 patients) among patients with intact aneurysms and 40% (12 of 30 patients) among those with ruptured aneurysms. In the 2 patients from the intact aneurysm group, proximal aortic clamps were applied at the hiatal level. In the ruptured aneurysm group, proximal aortic clamps were placed at the thoracic level in 10 patients, the infrarenal level in 1, and the hiatal level in 1. According to our study, the clinical status of the patient and the degree of operative urgency--as determined by the extent of the aneurysm--generally dictate the proximal clamp location. Patients who present with aneurysmal rupture or hypovolemic shock benefit from thoracic clamping, because it restores the blood pressure and allows time to replace the volume deficit. Infrarenal placement is advantageous in patients with intact aneurysms if there is sufficient space for the clamp between the renal arteries and the aortic aneurysm. In patients with juxtarenal aneurysms, hiatal clamping enables safe and easy anastomosis to the healthy aorta. Clamping at this level also helps prevent late anastomotic aneurysm formation, which is frequently encountered after inadvertent anastomosis of the graft to a diseased portion of the aorta. Further studies are needed in order to confirm these results.
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.
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.
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.
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.
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.
Hox genes are crucial for body axis specification during embryonic development. Hoxa11 has been previously reported to play a role in anterioposterior patterning of the axial skeleton, development of the urogenital tract of both sexes, and proximal-distal patterning of the limbs. Hoxa11 expression has also been observed in the neural tube. Herein, we report the generation of a Hoxa11eGFP targeted knock-in allele in mice in which eGFP replaces the first coding exon of Hoxa11 as an in-frame fusion. This allele closely recapitulates the reported mRNA expression patterns for Hoxa11. Hoxa11eGFP can be visualized in the tail, neural tube, limbs, kidneys, and reproductive tract of both sexes. Additionally, homozygous mutants recapitulate reported phenotypes for Hoxa11 loss of function mice, exhibiting loss of fertility in both males and females. This targeted mouse line will prove useful as a vital marker for Hoxa11 protein localization during control (heterozygous) or mutant organogenesis.
Hoxa11; eGFP; targeted mutation
Among the early events associated with atherosclerotic lesion development are increased macromolecular permeability of the endothelium and expression of genes that affect inflammation and oxidative state. The purpose of this study was to measure the expression of several atherosclerosis-related genes in endothelial cells scraped from arch and thoracic regions of the porcine aorta exhibiting elevated permeability. Aortae were collected from six swine that were exposed to circulating Evans blue dye (EBD), a marker of transendothelial albumin permeability. Endothelial cells were scraped from (1) white regions in the thoracic aorta, (2) light blue streaks and blue regions near ostia in the thoracic aorta, and (3) dark blue regions in the aortic arch. Expression levels of several genes were analyzed by real-time quantitative PCR. There were modest differences between the expression levels of several genes in cells from the light blue regions relative to those from white regions. In the dark blue regions, eNOS was drastically downregulated and MCP-1 was upregulated relative to their expression in both the white and light blue regions. The distinct levels of permeability and differences in gene expression profiles exhibited by cells from these different regions of the aorta may reflect corresponding differences in their hemodynamic environments.
atherosclerosis; aorta; porcine; permeability; gene expression; endothelium; nitric oxide synthase