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Analysis of in situ and ex vivo vascular endothelial growth factor receptor expression during experimental aortic aneurysm progression.

ABSTRACT: 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.

SUBMITTER: Tedesco MM

PROVIDER: S-EPMC2752726 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Analysis of in situ and ex vivo vascular endothelial growth factor receptor expression during experimental aortic aneurysm progression.

Tedesco Maureen M MM Terashima Masahiro M Blankenberg Francis G FG Levashova Zoia Z Spin Joshua M JM Backer Marina V MV Backer Joseph M JM Sho Mien M Sho Eiketsu E McConnell Michael V MV Dalman Ronald L RL

Arteriosclerosis, thrombosis, and vascular biology 20090702 10

<h4>Objective</h4>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.<h4>Methods and results</h4>Apo E(-/-) mice maintained on a high-fat diet underwent continuous infusion with angioten ...[more]

PMID: 19574559

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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: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:BACKGROUND:Near-infrared (NIR) imaging of liver segments provides substantial information for surgeons performing liver resection. It was hypothesized that ramucirumab, an endothelium-specific antibody approved by the Food and Drug Administration, could be used for liver segment imaging using the endothelium capture principle. METHODS:The capture efficacy of anti-vascular endothelial growth factor receptor (VEGFR) 2 monoclonal antibodies (mAbs) and segment imaging were studied in a mouse model. Binding of ramucirumab in human and porcine tissues was studied using immunofluorescence staining. Isolated porcine liver perfusion was used to analyse the labelling and NIR imaging of selected liver segments. RESULTS:VEGFR2 is well expressed on the endothelium of the smallest microvascular blood vessels in mouse, porcine and human liver tissues, as well as in human liver tumours. Perfusion of selected segments in the isolated liver model showed high capture of the anti-VEGFR2 (clone 522302) mAb and ramucirumab in mice and pigs respectively. NIR imaging of selected segments was achieved using isolated porcine liver perfusion with IRDye® 800CW-conjugated ramucirumab. CONCLUSION:VEGFR2 is well expressed on the smallest microvascular blood vessels and can capture antibodies during single intravascular passages with high efficacy. The ex?vivo imaging of a selected segment using endothelial capture of ramucirumab demonstrates the potential of this antibody for intraoperative navigation in liver surgery. Surgical relevance Imaging of liver segments provides substantial information for surgeons when performing liver resection. The antivascular endothelial growth factor receptor (VEGFR) 2 antibody ramucirumab conjugated with near-infrared dye could visualize selected liver segments using an endothelial capture-based approach in an isolated perfusion liver model. The ex?vivo imaging of a selected segment using endothelial capture of ramucirumab demonstrates the potential of this anti-VEGFR2 antibody for intraoperative navigation in liver surgery.

Project description:BackgroundTumor necrosis factor (TNF) is pathologically elevated in human abdominal aortic aneurysms (AAA). Non-selective TNF inhibition-based therapeutics are approved for human use but have been linked to several side effects. Compounds that target the proinflammatory soluble form of TNF (solTNF) but preserve the immunomodulatory capabilities of the transmembrane form of TNF (tmTNF) may prevent these side effects. We hypothesize that inhibition of solTNF signaling prevents AAA expansion.MethodsThe effect of the selective solTNF inhibitor, XPro1595, and the non-selective TNF inhibitor, Etanercept (ETN) was examined in porcine pancreatic elastase (PPE) induced AAA mice, and findings with XPro1595 was confirmed in angiotensin II (ANGII) induced AAA in hyperlipidemic apolipoprotein E (Apoe) -/- mice.ResultsXPro1595 treatment significantly reduced AAA expansion in both models, and a similar trend (p = 0.06) was observed in PPE-induced AAA in ETN-treated mice. In the PPE aneurysm wall, XPro1595 improved elastin integrity scores. In aneurysms, mean TNFR1 levels reduced non-significantly (p = 0.07) by 50% after TNF inhibition, but the histological location in murine AAAs was unaffected and similar to that in human AAAs. Semi-quantification of infiltrating leucocytes, macrophages, T-cells, and neutrophils in the aneurysm wall were unaffected by TNF inhibition. XPro1595 increased systemic TNF levels, while ETN increased systemic IL-10 levels. In ANGII-induced AAA mice, XPro1595 increased systemic TNF and IL-5 levels. In early AAA development, proteomic analyses revealed that XPro1595 significantly upregulated ontology terms including "platelet aggregation" and "coagulation" related to the fibrinogen complex, from which several proteins were among the top regulated proteins. Downregulated ontology terms were associated with metabolic processes.ConclusionIn conclusion, selective inhibition of solTNF signaling reduced aneurysm expansion in mice, supporting its potential as an attractive treatment option for AAA patients.

Dataset Information

Analysis of in situ and ex vivo vascular endothelial growth factor receptor expression during experimental aortic aneurysm progression.

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Analysis of in situ and ex vivo vascular endothelial growth factor receptor expression during experimental aortic aneurysm progression.

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