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Endothelium capture-based liver segment imaging using vascular endothelial growth factor receptor 2 in preclinical ex vivo models.

ABSTRACT: 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.

SUBMITTER: Kyuno D

PROVIDER: S-EPMC7093791 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Endothelium capture-based liver segment imaging using vascular endothelial growth factor receptor 2 in preclinical ex vivo models.

Kyuno D D Qian B B Groß W W Schäfer M M Ryschich E E

BJS open 20200122 2

<h4>Background</h4>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.<h4>Methods</h4>The capture efficacy of anti-vascular endothelial growth factor receptor (VEGFR) 2 monoclonal antibodies (mAbs) and segment imaging were studied in ...[more]

PMID: 31965760

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Project description:The lymphatic vascular system plays important roles in the maintenance of interstitial fluid pressure, the afferent immune response and the absorption of dietary lipids. However, the molecular mechanisms that control lymphatic vessel network maturation and function remain largely unknown. To identify novel players in lymphatic vessel function, we isolated pure populations of lymphatic and blood vascular endothelial cells from mouse intestine using fluorescence-activated high-speed cell sorting and performed transcriptional profiling. We found that the axonal guidance molecules semaphorin 3A (Sema3A) and Sema3D were specifically expressed by lymphatic vessels. Quantitative PCR of ex vivo isolated cells and immunohistochemical analysis confirmed these results. Importantly, we found that the semaphorin receptor neuropilin-1 (Nrp-1) is expressed on the valves of collecting lymphatic vessels. Treatment of mice in utero (E12.5-E16.5) with an antibody that blocks Sema3A binding to Nrp-1, but not with an antibody that blocks VEGFA binding to Nrp-1, resulted in abnormal development of collecting lymphatic vessels and valves, and aberrant smooth muscle cell coverage. Conversely, Sema3A-deficient mice displayed branching defects of collecting lymphatic vessels as well as impaired valve development. Together, these results reveal an unanticipated role of Sema3A/Nrp-1 signaling in the maturation of the lymphatic vascular network. Colon single-cell suspensions were prepared by a fast protocol that minimizes the RNA degradation. Fluorescence-activated cell sorting (FACS) was used to sort blood vascular endothelial cells (BEC) and lymphatic endothelial cells (LEC). 4 animal-matched pairs of LEC and BEC were chosen based on the quality of extracted and amplified material to provide homogenous groups of biological replicates. This gave 8 samples to analyze. Samples present LEC and BEC isolated from 4 healthy normal mice. The 4 mice used present the 4 biological replicates.

Project description:ObjectiveMural 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.Methods and resultsApo 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.ConclusionsMural 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.

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Endothelium capture-based liver segment imaging using vascular endothelial growth factor receptor 2 in preclinical ex vivo models.

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Endothelium capture-based liver segment imaging using vascular endothelial growth factor receptor 2 in preclinical ex vivo models.

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