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• W2073212944 abstract "Angiogenesis inhibitors are receiving increased attention as cancer therapeutics, but little is known of the cellular effects of these inhibitors on tumor vessels. We sought to determine whether two agents, AG013736 and VEGF-Trap, that inhibit vascular endothelial growth factor (VEGF) signaling, merely stop angiogenesis or cause regression of existing tumor vessels. Here, we report that treatment with these inhibitors caused robust and early changes in endothelial cells, pericytes, and basement membrane of vessels in spontaneous islet-cell tumors of RIP-Tag2 transgenic mice and in subcutaneously implanted Lewis lung carcinomas. Strikingly, within 24 hours, endothelial fenestrations in RIP-Tag2 tumors disappeared, vascular sprouting was suppressed, and patency and blood flow ceased in some vessels. By 7 days, vascular density decreased more than 70%, and VEGFR-2 and VEGFR-3 expression was reduced in surviving endothelial cells. Vessels in Lewis lung tumors, which lacked endothelial fenestrations, showed less regression. In both tumors, pericytes did not degenerate to the same extent as endothelial cells, and those on surviving tumor vessels acquired a more normal phenotype. Vascular basement membrane persisted after endothelial cells degenerated, providing a ghost-like record of pretreatment vessel number and location and a potential scaffold for vessel regrowth. The potent anti-vascular action observed is evidence that VEGF signaling inhibitors do more than stop angiogenesis. Early loss of endothelial fenestrations in RIP-Tag2 tumors is a clue that vessel phenotype may be predictive of exceptional sensitivity to these inhibitors. Angiogenesis inhibitors are receiving increased attention as cancer therapeutics, but little is known of the cellular effects of these inhibitors on tumor vessels. We sought to determine whether two agents, AG013736 and VEGF-Trap, that inhibit vascular endothelial growth factor (VEGF) signaling, merely stop angiogenesis or cause regression of existing tumor vessels. Here, we report that treatment with these inhibitors caused robust and early changes in endothelial cells, pericytes, and basement membrane of vessels in spontaneous islet-cell tumors of RIP-Tag2 transgenic mice and in subcutaneously implanted Lewis lung carcinomas. Strikingly, within 24 hours, endothelial fenestrations in RIP-Tag2 tumors disappeared, vascular sprouting was suppressed, and patency and blood flow ceased in some vessels. By 7 days, vascular density decreased more than 70%, and VEGFR-2 and VEGFR-3 expression was reduced in surviving endothelial cells. Vessels in Lewis lung tumors, which lacked endothelial fenestrations, showed less regression. In both tumors, pericytes did not degenerate to the same extent as endothelial cells, and those on surviving tumor vessels acquired a more normal phenotype. Vascular basement membrane persisted after endothelial cells degenerated, providing a ghost-like record of pretreatment vessel number and location and a potential scaffold for vessel regrowth. The potent anti-vascular action observed is evidence that VEGF signaling inhibitors do more than stop angiogenesis. Early loss of endothelial fenestrations in RIP-Tag2 tumors is a clue that vessel phenotype may be predictive of exceptional sensitivity to these inhibitors. Inhibitors of angiogenesis are now making their way through clinical trials.1Hlatky L Hahnfeldt P Folkman J Clinical application of antiangiogenic therapy: microvessel density, what it does and doesn't tell us.J Natl Cancer Inst. 2002; 94: 883-893Crossref PubMed Google Scholar, 2Herbst RS Mullani NA Davis DW Hess KR McConkey DJ Charnsangavej C O'Reilly MS Kim HW Baker C Roach J Ellis LM Rashid A Pluda J Bucana C Madden TL Tran HT Abbruzzese JL Development of biologic markers of response and assessment of antiangiogenic activity in a clinical trial of human recombinant endostatin.J Clin Oncol. 2002; 20: 3804-3814Crossref PubMed Scopus (179) Google Scholar, 3Ellis LM Antiangiogenic therapy: more promise and, yet again, more questions.J Clin Oncol. 2003; 21: 3897-3899Crossref PubMed Scopus (21) Google Scholar Some results with inhibitors of vascular endothelial growth factor (VEGF) are promising,4McCarthy M Antiangiogenesis drug promising for metastatic colorectal cancer.Lancet. 2003; 361: 1959Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 5Yang JC Haworth L Sherry RM Hwu P Schwartzentruber DJ Topalian SL Steinberg SM Chen HX Rosenberg SA A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer.N Engl J Med. 2003; 349: 427-434Crossref PubMed Scopus (2534) Google Scholar but challenges are faced in selecting the right patients, determining effective doses, and evaluating responses. Patient selection is made difficult by lack of understanding of which tumors have drug-sensitive blood vessels, and limited information is available on the cellular changes tumor vessels undergo in response to angiogenesis inhibitors. Conventional measurements of microvascular density, one of the most common microscopic methods used to quantify angiogenesis in tumors,6Weidner N Intratumor microvessel density as a prognostic factor in cancer.Am J Pathol. 1995; 147: 9-19PubMed Google Scholar is not always an accurate measure of efficacy because tumor mass may decrease in parallel with the number of blood vessels.1Hlatky L Hahnfeldt P Folkman J Clinical application of antiangiogenic therapy: microvessel density, what it does and doesn't tell us.J Natl Cancer Inst. 2002; 94: 883-893Crossref PubMed Google Scholar Other standard endpoints, such as tumor burden, provide little insight into whether drugs act on blood vessels or tumor cells and may not show whether tumor growth is stabilized by angiogenesis inhibition. Thus, new ways are needed for evaluating vascular effects of angiogenesis inhibitors.Blood vessels in tumors have multiple abnormalities. Tumor vessels express unique proteins7St. Croix B Rago C Velculescu V Traverso G Romans KE Montgomery E Lal A Riggins GJ Lengauer C Vogelstein B Kinzler KW Genes expressed in human tumor endothelium.Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1632) Google Scholar and have bizarre morphological features, including loss of arteriole-capillary-venule hierarchy, tortuosity, variable diameter, defective endothelial monolayer, and leakiness.8Hashizume H Baluk P Morikawa S McLean JW Thurston G Roberge S Jain RK McDonald DM Openings between defective endothelial cells explain tumor vessel leakiness.Am J Pathol. 2000; 156: 1363-1380Abstract Full Text Full Text PDF PubMed Scopus (1297) Google Scholar, 9McDonald DM Foss AJ Endothelial cells of tumor vessels: abnormal but not absent.Cancer Metastasis Rev. 2000; 19: 109-120Crossref PubMed Scopus (119) Google Scholar Even pericytes (mural cells) of tumor vessels are abnormal, as evidenced by altered gene expression and loss of intimate contact with endothelial cells.10Abramsson A Berlin O Papayan H Paulin D Shani M Betsholtz C Analysis of mural cell recruitment to tumor vessels.Circulation. 2002; 105: 112-117Crossref PubMed Scopus (147) Google Scholar, 11Morikawa S Baluk P Kaidoh T Haskell A Jain RK McDonald DM Abnormalities in pericytes on blood vessels and endothelial sprouts in tumors.Am J Pathol. 2002; 160: 985-1000Abstract Full Text Full Text PDF PubMed Scopus (789) Google Scholar Abnormalities of the basement membrane of tumor vessels are also present and reflect the disturbances of endothelial cells and pericytes.12Baluk P Morikawa S Haskell A Mancuso M McDonald DM Abnormalities of basement membrane on blood vessels and endothelial sprouts in tumors.Am J Pathol. 2003; 163: 1801-1815Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar We reasoned that a better understanding of these abnormalities and how they respond to treatment could give insight into the cellular effects of angiogenesis inhibitors.One approach to blocking angiogenesis involves inhibition of VEGF.4McCarthy M Antiangiogenesis drug promising for metastatic colorectal cancer.Lancet. 2003; 361: 1959Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 13Ferrara N Role of vascular endothelial growth factor in physiologic and pathologic angiogenesis: therapeutic implications.Semin Oncol. 2002; 29: 10-14PubMed Google Scholar VEGF and its receptors, VEGFR-1 (flt-1) and VEGFR-2 (flk-1/KDR), play key roles in the formation and growth of normal blood vessels and in tumor angiogenesis.14Ferrara N Gerber HP LeCouter J The biology of VEGF and its receptors.Nat Med. 2003; 9: 669-676Crossref PubMed Scopus (7742) Google Scholar There is compelling evidence that VEGF is a survival factor for some tumor vessels and that the growth of some tumors is dependent on VEGF-induced angiogenesis. The strength of the evidence stems in part from the use of multiple different approaches to inhibit VEGF signaling, including neutralizing antibodies against VEGF15Kim KJ Li B Winer J Armanini M Gillett N Phillips HS Ferrara N Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo.Nature. 1993; 362: 841-844Crossref PubMed Scopus (3328) Google Scholar or VEGFR-2,16Witte L Hicklin DJ Zhu Z Pytowski B Kotanides H Rockwell P Bohlen P Monoclonal antibodies targeting the VEGF receptor-2 (Flk1/KDR) as an anti-angiogenic therapeutic strategy.Cancer Metastasis Rev. 1998; 17: 155-161Crossref PubMed Scopus (301) Google Scholar anti-sense VEGF cDNA,17Im SA Gomez-Manzano C Fueyo J Liu TJ Ke LD Kim JS Lee HY Steck PA Kyritsis AP Yung WK Antiangiogenesis treatment for gliomas: transfer of antisense-vascular endothelial growth factor inhibits tumor growth in vivo.Cancer Res. 1999; 59: 895-900PubMed Google Scholar conditional expression of the VEGF gene,18Benjamin LE Keshet E Conditional switching of vascular endothelial growth factor (VEGF) expression in tumors: induction of endothelial cell shedding and regression of hemangioblastoma-like vessels by VEGF withdrawal.Proc Natl Acad Sci USA. 1997; 94: 8761-8766Crossref PubMed Scopus (443) Google Scholar soluble VEGF receptors,19Goldman CK Kendall RL Cabrera G Soroceanu L Heike Y Gillespie GY Siegal GP Mao X Bett AJ Huckle WR Thomas KA Curiel DT Paracrine expression of a native soluble vascular endothelial growth factor receptor inhibits tumor growth, metastasis, and mortality rate.Proc Natl Acad Sci USA. 1998; 95: 8795-8800Crossref PubMed Scopus (414) Google Scholar, 20Davidoff AM Leary MA Ng CY Vanin EF Gene therapy-mediated expression by tumor cells of the angiogenesis inhibitor flk-1 results in inhibition of neuroblastoma growth in vivo.J Pediatr Surg. 2001; 36: 30-36Abstract Full Text Full Text PDF PubMed Google Scholar chimeric proteins consisting of the extracellular domain of VEGFR-1 and VEGFR-2 joined to the Fc portion of IgG,21Holash J Davis S Papadopoulos N Croll SD Ho L Russell M Boland P Leidich R Hylton D Burova E Ioffe E Huang T Radziejewski C Bailey K Fandl JP Daly T Wiegand SJ Yancopoulos GD Rudge JS VEGF-Trap: a VEGF blocker with potent antitumor effects.Proc Natl Acad Sci USA. 2002; 99: 11393-11398Crossref PubMed Scopus (1463) Google Scholar adenoviral expression of soluble VEGF receptors22Tseng JF Farnebo FA Kisker O Becker CM Kuo CJ Folkman J Mulligan RC Adenovirus-mediated delivery of a soluble form of the VEGF receptor Flk1 delays the growth of murine and human pancreatic adenocarcinoma in mice.Surgery. 2002; 132: 857-865Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar or dominant-negative VEGFR-2,23Millauer B Shawver LK Plate KH Risau W Ullrich A Glioblastoma growth inhibited in vivo by a dominant-negative Flk-1 mutant.Nature. 1994; 367: 576-579Crossref PubMed Scopus (1158) Google Scholar and small molecules that inhibit VEGF receptor tyrosine kinase phosphorylation.24Fong TA Shawver LK Sun L Tang C App H Powell TJ Kim YH Schreck R Wang X Risau W Ullrich A Hirth KP McMahon G SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types.Cancer Res. 1999; 59: 99-106PubMed Google Scholar, 25Laird AD Vajkoczy P Shawver LK Thurnher A Liang C Mohammadi M Schlessinger J Ullrich A Hubbard SR Blake RA Fong TA Strawn LM Sun L Tang C Hawtin R Tang F Shenoy N Hirth KP McMahon G Cherrington JM SU6668 is a potent antiangiogenic and antitumor agent that induces regression of established tumors.Cancer Res. 2000; 60: 4152-4160PubMed Google Scholar, 26Drevs J Muller-Driver R Wittig C Fuxius S Esser N Hugenschmidt H Konerding MA Allegrini PR Wood J Hennig J Unger C Marme D PTK787/ZK 222584, a specific vascular endothelial growth factor-receptor tyrosine kinase inhibitor, affects the anatomy of the tumor vascular bed and the functional vascular properties as detected by dynamic enhanced magnetic resonance imaging.Cancer Res. 2002; 62: 4015-4022PubMed Google Scholar, 27Hu-Lowe D Hallin M Feeley R Zou H Rewolinski D Wickman G Chen E Kim Y Riney S Reed J Heller D Simmons B Kania R McTigue M Niesman M Gregory S Shalinsky D Bender S Characterization of potency and activity of the VEGF/PDGF receptor tyrosine kinase inhibitor AG013736.Proc Am Assoc Cancer Res. 2002; 43: A5357Google ScholarInhibition of VEGF signaling not only blocks angiogenesis in tumors but can also change or destroy tumor vessels.28Yuan F Chen Y Dellian M Safabakhsh N Ferrara N Jain RK Time-dependent vascular regression and permeability changes in established human tumor xenografts induced by an anti-vascular endothelial growth factor/vascular permeability factor antibody.Proc Natl Acad Sci USA. 1996; 93: 14765-14770Crossref PubMed Scopus (601) Google Scholar, 29Shaheen RM Davis DW Liu W Zebrowski BK Wilson MR Bucana CD McConkey DJ McMahon G Ellis LM Antiangiogenic therapy targeting the tyrosine kinase receptor for vascular endothelial growth factor receptor inhibits the growth of colon cancer liver metastasis and induces tumor and endothelial cell apoptosis.Cancer Res. 1999; 59: 5412-5416PubMed Google Scholar, 30Bruns CJ Liu W Davis DW Shaheen RM McConkey DJ Wilson MR Bucana CD Hicklin DJ Ellis LM Vascular endothelial growth factor is an in vivo survival factor for tumor endothelium in a murine model of colorectal carcinoma liver metastases.Cancer. 2000; 89: 488-499Crossref PubMed Scopus (187) Google Scholar, 31Huang J Frischer JS Serur A Kadenhe A Yokoi A McCrudden KW New T O'Toole K Zabski S Rudge JS Holash J Yancopoulos GD Yamashiro DJ Kandel JJ Regression of established tumors and metastases by potent vascular endothelial growth factor blockade.Proc Natl Acad Sci USA. 2003; 100: 7785-7790Crossref PubMed Scopus (223) Google Scholar VEGF/VEGFR inhibition can decrease the diameter, tortuosity, and permeability of tumor vessels28Yuan F Chen Y Dellian M Safabakhsh N Ferrara N Jain RK Time-dependent vascular regression and permeability changes in established human tumor xenografts induced by an anti-vascular endothelial growth factor/vascular permeability factor antibody.Proc Natl Acad Sci USA. 1996; 93: 14765-14770Crossref PubMed Scopus (601) Google Scholar and even transform surviving tumor vessels into a more normal phenotype.32Jain RK Normalizing tumor vasculature with anti-angiogenic therapy: a new paradigm for combination therapy.Nat Med. 2001; 7: 987-989Crossref PubMed Scopus (1794) Google Scholar, 33Erber R Thurnher A Katsen AD Groth G Kerger H Hammes HP Menger MD Ullrich A Vajkoczy P Combined inhibition of VEGF and PDGF signaling enforces tumor vessel regression by interfering with pericyte-mediated endothelial cell survival mechanisms.EMBO J. 2004; 18: 338-340Google Scholar Considering the important role of VEGF and its receptors in regulating vascular function, we sought to characterize the changes in tumor vessels produced by agents that block the action of this growth factor.In the present studies, we examined the cellular effects of two inhibitors of VEGF signaling, VEGF-Trap and AG013736, on blood vessels in spontaneous pancreatic islet tumors in RIP-Tag2 transgenic mice34Hanahan D Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes.Nature. 1985; 315: 115-122Crossref PubMed Scopus (1006) Google Scholar and implanted Lewis lung carcinoma (LLC) in syngeneic mice. VEGF-Trap is a decoy construct of VEGFR-1 and VEGFR-2 that inhibits VEGF signaling by selectively binding the ligand and has potent anti-angiogenic activity in preclinical tumor models.21Holash J Davis S Papadopoulos N Croll SD Ho L Russell M Boland P Leidich R Hylton D Burova E Ioffe E Huang T Radziejewski C Bailey K Fandl JP Daly T Wiegand SJ Yancopoulos GD Rudge JS VEGF-Trap: a VEGF blocker with potent antitumor effects.Proc Natl Acad Sci USA. 2002; 99: 11393-11398Crossref PubMed Scopus (1463) Google Scholar, 31Huang J Frischer JS Serur A Kadenhe A Yokoi A McCrudden KW New T O'Toole K Zabski S Rudge JS Holash J Yancopoulos GD Yamashiro DJ Kandel JJ Regression of established tumors and metastases by potent vascular endothelial growth factor blockade.Proc Natl Acad Sci USA. 2003; 100: 7785-7790Crossref PubMed Scopus (223) Google Scholar AG013736 is a small molecule inhibitor of VEGFR-1, VEGFR-2, VEGFR-3, and related tyrosine kinase receptors that has potent anti-angiogenic and anti-tumor effects in mice.27Hu-Lowe D Hallin M Feeley R Zou H Rewolinski D Wickman G Chen E Kim Y Riney S Reed J Heller D Simmons B Kania R McTigue M Niesman M Gregory S Shalinsky D Bender S Characterization of potency and activity of the VEGF/PDGF receptor tyrosine kinase inhibitor AG013736.Proc Am Assoc Cancer Res. 2002; 43: A5357Google Scholar, 35Wickman G Hallin M Dillon R Amundson K Acena A Grazzini M Herrmann M Vekich S McTigue M Kania R Bender S Shalinsky DR Hu-Lowe DD Further characterization of the potent VEGF/PDGF receptor tyrosine kinase inhibitor, AG013736, in preclinical tumor models for its antiangiogenesis and antitumor activity.Proc Am Assoc Cancer Res. 2003; 44: A3780Google Scholar We focused on changes occurring during the first week of treatment of established tumors to identify primary vascular effects of the inhibitors that precede or accompany the reduction in tumor growth documented by others.21Holash J Davis S Papadopoulos N Croll SD Ho L Russell M Boland P Leidich R Hylton D Burova E Ioffe E Huang T Radziejewski C Bailey K Fandl JP Daly T Wiegand SJ Yancopoulos GD Rudge JS VEGF-Trap: a VEGF blocker with potent antitumor effects.Proc Natl Acad Sci USA. 2002; 99: 11393-11398Crossref PubMed Scopus (1463) Google Scholar, 27Hu-Lowe D Hallin M Feeley R Zou H Rewolinski D Wickman G Chen E Kim Y Riney S Reed J Heller D Simmons B Kania R McTigue M Niesman M Gregory S Shalinsky D Bender S Characterization of potency and activity of the VEGF/PDGF receptor tyrosine kinase inhibitor AG013736.Proc Am Assoc Cancer Res. 2002; 43: A5357Google Scholar, 31Huang J Frischer JS Serur A Kadenhe A Yokoi A McCrudden KW New T O'Toole K Zabski S Rudge JS Holash J Yancopoulos GD Yamashiro DJ Kandel JJ Regression of established tumors and metastases by potent vascular endothelial growth factor blockade.Proc Natl Acad Sci USA. 2003; 100: 7785-7790Crossref PubMed Scopus (223) Google Scholar, 33Erber R Thurnher A Katsen AD Groth G Kerger H Hammes HP Menger MD Ullrich A Vajkoczy P Combined inhibition of VEGF and PDGF signaling enforces tumor vessel regression by interfering with pericyte-mediated endothelial cell survival mechanisms.EMBO J. 2004; 18: 338-340Google Scholar, 35Wickman G Hallin M Dillon R Amundson K Acena A Grazzini M Herrmann M Vekich S McTigue M Kania R Bender S Shalinsky DR Hu-Lowe DD Further characterization of the potent VEGF/PDGF receptor tyrosine kinase inhibitor, AG013736, in preclinical tumor models for its antiangiogenesis and antitumor activity.Proc Am Assoc Cancer Res. 2003; 44: A3780Google Scholar, 36Bergers G Song S Meyer-Morse N Bergsland E Hanahan D Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors.J Clin Invest. 2003; 111: 1287-1295Crossref PubMed Scopus (1223) Google Scholar Fluorescence, confocal, and electron microscopic approaches applied to tissues fixed in situ by vascular perfusion provided cellular and molecular readouts for assessing patency and blood flow of individual vessels, endothelial sprouts and fenestrations, VEGFR-2 and VEGFR-3 immunoreactivity, pericyte morphology, and changes in the vascular basement membrane. The orderly microvasculature of the mouse trachea was used to validate some of the methods in a simpler system.37Baffert F Thurston G Rochon-Duck M Le T Brekken R McDonald DM Age-related changes in VEGF-dependency and angiopoietin-1 induced plasticity of adult blood vessels.Circ Res. 2004; 94: 984-992Crossref PubMed Scopus (101) Google Scholar Cellular changes observed as soon as 24 hours after the onset of treatment reflected drug activity and suggested novel features that may help to identify types of blood vessels that are sensitive to inhibition of VEGF signaling.Materials and MethodsVEGF/VEGFR InhibitorsVEGF-Trap (also called VEGF-TrapR1R2), which consists of the second immunoglobulin (Ig) domain of VEGFR-1 and third Ig domain of VEGFR-2 fused to the constant region (Fc) of human IgG1,21Holash J Davis S Papadopoulos N Croll SD Ho L Russell M Boland P Leidich R Hylton D Burova E Ioffe E Huang T Radziejewski C Bailey K Fandl JP Daly T Wiegand SJ Yancopoulos GD Rudge JS VEGF-Trap: a VEGF blocker with potent antitumor effects.Proc Natl Acad Sci USA. 2002; 99: 11393-11398Crossref PubMed Scopus (1463) Google Scholar was supplied by Regeneron Pharmaceuticals, Inc., Tarrytown, NY. AG013736, which is a potent small molecule inhibitor of VEGF/platelet-derived growth factor (PDGF) receptor tyrosine kinases (IC50 = 1.2 nmol/L for VEGFR-1, 0.25 nmol/L for VEGFR-2, 0.29 nmol/L for VEGFR-3, 2.5 nmol/L for PDGFR-β, 2.0 for cKit, and 218 nmol/L for FGFR-1),35Wickman G Hallin M Dillon R Amundson K Acena A Grazzini M Herrmann M Vekich S McTigue M Kania R Bender S Shalinsky DR Hu-Lowe DD Further characterization of the potent VEGF/PDGF receptor tyrosine kinase inhibitor, AG013736, in preclinical tumor models for its antiangiogenesis and antitumor activity.Proc Am Assoc Cancer Res. 2003; 44: A3780Google Scholar was supplied by Pfizer Global Research and Development, San Diego, CA.Animals and TreatmentTumor-bearing RIP-Tag2 transgenic mice34Hanahan D Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes.Nature. 1985; 315: 115-122Crossref PubMed Scopus (1006) Google Scholar (C57BL/6 background, 10 to 12 weeks of age) were injected with VEGF-Trap (25 mg/kg in a volume of 5 μl/g i.p.) or its vehicle (5 μl/g; Chinese hamster ovary cell-derived human Fc domain in 40 mmol/L phosphate) on day 0 and studied on day 1 or 2 or were injected on days 0, 3, and 6 and studied on day 7. Alternatively, RIP-Tag2 mice were injected with AG013736 (25 mg/kg in a volume of 5 μl/g i.p.) or its vehicle (5 μl/g; 3 parts PEG 400 to 7 parts acidified H2O, pH 2 to 3) twice daily for 1, 2, or 7 days or once daily for 7 or 21 days. In addition, wild-type C57BL/6 mice, 10 weeks of age, with a 1-mm3 piece of LLC tumor implanted under the dorsal skin for 4 to 6 days, were treated with AG013736 (25 mg/kg i.p.) or vehicle twice daily for 7 days. These intervention treatment regimens38Bergers G Javaherian K Lo KM Folkman J Hanahan D Effects of angiogenesis inhibitors on multistage carcinogenesis in mice.Science. 1999; 284: 808-812Crossref PubMed Scopus (876) Google Scholar examined the effects of the agents on established RIP-Tag2 tumors and LLC tumors. Normal wild-type mice (FVB/n background, 8 weeks of age) were treated with AG013736 (25 mg/kg i.p.) twice daily for <10 days, VEGF-Trap for 7 days, or vehicle for developing the model using the tracheal microvasculature.Lectin Injection and Fixation by Vascular PerfusionAt the end of the treatment period, mice were anesthetized with ketamine (100 mg/kg i.m.) plus xylazine (10 mg/kg i.m.). In mice used for immunohistochemistry, blood flow and patency of individual tumor vessels were assessed by injection of 100 μg of fluorescein isothiocyanate-labeled Lycopersicon esculentum lectin in 0.9% NaCl (100 μl into femoral vein; Vector Laboratories, Burlingame, CA).8Hashizume H Baluk P Morikawa S McLean JW Thurston G Roberge S Jain RK McDonald DM Openings between defective endothelial cells explain tumor vessel leakiness.Am J Pathol. 2000; 156: 1363-1380Abstract Full Text Full Text PDF PubMed Scopus (1297) Google Scholar, 11Morikawa S Baluk P Kaidoh T Haskell A Jain RK McDonald DM Abnormalities in pericytes on blood vessels and endothelial sprouts in tumors.Am J Pathol. 2002; 160: 985-1000Abstract Full Text Full Text PDF PubMed Scopus (789) Google Scholar Two minutes later the chest was opened rapidly, and the vasculature was perfused for 2 minutes at a pressure of 120 mmHg with fixative [1% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4, Sigma, St. Louis, MO] from an 18-gauge cannula inserted into the aorta via an incision in the left ventricle. Blood and fixative exited through an opening in the right atrium. Tissues were removed, immersed in fixative for 1 hour at 4°C, and then processed for immunohistochemistry. In mice used for transmission electron microscopy (TEM), tissues were fixed by vascular perfusion of fixative containing 3% glutaraldehyde in 75 mmol/L cacodylate buffer, pH 7.1, 4% polyvinylpyrrolidone, 0.05% CaCl2, and 1% sucrose.8Hashizume H Baluk P Morikawa S McLean JW Thurston G Roberge S Jain RK McDonald DM Openings between defective endothelial cells explain tumor vessel leakiness.Am J Pathol. 2000; 156: 1363-1380Abstract Full Text Full Text PDF PubMed Scopus (1297) Google Scholar, 11Morikawa S Baluk P Kaidoh T Haskell A Jain RK McDonald DM Abnormalities in pericytes on blood vessels and endothelial sprouts in tumors.Am J Pathol. 2002; 160: 985-1000Abstract Full Text Full Text PDF PubMed Scopus (789) Google Scholar The fixative for scanning electron microscopy (SEM) contained 2% glutaraldehyde in 100 mmol/L phosphate buffer. Fixative was perfused at 120 mmHg for 2 minutes and then 100 mmHg for 3 minutes. Tissues were removed, immersed in fixative for at least 18 hours (4°C), and then processed for TEM or SEM.Electron MicroscopyFor TEM, RIP-Tag2 tumors, LLC tumors, pancreas, thyroid, and tongue were fixed by vascular perfusion and then were trimmed to a maximal dimension of 5 mm, embedded in 7% agarose, and 0.25- to 1-mm slices were cut with a tissue chopper or razor blade. Specimens measuring ∼1 to 3 mm on a side were cut from the slices, rinsed with 100 mmol/L cacodylate buffer, fixed with OsO4 (1% in 100 mmol/L cacodylate buffer at 4°C, 2 hours), rinsed with water, en bloc stained with uranyl acetate (2% aqueous for 48 hours at 38°C), dehydrated with acetone, and embedded in epoxy resin.8Hashizume H Baluk P Morikawa S McLean JW Thurston G Roberge S Jain RK McDonald DM Openings between defective endothelial cells explain tumor vessel leakiness.Am J Pathol. 2000; 156: 1363-1380Abstract Full Text Full Text PDF PubMed Scopus (1297) Google Scholar, 11Morikawa S Baluk P Kaidoh T Haskell A Jain RK McDonald DM Abnormalities in pericytes on blood vessels and endothelial sprouts in tumors.Am J Pathol. 2002; 160: 985-1000Abstract Full Text Full Text PDF PubMed Scopus (789) Google Scholar Sections 0.5 μm in thickness were stained with toluidine blue for light microscopy, and sections 60 to 80 nm in thickness were stained with lead citrate and examined with a Zeiss EM-10C electron microscope. For SEM, RIP-Tag2 tumors and thyroid glands were fixed by vascular perfusion, trimmed to a maximal dimension of 7 mm, and treated with 30% potassium hydroxide in distilled water for 8 minutes at 60°C to remove the extracellular matrix around blood vessels and tumor cells.39Hashizume H Ushiki T Three-dimensional cytoarchitecture of angiogenic blood vessels in a gelatin sheet implanted in the rat skeletal muscular layers.Arch Histol Cytol. 2002; 65: 347-357Crossref PubMed Scopus (13) Google Scholar The specimens were stained with a solution of 2% tannic acid and 1% OsO4, dehydrated with a graded series of ethanol, transferred to isoamyl acetate, and critical point-dried in liquid CO2. Some tumors were cracked with fine forceps under a dissection microscope to expose blood vessels within the specimen. Dried specimens were put on aluminum stubs, coated with OsO4 in an Osmium Plasma Coater (Vacuum Device Corp., Japan), and examined with a Hitachi S-4300N scanning electron microscope.ImmunohistochemistryEndothelial cells of tumor vessels were evaluated by immunohistochemistry using six primary antibodies: rat monoclonal anti-CD31 (PECAM-1, clone MEC 13.3, 1:1000; Pharmingen, San Diego, CA), hamster monoclonal anti-CD31 (clone 2H8, 1:1000; Chemicon, Temecula, CA), rabbit polyclonal anti-VEGFR-2 (VEGF receptor-2, antibody T014, 1:2000; gift from Rolf Brekken and Philip Thorpe, University of Texas Southwestern Medical Center), goat polyclonal anti-VEGFR-3 (VEGF receptor-3, 1:1000; R&D Systems, Minneapolis, MN), rat monoclonal anti-CD105 (endoglin, clone MJ7/18, 1:500; Pharmingen), and rat monoclonal anti-α5 integrin [CD49e, clone 5H10-27 (MFR5), 1:400; Pharmingen). Pericytes were examined with two primary antibodies: Cy3-conjugated mouse monoclonal anti-α-smooth muscle actin (SMA) (clone 1A4, 1:1000; Sigma Chemical Co., St. Louis, MO) and rat monoclonal anti-PDGFR-β (PDGF receptor-β, clone APB5, 1:2000; gift from Akiyoshi Uemura, Kyoto University, Japan). Vascular basement membrane was examined with rabbit polyclonal anti-type IV collagen antibody (1:10,000; Cosmo Bio Co., Tokyo, Japan).12Baluk P Morikawa S Haskell A Mancuso M McDonald DM Abnormalit" @default.
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• W2073212944 title "Inhibition of Vascular Endothelial Growth Factor (VEGF) Signaling in Cancer Causes Loss of Endothelial Fenestrations, Regression of Tumor Vessels, and Appearance of Basement Membrane Ghosts" @default.
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