Hypoxia-inducible factor 1 in clinical and experimental aortic aneurysm disease.
ABSTRACT: OBJECTIVE:Mural angiogenesis and macrophage accumulation are two pathologic hallmarks of abdominal aortic aneurysm (AAA) disease. The heterodimeric transcription factor hypoxia-inducible factor 1 (HIF-1) is an essential regulator of angiogenesis and macrophage function. In this study, we investigated HIF-1 expression and activity in clinical and experimental AAA disease. METHODS:Human aortic samples were obtained from 24 AAA patients and six organ donors during open abdominal surgery. Experimental AAAs were created in 10-week-old male C57BL/6J mice by transient intra-aortic infusion of porcine pancreatic elastase (PPE). Expression of HIF-1? and its target gene messenger RNA (mRNA) levels were assessed in aneurysmal and control aortae. The HIF-1? inhibitors 2-methoxyestradiol and digoxin, the prolyl hydroxylase domain-containing protein (PHD) inhibitors cobalt chloride and JNJ-42041935, and the vehicle alone as control were administered daily to mice at varying time points beginning before or after PPE infusion. Influences on experimental AAA formation and progression were assessed by serial transabdominal ultrasound measurements of aortic diameter and histopathologic analysis at sacrifice. RESULTS:The mRNA levels for HIF-1?, vascular endothelial growth factor A, glucose transporter 1, and matrix metalloproteinase 2 were significantly increased in both human and experimental aneurysm tissue. Tissue immunostaining detected more HIF-1? protein in both human and experimental aneurysmal aortae compared with respective control aortae. Treatment with either HIF-1? inhibitor, beginning before or after PPE infusion, prevented enlargement of experimental aneurysms. Both HIF-1? inhibition regimens attenuated medial elastin degradation, smooth muscle cell depletion, and mural angiogenesis and the accumulation of macrophages, T cells, and B cells. Whereas mRNA levels for PHD1 and PHD2 were elevated in experimental aneurysmal aortae, pharmacologic inhibition of PHDs had limited effect on experimental aneurysm progression. CONCLUSIONS:Expression of HIF-1? and its target genes is increased in human and experimental AAAs. Treatment with HIF-1? inhibitors limits experimental AAA progression, with histologic evidence of attenuated mural leukocyte infiltration and angiogenesis. These findings underscore the potential significance of HIF-1? in aneurysm pathogenesis and as a target for pharmacologic suppression of AAA disease.
Project description:Macrophages are critical contributors to 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.AAAs were created in 10-week-old male C57BL/6J mice by transient infrarenal aortic porcine pancreatic elastase infusion. Mice were treated with MKEY via intravenous injection either (1) before porcine pancreatic elastase infusion or (2) after aneurysm initiation. Immunostaining demonstrated CCL5 and CCR5 expression on aneurysmal aortae and mural monocytes/macrophages, respectively. MKEY treatment partially inhibited migration of adaptively transferred leukocytes into aneurysmal aortae in recipient mice. Although all vehicle-pretreated mice developed AAAs, 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 metalloproteinase 2 and 9 expression after porcine pancreatic elastase infusion. MKEY initiated after porcine pancreatic elastase infusion also stabilized or reduced enlargement of existing AAAs. Finally, MKEY treatment was effective in limiting AAA formation after angiotensin II infusion in apolipoprotein E-deficient mice.MKEY suppresses AAA formation and progression in 2 complementary experimental models. Peptide inhibition of CXCL4-CCL5 interactions may represent a viable translational strategy to limit progression of human AAA disease.
Project description:OBJECTIVE:Angiotensin (Ang) II type 1 receptor (AT1) activation is essential for the development of exogenous Ang II-induced abdominal aortic aneurysms (AAAs) in hyperlipidemic animals. Experimental data derived from this modeling system, however, provide limited insight into the role of endogenous Ang II in aneurysm pathogenesis. Consequently, the potential translational value of AT1 inhibition in clinical AAA disease management remains incompletely understood on the basis of the existing literature. METHODS:AAAs were created in wild-type (WT) and AT1a knockout (KO) mice by intra-aortic infusion of porcine pancreatic elastase (PPE). WT mice were treated with the AT1 receptor antagonist telmisartan, 10 mg/kg/d in chow, or the peroxisome proliferator-activated receptor ? (PPAR?) antagonist GW9662, 3 mg/kg/d through oral gavage, beginning 1 week before or 3 days after PPE infusion. Influences on aneurysm progression as well as mechanistic insights into AT1-mediated pathogenic processes were determined using noninvasive ultrasound imaging, histopathology, aortic gene expression profiling, and flow cytometric analysis. RESULTS:After PPE infusion, aortic enlargement was almost completely abrogated in AT1a KO mice compared with WT mice. As defined by a ?50% increase in aortic diameter, no PPE-infused, AT1a KO mouse actually developed an AAA. On histologic evaluation, medial smooth muscle cellularity and elastic lamellae were preserved in AT1a KO mice compared with WT mice, with marked attenuation of mural angiogenesis and leukocyte infiltration. In WT mice, telmisartan administration effectively suppressed aneurysm pathogenesis after PPE infusion as well, regardless of whether treatment was initiated before or after aneurysm creation or continued for a limited or extended time. Telmisartan treatment was associated with reduced messenger RNA levels for CCL5 and matrix metalloproteinases 2 and 9 in aneurysmal aortae, with no apparent effect on PPAR?-regulated gene expression. Administration of the PPAR? antagonist GW9662 failed to "rescue" the aneurysm phenotype in telmisartan-treated, PPE-infused WT mice. Neither effector T-cell differentiation nor regulatory T-cell cellularity was affected by telmisartan treatment status. CONCLUSIONS:Telmisartan effectively suppresses the progression of elastase-induced AAAs without apparent effect on PPAR? activation or T-cell differentiation. These findings reinforce the critical importance of endogenous AT1 activation in experimental AAA pathogenesis and reinforce the translational potential of AT1 inhibition in medical aneurysm disease management.
Project description:Mural inflammation and neovascularization are characteristic pathological 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.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 to 4 time points per 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 Cy 5.5 fluorescent tracer (7 to 18 microg/mouse IV). NIRF with inactivated single chain VEGF/Cy tracer (scVEGF/In, 18 microg/mouse IV) was performed on 2 additional Ang II mice to control for nonreceptor-mediated tracer binding and uptake. After 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. 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.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.
Project description:OBJECTIVE:We examined the pathogenic significance of VEGF (vascular endothelial growth factor)-A in experimental abdominal aortic aneurysms (AAAs) and the translational value of pharmacological VEGF-A or its receptor inhibition in aneurysm suppression. Approaches and Results: AAAs were created in male C57BL/6J mice via intra-aortic elastase infusion. Soluble VEGFR (VEGF receptor)-2 extracellular ligand-binding domain (delivered in Ad [adenovirus]-VEGFR-2), anti-VEGF-A mAb (monoclonal antibody), and sunitinib were used to sequester VEGF-A, neutralize VEGF-A, and inhibit receptor tyrosine kinase activity, respectively. Influences on AAAs were assessed using ultrasonography and histopathology. In vitro transwell migration and quantitative reverse transcription polymerase chain reaction assays were used to assess myeloid cell chemotaxis and mRNA expression, respectively. Abundant VEGF-A mRNA and VEGF-A-positive cells were present in aneurysmal aortae. Sequestration of VEGF-A by Ad-VEGFR-2 prevented AAA formation, with attenuation of medial elastolysis and smooth muscle depletion, mural angiogenesis and monocyte/macrophage infiltration. Treatment with anti-VEGF-A mAb prevented AAA formation without affecting further progression of established AAAs. Sunitinib therapy substantially mitigated both AAA formation and further progression of established AAAs, attenuated aneurysmal aortic MMP2 (matrix metalloproteinase) and MMP9 protein expression, inhibited inflammatory monocyte and neutrophil chemotaxis to VEGF-A, and reduced MMP2, MMP9, and VEGF-A mRNA expression in macrophages and smooth muscle cells in vitro. Additionally, sunitinib treatment reduced circulating monocytes in aneurysmal mice. CONCLUSIONS:VEGF-A and its receptors contribute to experimental AAA formation by suppressing mural angiogenesis, MMP and VEGF-A production, myeloid cell chemotaxis, and circulating monocytes. Pharmacological inhibition of receptor tyrosine kinases by sunitinib or related compounds may provide novel opportunities for clinical aneurysm suppression.
Project description:Background The protective effects of polyamines on cardiovascular disease have been demonstrated in many studies. However, the roles of spermidine, a natural polyamine, in abdominal aortic aneurysm (AAA) disease have not been studied. In this study, we investigated the influence and potential mechanisms of spermidine treatment on experimental AAA disease. Methods and Results Experimental AAAs were induced in 8- to 10-week-old male C57BL/6J mice by transient intra-aortic infusion of porcine pancreatic elastase. Spermidine was administered via drinking water at a concentration of 3 mmol/L. Spermidine treatment prevented experimental AAA formation with preservation of medial elastin and smooth muscle cells. In immunostaining, macrophages, T cells, neutrophils, and neovessels were significantly reduced in aorta of spermidine-treated, as compared with vehicle-treated elastase-infused mice. Additionally, flow cytometric analysis showed that spermidine treatment reduced aortic leukocyte infiltration and circulating inflammatory cells. Furthermore, we demonstrated that spermidine treatment promoted autophagy-related proteins in experimental AAAs using Western blot analysis, immunostaining, and transmission electron microscopic examination. Autophagic function was evaluated for human abdominal aneurysmal and nonaneurysmal adjacent aortae from AAA patients using Western blot analysis and immunohistochemistry. Dysregulated autophagic function, as evidenced by increased SQSTM1/p62 protein and phosphorylated mTOR, was found in aneurysmal, as compared with nonaneurysmal, aortic segments. Conclusions Our results suggest that spermidine supplementation limits experimental AAA formation associated with preserved aortic structural integrity, attenuated aortic inflammatory infiltration, reduced circulating inflammatory monocytes, and increased autophagy-related proteins. These findings suggest that spermidine may be a promising treatment for AAA disease.
Project description:Hypoxia inducible factor-1? (HIF-1?) pathway is associated with many vascular diseases, including atherosclerosis, arterial aneurysms, pulmonary hypertension and chronic venous diseases. Significant HIF-1? expression could be found at the rupture edge at human abdominal aortic aneurysm (AAA) tissues. While our initial in vitro experiments had shown that deferoxamine (DFO) could attenuate angiotensin II (AngII) induced endothelial activations; we unexpectedly found that DFO augmented the severity of AngII-induced AAA, at least partly through increased accumulation of HIF-1?. The findings promoted us to test whether aneurysmal prone factors could up-regulate the expression of MMP-2 and MMP-9 through aberrantly increased HIF-1? and promote AAA development. AngII induced AAA in hyperlipidemic mice model was used. DFO, as a prolyl hydroxylase inhibitor, stabilized HIF-1? and augmented MMPs activities. Aneurysmal-prone factors induced HIF-1? can cause overexpression of MMP-2 and MMP-9 and promote aneurysmal progression. Pharmacological HIF-1? inhibitors, digoxin and 2-ME could ameliorate AngII induced AAA in vivo. HIF-1? is pivotal for the development of AAA. Our study provides a rationale for using HIF-1? inhibitors as an adjunctive medical therapy in addition to current cardiovascular risk-reducing regimens.
Project description:Objective:Abdominal aortic aneurysm (AAA) development has been characterized by increased expression of vascular endothelial growth factor (VEGF), which contributes to angiogenesis via cyclooxygenase-2 (COX-2). Quercetin, one of the most common and well-researched flavonoids and abundant in vegetables and fruits, has beneficial effects in inhibiting angiogenesis. This study investigated the antiangiogenic effects of quercetin on experimental aneurysms. Methods:We utilized the in vivo AAA mouse model induced by the periaortic application of CaCl2 to examine the effectiveness of quercetin in blocking angiogenesis. Quercetin was administered at 60?mg/kg once daily on the day of the AAA induction and then continued for 6 weeks. Celecoxib, a selective COX-2 inhibitor, was used as the positive control. Results:Our results demonstrated that quercetin significantly attenuated aneurysm growth in AAA mice and medial neovascularization. Accordingly, quercetin decreased the expression of proangiogenic mediators, including VEGF-A, intercellular adhesion molecule-1, vascular cell adhesion molecule 1, and vascular endothelial cadherin. Quercetin treatment also inhibited the expression of COX-2 and hypoxia-inducible factor 1? (HIF-1?). It was also found that quercetin-3-glucuronide, a major quercetin metabolite, downregulated the expression of COX-2, HIF-1?, VEGF-A, and matrix metalloproteinase activities in aortic vascular smooth muscle cells isolated from AAA mice. Conclusion:Quercetin attenuates neovascularization during AAA growth, and this effect is mediated via the inhibition of COX-2, which decreases HIF-1?/VEGF signaling-related angiogenesis.
Project description:Apoptosis of smooth muscle cells (SMCs) is a prominent pathological characteristic of abdominal aortic aneurysm (AAA). We have previously shown that SMC apoptosis stimulates proinflammatory signaling in a mouse model of AAA. Here, we test whether protein kinase C-? (PKC?), an apoptotic mediator, participates in the pathogenesis of AAA by regulating apoptosis and proinflammatory signals.Mouse experimental AAA is induced by perivascular administration of CaCl(2). Mice deficient in PKC? exhibit a profound reduction in aneurysmal expansion, SMC apoptosis, and transmural inflammation as compared with wild-type littermates. Delivery of PKC? to the aortic wall of PKC?(-/-) mice restores aneurysm, whereas overexpression of a dominant negative PKC? mutant in the aorta of wild-type mice attenuates aneurysm. In vitro, PKC?(-/-) aortic SMCs exhibit significantly impaired monocyte chemoattractant protein-1 production. Ectopic administration of recombinant monocyte chemoattractant protein-1 to the arterial wall of PKC?(-/-) mice restores inflammatory response and aneurysm development.PKC? is an important signaling mediator for SMC apoptosis and inflammation in a mouse model of AAA. By stimulating monocyte chemoattractant protein-1 expression in aortic SMCs, upregulated PKC? exacerbates the inflammatory process, in turn perpetuating elastin degradation and aneurysmal dilatation. Inhibition of PKC? may serve as a potential therapeutic strategy for AAA.
Project description:Abdominal aortic aneurysm (AAA) is characterized by transmural infiltration of myeloid cells at the vascular injury site. Previously, we reported preventive effects of Notch deficiency on the development of AAA by reduction of infiltrating myeloid cells. In this study, we examined if Notch inhibition attenuates the progression of pre-established AAA and potential implications. Pharmacological Notch inhibitor (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester; DAPT) was administered subcutaneously three times a week starting at day 28 of angiotensin II (AngII) infusion. Progressive increase in pulse wave velocity (PWV), maximal intra-luminal diameter (MILD) and maximal external aortic diameter (MEAD) were observed at day 56 of the AngII. DAPT prevented such increase in MILD, PWV and MEAD (P < 0.01). Histologically, the aortae of DAPT-treated Apoe<sup>-/-</sup> mice had significant reduction in inflammatory response and elastin fragmentation. Naked collagen microfibrils and weaker banded structure observed in the aortae of Apoe<sup>-/-</sup> mice in response to AngII, were substantially diminished by DAPT. A significant decrease in the proteolytic activity in the aneurysmal tissues and vascular smooth muscle cells (vSMCs) was observed with DAPT (P < 0.01). In human and mouse AAA tissues, increased immunoreactivity of activated Notch signaling correlated strongly with CD38 expression (R<sup>2</sup> = 0.61). Collectively, we propose inhibition of Notch signaling as a potential therapeutic target for AAA progression.
Project description:Background Development of abdominal aortic aneurysm (AAA) is associated with proinflammatory cytokines including interleukin-12 (IL12). Deficiency of interleukin 12p40 (IL12p40) increases localized fibrotic events by promoting TGF?2 (transforming growth factor ?)-dependent anti-inflammatory response. Here, we determined whether IL12p40 deficiency in apolipoprotein E<sup>-/-</sup> mice attenuates the development of AAA by antagonizing proinflammatory response. Methods and Results Double knockout (DKO) mice were generated by crossbreeding IL12p40<sup>-/-</sup> mice with apolipoprotein E<sup>-/-</sup> mice (n=12). Aneurysmal studies were performed using angiotensin II (1 µg/kg/min; subcutaneous). Surprisingly, DKO mice did not prevent the development of AAA with angiotensin II infusion. Immunohistological analysis, however, showed distinct pathological features between apolipoprotein E<i><sup>-/-</sup></i> and DKO mice. Polymerase chain reaction (7 day) and cytokine arrays (28 day) of the aortic tissues from DKO mice showed significantly increased expression of cytokines related to anti-inflammatory response (interleukin 5 and interleukin 13), synthetic vascular smooth muscle cell phenotype (Activin receptor-like kinase-1 (ALK-1), artemin, and betacellulin) and T helper 17-associated response (4-1BB, interleukin-17e (Il17e) and Cd40 ligand (Cd-40L)). Indeed, DKO mice exhibited increased expression of the fibro-proteolytic pathway in the medial layer of aortae induced by cellular communication network factor 2 (CCN2) and Cd3<sup>+</sup>IL17<sup>+</sup> cells compared with apolipoprotein E<i><sup>-/-</sup></i> mice. Laser capture microdissection showed predominant expression of CCN2/TGF?2 in the medial layer of human AAA. Finally, <i>Ccn2</i> haploinsufficiency in the mice showed decreased AAA incidence in response to elastase infusion, associated with decreased matrix metalloproteinase-2 expression. Conclusions Our study reveals novel roles for <i>IL12p40</i> deficiency in inducing fibro-proteolytic activities in the aneurysmal mouse model. Mechanistically, these effects of IL12p40 deficiency are mediated by CCN2/matrix metalloproteinase-2 crosstalk in the medial layer of aneurysmal aortae.