Abnormalities of Basement Membrane on Blood Vessels and Endothelial Sprouts in Tumors
2003; Elsevier BV; Volume: 163; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)63540-7
ISSN1525-2191
AutoresPeter Bałuk, Shunichi Morikawa, Amy Haskell, Michael R. Mancuso, Donald M. McDonald,
Tópico(s)Platelet Disorders and Treatments
ResumoOften described as incomplete or absent, the basement membrane of blood vessels in tumors has attracted renewed attention as a source of angiogenic and anti-angiogenic molecules, site of growth factor binding, participant in angiogenesis, and potential target in cancer therapy. This study evaluated the composition, extent, and structural integrity of the basement membrane on blood vessels in three mouse tumor models: spontaneous RIP-Tag2 pancreatic islet tumors, MCa-IV mammary carcinomas, and Lewis lung carcinomas. Tumor vessels were identified by immunohistochemical staining for the endothelial cell markers CD31, endoglin (CD105), vascular endothelial growth factor receptor-2, and integrin alpha5 (CD49e). Confocal microscopic studies revealed that basement membrane identified by type IV collagen immunoreactivity covered >99.9% of the surface of blood vessels in the three tumors, just as in normal pancreatic islets. Laminin, entactin/nidogen, and fibronectin immunoreactivities were similarly ubiquitous on tumor vessels. Holes in the basement membrane, found by analyzing 1-μm confocal optical sections, were 99.9% of the surface of blood vessels in the three tumors, just as in normal pancreatic islets. Laminin, entactin/nidogen, and fibronectin immunoreactivities were similarly ubiquitous on tumor vessels. Holes in the basement membrane, found by analyzing 1-μm confocal optical sections, were <2.5 μm in diameter and involved only 0.03% of the vessel surface. Despite the extensive vessel coverage, the basement membrane had conspicuous structural abnormalities, including a loose association with endothelial cells and pericytes, broad extensions away from the vessel wall, and multiple layers visible by electron microscopy. Type IV collagen-immunoreactive sleeves were also present on endothelial sprouts, supporting the idea that basement membrane is present where sprouts grow and regress. These findings indicate that basement membrane covers most tumor vessels but has profound structural abnormalities, consistent with the dynamic nature of endothelial cells and pericytes in tumors. Blood vessels of tumors have multiple structural and functional abnormalities. Their unusual leakiness, potential for rapid growth and remodeling, and expression of distinctive surface molecules not only are responsible for mediating hematogenous spread of tumor cells and maintaining the unusual microenvironment of tumors but also are key to the efficacy of targeted tumor therapy.1Pasqualini R Arap W McDonald DM Probing the structural and molecular diversity of tumor vasculature.Trends Mol Med. 2002; 8: 563-571Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 2St. 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 (1668) Google Scholar, 3Folkman J Angiogenesis-dependent diseases.Semin Oncol. 2001; 28: 536-542Abstract Full Text PDF PubMed Scopus (288) Google Scholar, 4Jain RK Normalizing tumor vasculature with anti-angiogenic therapy: a new paradigm for combination therapy.Nat Med. 2001; 7: 987-989Crossref PubMed Scopus (1875) Google Scholar Like normal blood vessels, tumor vessels consist of endothelial cells, mural cells (pericytes or smooth muscle cells), and their enveloping basement membrane. The nature and severity of the abnormalities in the endothelial cells and pericytes are described in numerous reports,5Warren BA The vascular morphology of tumors.in: Peterson HE Tumor Blood Circulation. 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The identification of basement membrane as a source of angiogenic and anti-angiogenic factors and as a potential diagnostic or therapeutic target in cancer further increases the importance of this component of the wall of tumor vessels.12Neri D Carnemolla B Nissim A Leprini A Querze G Balza E Pini A Tarli L Halin C Neri P Zardi L Winter G Targeting by affinity-matured recombinant antibody fragments of an angiogenesis associated fibronectin isoform.Nature Biotechnol. 1997; 15: 1271-1275Crossref Scopus (287) Google Scholar, 13Marneros AG Olsen BR The role of collagen-derived proteolytic fragments in angiogenesis.Matrix Biol. 2001; 20: 337-345Crossref PubMed Scopus (181) Google Scholar, 14Xu J Rodriguez D Petitclerc E Kim JJ Hangai M Moon YS Davis GE Brooks PC Yuen SM Proteolytic exposure of a cryptic site within collagen type IV is required for angiogenesis and tumor growth in vivo.J Cell Biol. 2001; 154: 1069-1079Crossref PubMed Scopus (406) Google Scholar, 15Kalluri R Basement membranes: structure, assembly and role in tumor angiogenesis.Nat Rev Cancer. 2003; 3: 422-433Crossref PubMed Scopus (1356) Google Scholar The vascular basement membrane is a dynamic, self-assembled layer of proteins, glycoproteins, and proteoglycans formed by and enveloping endothelial cells and pericytes of blood vessels. Major constituents include type IV collagen, laminin, entactin/nidogen, fibronectin, and the heparan-sulfate proteoglycan perlecan.16Yurchenco PD O'Rear J Supramolecular organization of basement membranes.in: Rohrbach DH Timpl R Molecular and Cellular Aspects of Basement Membranes. Academic Press, San Diego1993: 19-47Crossref Google Scholar Basement membrane on blood vessels can be readily identified by immunohistochemical staining of the component proteins.17Paulus W Roggendorf W Schuppan D Immunohistochemical investigation of collagen subtypes in human glioblastomas.Virchows Arch A Pathol Anat Histopathol. 1988; 413: 325-332Crossref PubMed Scopus (74) Google Scholar, 18Hewitt RE Powe DG Morrell K Balley E Leach IH Ellis IO Turner DR Laminin and collagen IV subunit distribution in normal and neoplastic tissues of colorectum and breast.Br J Cancer. 1997; 75: 221-229Crossref PubMed Scopus (62) Google Scholar Fine structural features of basement membrane (basal lamina), including the ∼30- to 40-nm-thick electron-lucent lamina rara next to the cell membrane and the ∼50- to 80-nm-thick outer electron dense lamina densa, are evident by electron microscopy (EM).19Inoue S Ultrastructure of basement membranes.Int Rev Cytol. 1989; 117: 57-98Crossref PubMed Scopus (116) Google Scholar Conventional wisdom predicts that the basement membrane of endothelial cells is degraded during angiogenesis to enable sprout formation and endothelial cell migration.20Kalebic T Garbisa S Glaser B Liotta LA Basement membrane collagen: degradation by migrating endothelial cells.Science. 1983; 221: 281-283Crossref PubMed Scopus (267) Google Scholar Alternatively, it is possible that the basement membrane does not disappear during angiogenesis but instead remodels continuously as endothelial sprouts form and new vessels grow.21Jerdan JA Michels RG Glaser BM Extracellular matrix of newly forming vessels—an immunohistochemical study.Microvasc Res. 1991; 42: 255-265Crossref PubMed Scopus (35) Google Scholar, 22Gusterson BA Warburton MJ Mitchell D Kraft N Hancock WW Invading squamous cell carcinoma can retain a basal lamina. 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Many morphological studies have reported that the basement membrane of tumor vessels is incomplete or absent,17Paulus W Roggendorf W Schuppan D Immunohistochemical investigation of collagen subtypes in human glioblastomas.Virchows Arch A Pathol Anat Histopathol. 1988; 413: 325-332Crossref PubMed Scopus (74) Google Scholar, 24Steinberg F Konerding MA Streffer C The vascular architecture of human xenotransplanted tumors: histological, morphometrical, and ultrastructural studies.J Cancer Res Clin Oncol. 1990; 116: 517-524Crossref PubMed Scopus (38) Google Scholar, 25Paku S Paweletz N First steps of tumor-related angiogenesis.Lab Invest. 1991; 65: 334-346PubMed Google Scholar, 26Nakanishi H Okayama M Oguri K Hayashi K Tateno H Hosoda S Close association between tumour cells and vascular basement membrane in gastric cancers with liver metastasis. An immunohistochemical and electron microscopic study with special attention to extracellular matrices.Virchows Arch A Pathol Anat Histopathol. 1991; 418: 531-538Crossref PubMed Scopus (11) Google Scholar, 27Paku S Current concepts of tumor-induced angiogenesis.Pathol Oncol Res. 1998; 4: 62-75Crossref PubMed Scopus (40) Google Scholar yet other studies suggest that it is present but morphologically abnormal.28Farnoud MR Lissak B Kujas M Peillon F Racadot J Li JY Specific alterations of the basement membrane and stroma antigens in human pituitary tumours in comparison with the normal anterior pituitary. An immunocytochemical study.Virchows Arch A Pathol Anat Histopathol. 1992; 421: 449-455Crossref PubMed Scopus (25) Google Scholar These discrepancies probably result from the use of different approaches. Some reports of defective or absent basement membrane on tumor vessels are based on observations made by transmission EM. The high resolution of this approach can reveal tiny abnormalities but may miss unstained components and the overall amount of coverage. Light microscopic immunohistochemistry can detect the specific protein components of basement membrane and readily provide an overview despite its lower spatial resolution. Although this approach is frequently used to visualize basement membrane in tumors,17Paulus W Roggendorf W Schuppan D Immunohistochemical investigation of collagen subtypes in human glioblastomas.Virchows Arch A Pathol Anat Histopathol. 1988; 413: 325-332Crossref PubMed Scopus (74) Google Scholar, 18Hewitt RE Powe DG Morrell K Balley E Leach IH Ellis IO Turner DR Laminin and collagen IV subunit distribution in normal and neoplastic tissues of colorectum and breast.Br J Cancer. 1997; 75: 221-229Crossref PubMed Scopus (62) Google Scholar, 29Murray JC Smith KA Lauk S Vascular markers for murine tumours.Radiother Oncol. 1989; 16: 221-234Abstract Full Text PDF PubMed Scopus (17) Google Scholar, 30Eyden B Yamazaki K Menasce LP Charchanti A Agnantis NJ Basement-membrane-related peri-vascular matrices not organised as a basal lamina: distribution in malignant tumours and benign lesions.J Submicrosc Cytol Pathol. 2000; 32: 515-523PubMed Google Scholar one of the challenges is to distinguish the basement membrane of blood vessels from that associated with tumor cells or other components. Co-localization of markers for basement membrane and endothelial cells helps in this regard. However, the distribution and extent of coverage of basement membrane proteins specifically associated with endothelial cells and pericytes has been examined in relatively few tumors.28Farnoud MR Lissak B Kujas M Peillon F Racadot J Li JY Specific alterations of the basement membrane and stroma antigens in human pituitary tumours in comparison with the normal anterior pituitary. An immunocytochemical study.Virchows Arch A Pathol Anat Histopathol. 1992; 421: 449-455Crossref PubMed Scopus (25) Google Scholar The goal of the present study was to characterize the completeness and abnormalities of the basement membrane of blood vessels and endothelial sprouts in mouse tumor models. Specifically, we sought to: 1) determine the distribution and extent of coverage of vascular basement membrane in tumors by using immunohistochemistry to co-localize multiple markers of endothelial cells and basement membrane; 2) examine the relationship between vascular basement membrane and endothelial cells and pericytes in tumors; and 3) determine whether basement membrane is associated with endothelial sprouts. To this end, we studied three tumor models in mice: spontaneous pancreatic islet tumors in RIP-Tag2 transgenic mice and implanted syngeneic MCa-IV mouse mammary carcinomas and Lewis lung carcinomas.10Morikawa 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 (816) Google Scholar Basement membrane, identified by type IV collagen, laminin, fibronectin, or entactin/nidogen immunoreactivity, was co-localized with endothelial cells identified by CD31, endoglin (CD105), vascular endothelial growth factor receptor-2 (VEGFR-2), or integrin alpha5 (CD49e) immunoreactivity and with pericytes marked by α-smooth muscle actin (α-SMA) immunoreactivity. Three-dimensional views were obtained by examining 80-μm-thick sections by confocal microscopy. Ultrastructural relationships were examined by transmission EM. Spontaneous pancreatic islet cell tumors were examined in 10-week-old RIP-Tag2 transgenic mice with a C57BL/6 background.10Morikawa 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 (816) Google Scholar Normal pancreatic islets were examined in wild-type C57BL/6 mice. Implanted MCa-IV mouse mammary carcinomas (Massachusetts General Hospital, Boston, MA) and Lewis lung carcinomas (American Type Culture Collection, Rockville, MD) were studied in syngeneic male C3H and C57BL/6 mice, respectively, 2 to 3 weeks after implantation when they were 5 to 10 mm in diameter.10Morikawa 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 (816) Google Scholar All experimental procedures were approved by the Committee on Animal Research at the University of California, San Francisco, CA. Mice were anesthetized with ketamine (87 mg/kg) plus xylazine (13 mg/kg) injected intramuscularly. The chest was opened rapidly, and the vasculature was perfused for 3 minutes at a pressure of 120 mmHg with fixative [4% paraformaldehyde in 0.1 mol/L phosphate-buffered saline (PBS), pH 7.4; Sigma Chemical Co., 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. After the perfusion, the entire pancreas or implanted tumor with overlying dorsal skin was removed and placed into fixative for 2 hours at 4°C. Specimens were then rinsed several times with PBS, embedded in 10% agarose for Vibratome sectioning or infiltrated overnight with 30% sucrose, and frozen for cryostat sectioning. Vibratome or cryostat sections 80 μm in thickness were incubated in 5% normal goat serum (Jackson ImmunoResearch, Inc., West Grove, PA) in PBS containing 0.3% Triton X-100 (Sigma), 0.2% bovine serum albumin (Sigma), and 0.01% thimerosal (Sigma) for 1 hour at room temperature to block nonspecific antibody binding. Next, the sections were incubated for 12 to 15 hours at room temperature in humidified chambers in combinations of two or three primary antibodies diluted in the medium described above. Endothelial cells were identified with antibodies to CD31 [PECAM-1, rat monoclonal, clone MEC 13.3, 1:500 (BD Pharmingen, San Diego, CA) or hamster monoclonal, clone 2H8, 1:500 (Chemicon, Temecula, CA)], endoglin (CD105, rat monoclonal, clone MJ7/18, 1:400; BD Pharmingen), VEGFR-2 (rabbit polyclonal T014, 1:2000; a kind gift from Dr. Rolf A. Brekken, Hope Heart Institute, Seattle, WA), or integrin alpha5 subunit (CD49e, rat monoclonal, clone 5H10-17, 1:400; BD Pharmingen). Pericytes were identified with anti-α-SMA (α-SMA, Cy3-conjugated mouse monoclonal, clone 1A4, 1:1000; Sigma). Basement membrane was identified with antibodies to type IV collagen (rabbit polyclonal, 1:10,000; Cosmo Bio Co. Ltd., Tokyo, Japan), laminin (rabbit polyclonal antibody that recognizes most laminin isoforms, 1:2000; Chemicon), entactin/nidogen (rat monoclonal, clone MAB1946, 1:1000; Chemicon), or fibronectin (rabbit polyclonal, 1:2000; Sigma). After several rinses with PBS, specimens were incubated for 6 hours at room temperature with fluorescent (fluorescein isothiocyanate, Cy3, or Cy5) secondary antibodies (goat anti-rat, anti-hamster, or anti-rabbit; Jackson ImmunoResearch) diluted 1:400 in PBS containing 0.3% Triton X-100 followed by several rinses in PBS. Finally specimens were mounted in Vectashield (Vector Laboratories, Burlingame, CA) and were examined with a Zeiss Axiophot fluorescence microscope equipped with single, dual, and triple fluorescence filters and a low-light, three-chip CoolCam CCD camera (SciMeasure Analytical Systems, Atlanta, GA) and a Zeiss LSM 410 or LSM 510 confocal microscope (Carl Zeiss, Thornwood, NY) with three photomultiplier tubes. Images were saved as digital files. Tumors were fixed for transmission EM by vascular perfusion as for immunohistochemistry except the fixative contained 3% glutaraldehyde, 0.05% calcium chloride, 4% polyvinylpyrrolidone, and 1% sucrose in 75 mmol/L sodium cacodylate buffer, pH 7.1. After the 5-minute perfusion, tissues were removed and fixed overnight or longer at 4°C, and then sections 80 μm in thickness were cut with a Vibratome (TPI, St. Louis, MO). Sections were treated with 1% osmium tetroxide in 100 mmol/L cacodylate buffer (pH 7.2) for 2 hours at 4°C and then with 2% aqueous uranyl acetate for 48 hours at 37°C, dehydrated in acetone, and embedded in epoxy resin. Sections, 0.5 μm in thickness, were stained with toluidine blue for light microscopy, and sections 50 to 100 nm in thickness were stained with 0.8% lead citrate in 0.2 N NaOH and examined with a Zeiss EM 10-C electron microscope. The extent of coverage of blood vessels by type IV collagen immunoreactivity was examined in 1-μm-thick optical sections made by confocal microscopy of 80-μm-thick tissue sections. Confocal images of cross-sections of 25 vessels were prepared from specimens of normal pancreatic islets and non-necrotic regions of each type of tumor (n = 4 mice per group). Regions of blood vessel profiles lacking type IV collagen immunoreactivity were counted and measured, and the percentage of profiles with such defects was calculated. The perimeters of CD31-immunoreactive endothelial cells and type IV collagen-immunoreactive basement membrane were measured using a digitizing tablet.10Morikawa 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 (816) Google Scholar The percentage of vessel wall coverage by basement membrane was calculated as the ratio of the length of type IV collagen staining to total vessel perimeter reflected by CD31 staining. The length of CD31-immunoreactive endothelial sprouts, identified as thin, tapered, blind-ending projections away from the main axis of vessels, was measured, as was the length of their coat of type IV collagen immunoreactivity. Values are expressed as means ± SEM (SE). The significance of differences between means was assessed by analysis of variance followed by the Bonferroni/Dunn test for multiple comparisons, with statistical significance judged as P < 0.05. Vascular basement membrane was identified by co-localization of immunohistochemical markers of endothelial cells and basement membrane. To determine which immunohistochemical marker was most effective for staining endothelial cells in the tumor models, we first compared two different antibodies to CD31 and then paired the better one with antibodies to endoglin (CD105), VEGFR-2, or integrin alpha5 (CD49e). Both antibodies to CD31 stained the same population of tumor vessels. Similarly, antibodies to CD31 primarily co-localized with the other three markers, but scattered vessels stained more strongly with one antibody or another (Figure 1). Overall, antibodies to CD31 stained the largest number of vessels. CD105 and VEGFR-2 were approximately the same as CD31 (Figure 1; A to C and D to F); CD49e stained slightly fewer vessels (Figure 1; G to I). Immunohistochemical staining of type IV collagen, laminin, fibronectin, and entactin/nidogen immunoreactivities uniformly stained the blood vessels in RIP-Tag2 pancreatic tumors, MCa-IV mammary carcinomas, and Lewis lung carcinomas (Figure 2; A to C, G, H). Double staining documented the uniform co-localization of basement membrane proteins (Figure 2; A to C) and CD31 immunoreactivity of endothelial cells (Figure 2; D to F). Adjacent sections stained with different antibodies showed that the immunoreactivities of multiple basement membrane proteins co-localized with one another on tumor vessels (Figure 2; A to C). Type IV collagen immunoreactivity strongly defined the vascular basement membrane (Figure 2A); other regions of the tumors were stained weakly or not at all. Laminin (Figure 2B), fibronectin (Figure 2C), and entactin/nidogen (Figure 2H) immunoreactivities clearly marked the vascular basement membrane but also stained some tumor cells or stromal elements. Because type IV collagen immunoreactivity, of the four markers used, was the most selective for vascular basement membrane in these tumors, it was used for further studies. In normal pancreatic islets, type IV collagen immunoreactivity co-localized with CD31 immunoreactivity in all blood vessels and thus gave an accurate representation of the vascular architecture (Figure 3, A and B); however, type IV collagen immunoreactivity also surrounded acini of the normal exocrine pancreas (Figure 3A, arrows). In 80-μm-thick sections of the three tumors, the type IV collagen sleeves disclosed the irregularity and tortuosity of the tumor vessels (Figure 3, C and D). On some tumor vessels, the region of type IV collagen immunoreactivity was appreciably larger than the region CD31 immunoreactivity, suggesting that some regions of vascular basement membrane were not tightly associated with endothelial cells (Figure 3, C and D, arrowheads). No blood vessel identified by CD31 immunoreactivity in any of the tumors completely lacked type IV collagen immunoreactivity. Confocal microscopic examination of 1-μm optical cross-sections of blood vessels in normal pancreatic islets showed a continuous, smooth and uniform layer of type IV collagen immunoreactivity that faithfully matched the CD31 immunoreactivity of endothelial cells (Figure 4A). Quantitative analysis of 100 vessels in normal islets found only one small defect in the type IV collagen immunoreactivity and >99.9% coverage (Table 1). Corresponding 1-μm optical sections of RIP-Tag2 tumor vessels showed the layer of type IV collagen to be variable in thickness and to have conspicuous surface projections and other irregularities that were never seen in normal vessels (Figure 4, B and C, arrows). Yet the layer of type IV collagen on tumor vessels was nearly complete (Figure 4, B and C). Measurements revealed that type IV collagen immunoreactivity covered >99.9% of the endothelial surface; only 0.03% of the vessel surface lacked staining (Table 1). One defect, ∼0.4 μm in diameter, was found in sections of 100 vessel profiles in RIP-Tag2 tumors (Figure 4, B and C). MCa-IV breast carcinomas had more basement membrane abnormalities (Figure 4; D to F); but even here defects were detected in only 2% of the 1-μm optical sections examined, and the largest was 2.5 μm in diameter (Table 1). Blood vessels of Lewis lung carcinomas had basement membrane defects approximately the same size and frequency as those in RIP-Tag tumors (Figure 4G, Table 1). Basement membrane defects smaller than ∼0.1 μm probably would not be detected by this approach.Table 1Extent of Basement Membrane Coverage of Normal Blood Vessels and Tumor VesselsNormal pancreatic isletsRIP-Tag2 pancreatic islet tumorsMCa-IV mammary carcinomasLewis lung carcinomasPercent of vessel profiles entirely covered by type IV collagen immunoreactivity99 ± 1%99 ± 1%98 ± 1%99 ± 1%Number of defects in type IV collagen immunoreactivity in 100-vessel sample1131Size of defects in type IV collagen immunoreactivity1.0 μm0.3 μm1.3, 2.1, 2.5 μm0.4 μmPercent of vessel coverage by type IV collagen immunoreactivity> 99.9%> 99.9%> 99.9%> 99.9%Basement membrane was identified by type IV collagen immunoreactivity and corresponding endothelial cells were visualized by CD31 immunoreactivity in 80-μm-thick sections of normal pancreatic islets and three mouse tumor models. Perimeters of type IV collagen and CD31 staining on vessels cut perpendicular to their longitudinal axis were measured in 1-μm confocal optical sections. Proportion of vessels without basement membrane defects was determined from the percent of profiles entirely surrounded by type IV collagen staining. Percent of basement membrane coverage was calculated as the ratio of the length of type IV collagen staining to total vessel perimeter reflected by CD31 staining. One hundred vessel profiles were measured in each model (25 profiles per mouse; four mice per model). Values are expressed as means ± SE, (n = 4). Cumulative vessel perimeters for each model were 2070 μm for normal pancreatic islets, 2320 μm for RIP-Tag2 tumors, 11315 μm for MCa-IV carcinomas, and 8343 μm for Lewis lung carcinomas. Differences in vessel perimeter among the three tumors reflect documented differences in mean vessel size.7Hashizume 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 (1350) Google Scholar, 10Morikawa 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 (816) Google Scholar Defects in type IV collagen staining, measured in 1-μm optical sections, constituted ≈ 0.05% in normal islet blood vessels and 0.01%, 0.05%, and 0.005% (overall value = 0.03%) of the vessel surface in the three tumors. Open table in a new tab Basement membrane was identified by type IV collagen immunoreactivity and corresponding endothelial cells were visualized by CD31 immunoreactivity in 80-μm-thick sections of normal pancreatic islets and three mouse tumor models. Perimeters of type IV collagen and CD31 staining on vessels cut perpendicular to their longitudinal axis were measured in 1-μm confocal optical sections. Proportion of vessels without basement membrane defects was determined from the percent of profiles entirely surrounded by type IV collagen staining. Percent of basement membrane coverage was calculated as the ratio of the length of type IV co
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