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• W2019200520 abstract "One of the hallmarks of intussusceptive angiogenesis is the development of intraluminal connective tissue pillars. The exact mechanism of pillar formation has not yet been elucidated. By using electron and confocal microscopy, we observed intraluminal nascent pillars that contain a collagen bundle covered by endothelial cells (ECs) in the vasculature of experimental tumors. We proposed a new mechanism for the development of these pillars. First, intraluminal endothelial bridges are formed. Second, localized dissolution of the basement membrane occurs and a bridging EC attaches to a collagen bundle in the underlying connective tissue. A pulling force is then exerted by the actin cytoskeleton of the ECs via specific attachment points, which contain vinculin, to the collagen bundle, resulting in suction and subsequent transport of the collagen bundle into and through the vessel lumen. Third, the pillar matures through the immigration of connective tissue cells and the deposition of new collagenous connective tissue. The proposed simple mechanism generates a connection between the processes of endothelial bridging and intussusceptive angiogenesis and identifies the source of the force behind pillar formation. Moreover, it ensures the rapid formation of pillars from pre-existing building blocks and the maintenance of EC polarity. To describe it, we coined the term inverse sprouting. One of the hallmarks of intussusceptive angiogenesis is the development of intraluminal connective tissue pillars. The exact mechanism of pillar formation has not yet been elucidated. By using electron and confocal microscopy, we observed intraluminal nascent pillars that contain a collagen bundle covered by endothelial cells (ECs) in the vasculature of experimental tumors. We proposed a new mechanism for the development of these pillars. First, intraluminal endothelial bridges are formed. Second, localized dissolution of the basement membrane occurs and a bridging EC attaches to a collagen bundle in the underlying connective tissue. A pulling force is then exerted by the actin cytoskeleton of the ECs via specific attachment points, which contain vinculin, to the collagen bundle, resulting in suction and subsequent transport of the collagen bundle into and through the vessel lumen. Third, the pillar matures through the immigration of connective tissue cells and the deposition of new collagenous connective tissue. The proposed simple mechanism generates a connection between the processes of endothelial bridging and intussusceptive angiogenesis and identifies the source of the force behind pillar formation. Moreover, it ensures the rapid formation of pillars from pre-existing building blocks and the maintenance of EC polarity. To describe it, we coined the term inverse sprouting. Angiogenesis is the formation of new blood vessels from pre-existing ones. Several different forms exist,1Dome B. Hendrix M.J. Paku S. Tovari J. Timar J. Alternative vascularization mechanisms in cancer: pathology and therapeutic implications.Am J Pathol. 2007; 170: 1-15Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar but so far endothelial sprouting2Paku S. Paweletz N. First steps of tumor-related angiogenesis.Lab Invest. 1991; 65: 334-346PubMed Google Scholar, 3van Hinsbergh V.W. Koolwijk P. Endothelial sprouting and angiogenesis: matrix metalloproteinases in the lead.Cardiovasc Res. 2008; 78: 203-212Crossref PubMed Scopus (296) Google Scholar and intussusceptive angiogenesis4Makanya A.N. Hlushchuk R. Djonov V.G. Intussusceptive angiogenesis and its role in vascular morphogenesis, patterning, and remodeling.Angiogenesis. 2009; 12: 113-123Crossref PubMed Scopus (159) Google Scholar, 5Djonov V. Baum O. Burri P.H. Vascular remodeling by intussusceptive angiogenesis.Cell Tissue Res. 2003; 314: 107-117Crossref PubMed Scopus (224) Google Scholar, 6Burri P.H. Hlushchuk R. Djonov V. Intussusceptive angiogenesis: its emergence, its characteristics, and its significance.Dev Dyn. 2004; 231: 474-488Crossref PubMed Scopus (268) Google Scholar, 7Patan S. Tanda S. Roberge S. Jones R.C. Jain R.K. Munn L.L. Vascular morphogenesis and remodeling in a human tumor xenograft: blood vessel formation and growth after ovariectomy and tumor implantation.Circ Res. 2001; 89: 732-739Crossref PubMed Scopus (83) Google Scholar, 8Patan S. Munn L.L. Jain R.K. Intussusceptive microvascular growth in a human colon adenocarcinoma xenograft: a novel mechanism of tumor angiogenesis.Microvasc Res. 1996; 51: 260-272Crossref PubMed Scopus (211) Google Scholar have been investigated the most intensively. Endothelial sprouting is characterized by the parallel migration of capillary bud endothelial cells (ECs). During this process, proliferating ECs maintain their basal-luminal polarity and form a slit-like lumen that is continuous with the lumen of the so-called mother vessel. Basement membrane material is deposited continuously by the sprout ECs, whereas only the tip of the growing bud is in contact with the collagenous connective tissue matrix. As the final step, proliferating pericytes of the mother vessel migrate along the basement membrane of the sprout, resulting in the maturation of the new vessel.2Paku S. Paweletz N. First steps of tumor-related angiogenesis.Lab Invest. 1991; 65: 334-346PubMed Google Scholar In contrast to endothelial sprouting, the other major angiogenic mechanism, intussusceptive microvascular growth, or intussusceptive angiogenesis, which has been described in a wide variety of normal and pathological conditions, is faster and does not depend primarily on EC proliferation. The most characteristic feature of intussusceptive angiogenesis is the insertion of connective tissue columns, called tissue pillars, into the lumen and the subsequent growth of these pillars, resulting in partitioning of the vessel lumen and the consequent increase in the density of the given capillary network. According to the current view, the development of tissue pillars is preceded by the formation of vessel wall folds or the protrusions of the opposite points of the vessel wall into the lumen.4Makanya A.N. Hlushchuk R. Djonov V.G. Intussusceptive angiogenesis and its role in vascular morphogenesis, patterning, and remodeling.Angiogenesis. 2009; 12: 113-123Crossref PubMed Scopus (159) Google Scholar, 5Djonov V. Baum O. Burri P.H. Vascular remodeling by intussusceptive angiogenesis.Cell Tissue Res. 2003; 314: 107-117Crossref PubMed Scopus (224) Google Scholar, 6Burri P.H. Hlushchuk R. Djonov V. Intussusceptive angiogenesis: its emergence, its characteristics, and its significance.Dev Dyn. 2004; 231: 474-488Crossref PubMed Scopus (268) Google Scholar, 7Patan S. Tanda S. Roberge S. Jones R.C. Jain R.K. Munn L.L. Vascular morphogenesis and remodeling in a human tumor xenograft: blood vessel formation and growth after ovariectomy and tumor implantation.Circ Res. 2001; 89: 732-739Crossref PubMed Scopus (83) Google Scholar, 8Patan S. Munn L.L. Jain R.K. Intussusceptive microvascular growth in a human colon adenocarcinoma xenograft: a novel mechanism of tumor angiogenesis.Microvasc Res. 1996; 51: 260-272Crossref PubMed Scopus (211) Google Scholar However, the origin of the force generating these invaginations has not yet been clarified. Although it is believed that perivascular cells or pericytes may play a role in this initial step by exerting a pushing force on the vessel wall, this concept is questionable because the structure of the cellular cytoskeleton allows only pulling forces at high strength, whereas pushing forces are several hundredfold lower in magnitude.9Cojoc D. Difato F. Ferrari E. Shahapure R.B. Laishram J. Righi M. Di Fabrizio E.M. Torre V. Properties of the force exerted by filopodia and lamellipodia and the involvement of cytoskeletal components.PLoS One. 2007; 2: e1072Crossref PubMed Scopus (109) Google Scholar, 10Burton K. Park J.H. Taylor D.L. Keratocytes generate traction forces in two phases.Mol Biol Cell. 1999; 10: 3745-3769Crossref PubMed Scopus (158) Google Scholar Another phenomenon thought to be different from intussusceptive angiogenesis, but also leading to vascular division, was described as well. This process is characterized by the development of intraluminal bridges formed by ECs, followed by the development of connective tissue by an unknown mechanism within these bridges.11Nagy J.A. Chang S.H. Dvorak A.M. Dvorak H.F. Why are tumour blood vessels abnormal and why is it important to know?.Br J Cancer. 2009; 100: 865-869Crossref PubMed Scopus (372) Google Scholar, 12Dvorak H.F. Rous-Whipple Award Lecture: how tumors make bad blood vessels and stroma.Am J Pathol. 2003; 162: 1747-1757Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 13Zhou A. Egginton S. Hudlická O. Brown M.D. Internal division of capillaries in rat skeletal muscle in response to chronic vasodilator treatment with alpha1-antagonist prazosin.Cell Tissue Res. 1998; 293: 293-303Crossref PubMed Scopus (100) Google Scholar Based on our observation of the vascularization of s.c. growing tumors in mice, we present herein the detailed mechanism of intraluminal pillar formation, which offers a rationale for the puzzles previously discussed and incorporates the previous two concepts. The C38 mouse colorectal carcinoma line was maintained by serial s.c. transplantations in C57Bl/6 mice, as previously described.14Paku S. Kopper L. Nagy P. Development of the vasculature in “pushing-type” liver metastases of an experimental colorectal cancer.Int J Cancer. 2005; 115: 893-902Crossref PubMed Scopus (35) Google Scholar, 15Dezso K. Bugyik E. Papp V. László V. Döme B. Tóvári J. Tímár J. Nagy P. Paku S. Development of arterial blood supply in experimental liver metastases.Am J Pathol. 2009; 175: 835-843Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar Tumor tissue was cut into cubes measuring 5 × 5 × 5 mm. Animals were anesthetized with ketamine, 80 mg/kg, and xylazine, 12 mg/kg (Sigma Chemical Co, St Louis, MO); one piece of tumor tissue was transplanted into the back of each mouse. Animals were sacrificed 3 weeks after tumor inoculation. For analysis of immunofluorescence labeling with monoclonal antibodies, the tumors were transplanted into mice with severe combined immunodeficiency to reduce non-specific staining. HT25 human colon carcinoma cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Sigma Chemical Co). The s.c. tumors were produced by injecting 2 × 106 tumor cells into the back of anesthetized male mice with severe combined immunodeficiency, as previously described.16Tóvári J. Gilly R. Rásó E. Paku S. Bereczky B. Varga N. Vágó A. Tímár J. Recombinant human erythropoietin alpha targets intratumoral blood vessels, improving chemotherapy in human xenograft models.Cancer Res. 2005; 65: 7186-7193Crossref PubMed Scopus (49) Google Scholar Animals were sacrificed 4 weeks after tumor inoculation. Preparation of tumor samples for electron microscopy was performed as previously described.14Paku S. Kopper L. Nagy P. Development of the vasculature in “pushing-type” liver metastases of an experimental colorectal cancer.Int J Cancer. 2005; 115: 893-902Crossref PubMed Scopus (35) Google Scholar In brief, the anesthetized animals (three mice for each tumor line) were perfused via the left ventricle with PBS for 10 minutes and with 4% paraformaldehyde and 1% glutaraldehyde in PBS (pH 7.2) for 15 minutes at room temperature. The s.c. tumors were removed, cut into 1 × 2-mm pieces, and immersed in the same fixative for an additional 2 hours. The pieces were post-fixed in 1% OsO4, 0.5% K-ferrocyanide in PBS for 2 hours, dehydrated in a graded series of acetone, and embedded in Spurr's mixture. A total of 8 to 10 serial semithin sections were cut, stained by 0.5% toluidine blue (pH 8.5), and analyzed for the presence of pillars. The structures identified on the last semithin section were followed backward to ensure that they represented pillars and were not simply vessel bifurcations or other structures. Areas of interest were trimmed out by comparing the structures on the cut surface of the tissue blocks with the semithin sections and then serially sectioned by an RMC MT-7 ultramicrotome (Research and Manufacturing Co, Tucson, AZ). The sections were placed on thin bar grids, stained with 2% uranyl acetate and lead citrate, and analyzed using a Philips CM10 electron microscope (Eindhoven, The Netherlands). Pillars cut lengthwise were also examined during analysis of serial ultrathin sections. In this case, the entire thickness of the pillar was available for analysis at the ultrastructural level. Serial semithin sections were captured by an Olympus DP50 camera (Olympus, Tokyo, Japan). Digitized images were transferred to the Biovis3D software program (BioVis3D, Montevideo, Uruguay). Three-dimensional (3D) reconstructions were performed using color contouring to highlight the recreated structures. Frozen sections (15-μm thick) were fixed in methanol and were incubated at room temperature (for 1 hour) with a mixture of the following primary antibodies: monoclonal anti-mouse CD31 (dilution, 1:100; catalogue no. 01951D; Pharmingen, San Diego, CA), polyclonal anti-collagen I (dilution, 1:100; catalogue no. AB765P; Chemicon, Billerica, MA), monoclonal anti-vinculin (dilution, 1:100; catalogue no. V4505; Sigma Chemical Co), monoclonal anti-integrin α-1 (dilution, 1:20; catalogue no. 555001; BD Pharmingen, San Jose, CA), polyclonal anti-integrin α-1 (dilution, 1:50; catalogue no. sc-10728; Santa-Cruz Biotechnology, Santa-Cruz, CA), monoclonal anti-integrin α-2 (dilution, 1:100; catalogue no. 108901; Biolegend, San Diego), polyclonal anti-integrin α-2 (dilution, 1:200; catalogue no. AB1936; Millipore, Billerica, MA), polyclonal anti-integrin α-11 (dilution, 1:100; catalogue no. sc-98740; Santa-Cruz Biotechnology), and monoclonal anti-mouse CD29 (dilution, 1:100; catalogue no. 550531; Pharmingen). After washing, appropriate secondary antibodies conjugated with fluorescein isothiocyanate, tetra rhodamine isothiocyanate, or Cy5 were used (all from Jackson Immunoresearch Inc., Suffolk, UK). The vinculin and integrin α-2 signals were amplified by using an appropriate biotinylated secondary antibody (dilution, 1:200; Vector Laboratories, Burlingame, CA), followed by streptavidin fluorescein isothiocyanate (Jackson Immunoresearch Inc.). To analyze the localization of actin filaments within the pillars, the sections were reacted with phalloidin–tetra rhodamine isothiocyanate (dilution, 1:500; catalogue no. P1951; Sigma Chemical Co). Sections were scanned by eye for the presence of pillars using a ×100 objective. Only pillars running parallel and lying completely within the sectioning plane were analyzed by a Bio-Rad MRC 1024 confocal microscope (Bio-Rad, Richmond, CA). For 3D reconstructions, 30 to 40 optical sections were generated. The size of the collagen bundles was determined at the ultrastructural level in the peritumoral connective tissue and within the pillars. Measurements were made on digitalized electron micrographs (original magnification, ×1500 to ×7000) taken from s.c. tumors of both cancer cell lines using Olympus Quick Photo Micro software (Olympus). In the peritumoral connective tissue, collagen bundles tightly packed with collagen fibers were chosen randomly (>250). In cross sections, the smallest diameter of the bundle was measured. After their identification in semithin sections, >50 pillars were analyzed at the ultrastructural level. Only pillars exclusively containing collagen fibers, but no pillars with connective tissue cells, were chosen. The total thickness of the pillars (including ECs) was also measured. To study the effects of angiogenesis-modulating agents on tumor vascularization and pillar formation, groups of six mice bearing C38 tumors received recombinant human erythropoietin α (rHuEPO, epoetinum α; Jannsen-Cilag, Shaffhausen, Switzerland),16Tóvári J. Gilly R. Rásó E. Paku S. Bereczky B. Varga N. Vágó A. Tímár J. Recombinant human erythropoietin alpha targets intratumoral blood vessels, improving chemotherapy in human xenograft models.Cancer Res. 2005; 65: 7186-7193Crossref PubMed Scopus (49) Google Scholar PTK787/ZK22854 (vatalanib; Novartis/Schering AG, Berlin, Germany)17Wood J.M. Bold G. Buchdunger E. Cozens R. Ferrari S. Frei J. Hofmann F. Mestan J. Mett H. O'Reilly T. Persohn E. Rösel J. Schnell C. Stover D. Theuer A. Towbin H. Wenger F. Woods-Cook K. Menrad A. Siemeister G. Schirner M. Thierauch K.H. Schneider M.R. Drevs J. Martiny-Baron G. Totzke F. PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration.Cancer Res. 2000; 60: 2178-2189PubMed Google Scholar (obtained from Selleck Chemicals LLC, Houston, TX), or the vehicle as a control. In mice treated with vatalanib, tumors were allowed to grow for 12 days before treatment. Then, mice were treated orally with 100 mg/kg vatalanib (PTK787/ZK222584, dissolved in water containing 5% dimethyl sulfoxide and 1% Tween-80) for 4 days, as in a previous study.18Hlushchuk R. Riesterer O. Baum O. Wood J. Gruber G. Pruschy M. Djonov V. Tumor recovery by angiogenic switch from sprouting to intussusceptive angiogenesis after treatment with PTK787/ZK222584 or ionizing radiation.Am J Pathol. 2008; 173: 1173-1185Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar Mice treated with rHuEPO were given 150 IU/kg in physiological salt solution three times per week i.p. from day 1 after tumor inoculation, as previously described.16Tóvári J. Gilly R. Rásó E. Paku S. Bereczky B. Varga N. Vágó A. Tímár J. Recombinant human erythropoietin alpha targets intratumoral blood vessels, improving chemotherapy in human xenograft models.Cancer Res. 2005; 65: 7186-7193Crossref PubMed Scopus (49) Google Scholar The mice in all groups were sacrificed on day 17, and tumors were removed, weighed, and frozen. CD31-labeled frozen sections were scanned by eye using a ×100 objective to determine the number of pillars within the entire section. The total area of the sections was determined using Olympus Quick Photo Micro software. To determine the area fraction of CD31-positive blood vessels in tumor sections, two to three confocal images were taken from each tumor section using a ×4 objective (area, 11.3 mm2). The micrographs were analyzed using ImageJ software (NIH, Bethesda, MD). Results are expressed as the number of pillars per squared millimeter of tumor tissue or microvessel area. Animals were anesthetized and shaved. A 1-cm-long full-thickness incision was made in the dorsal skin of C57 black mice. The wounds were partially closed by a single nylon suture. The mice were euthanized on days 3, 5, 7, and 10 after wounding. Two mice were sacrificed at each time point. The wounds and the surrounding intact skin, measuring 2 × 2 mm, were removed and cut further into 1 × 2-mm pieces, with the long axis running perpendicular to the wound. These pieces were fixed and embedded for electron microscopy, as previously described. Eighty-six tissue blocks were semithin sectioned and analyzed (total area, approximately 250 mm2) for the presence of pillars using a ×63 objective. Statistical analysis was performed using the Student's t-test. Intussusceptive angiogenesis was observed in s.c. tumors of both cancer cell lines. This type of angiogenesis was the main means of new vessel formation. Endothelial sprouting with characteristic slit-like lumen-containing capillaries2Paku S. Paweletz N. First steps of tumor-related angiogenesis.Lab Invest. 1991; 65: 334-346PubMed Google Scholar was scarcely detected. Intussusception was mainly detected in angiogenic hot spots peritumorally, but it also occurred within the tumor mass. The first step of intussusception is thought to be the development of protrusions or infoldings of the vessel wall within the lumen.6Burri P.H. Hlushchuk R. Djonov V. Intussusceptive angiogenesis: its emergence, its characteristics, and its significance.Dev Dyn. 2004; 231: 474-488Crossref PubMed Scopus (268) Google Scholar We analyzed 89 infolds sharply intruding into the vessel lumens in >172 serially sectioned areas (semithin sections) altogether. None of these structures projected into the lumen by themselves. By tracing them over several serial sections, we found that each capillary infold was connected to a different part of the vessel lumen (on the opposite or the same side). These infolds proved to be pillars, part of blind-ending lumens or simple vessel ramifications (Figure 1; see also Supplemental Figure S1 at http://ajp.amjpathol.org). In areas of intensive intussusception, proliferating ECs (Figure 1, H and L; see also Supplemental Figure S1H at http://ajp.amjpathol.org) and intraluminal endothelial bridges were frequently observed (Figure 1, H–M and K–O; and Figure 2, A–E). These bridges either were simple EC processes projecting into the vessel lumen and attaching to the endothelial tube in a different position (Figure 2F) or were formed by the participation of cellular processes of different ECs (Figure 2G). However, the most characteristic phenomenon of this type of tumor-induced intussusceptive angiogenesis was the development of transluminal pillars containing tightly packed collagen fibers covered by ECs (Figure 2H). The pillars either spanned the vessel lumen or originated and terminated on the same side of the vessel (Figure 1, Figure 3). The diameters of these collagen bundles did not differ significantly from those within the peritumoral connective tissue (Table 1). The overall diameter (including the EC layer) of the pillars corresponded well with those observed earlier in other studies (2.5 μm).4Makanya A.N. Hlushchuk R. Djonov V.G. Intussusceptive angiogenesis and its role in vascular morphogenesis, patterning, and remodeling.Angiogenesis. 2009; 12: 113-123Crossref PubMed Scopus (159) Google Scholar The fibers were oriented parallel to the axis of the pillars (Figure 3, B–D) and were covered by several ECs. However, the basement membrane under these cells was generally absent (Figures 2H and 3G). Moreover, neither pericytes nor other cells were present in these small nascent pillars. Along the pillars, cut parallel to their axis, high electron density areas could be observed in the membrane of the ECs, suggesting specific adhesion between the ECs and the collagen bundle. In accordance with this observation, immunofluorescence analysis revealed vinculin-containing adhesion spots along the pillars (Figure 4, A–D). However, although immunolabeling with antibodies against integrin α-1, α-2, or α-11 demonstrated high α-2 and α-11 expression levels in the pericapillary connective tissue, these collagen-binding integrin subunits were either occasionally present as small dots at a low density at the abluminal surface of pillar-forming ECs (as in the case of α-2 labeling; see Supplemental Figure S2, A and B, at http://ajp.amjpathol.org) or totally absent (as in the case of α-1 or α-11 labeling; see Supplemental Figure S2, E and F, at http://ajp.amjpathol.org). Nevertheless, in more developed pillars, we could detect large integrin α-2–containing adhesion spots (see Supplemental Figure S2, C and D, at http://ajp.amjpathol.org). Staining for integrin β-1 showed no specific localization of this subunit that was distributed evenly under the ECs of the vessel and pillars (data not shown).Figure 23D and ultrastructure of endothelial bridges and transcapillary pillars. A–D: Serial semithin sections of an endothelial bridge. The bridge (arrows) is present only in two consecutive sections. The light blue staining (collagen core) within the bridge is absent. Scale bar = 20 μm. E: 3D reconstruction of 13 semithin sections. The endothelial bridge and the nuclear area of the ECs are highlighted in yellow, and the vessel wall and the surrounding tissue are shown in red. F: An endothelial bridge formed by a single cellular process (arrow). The process is attached to the EC itself (arrowhead). Scale bar = 1 μm. G: Endothelial bridges formed by cellular processes of several ECs. Arrows point at intercellular junctions. The collagen bundle is located close to the vessel (arrowhead). Scale bar = 5 μm. H: Cross section of a transluminal pillar. The pillar is formed by a collagen bundle tightly packed with fibers and by two covering ECs. Arrows point at interendothelial junctions. Some basement membrane material is visible below the ECs at the upper part of the pillar (arrowhead). Scale bar = 1 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Ultrastructure of the pillar. A–E: Serial ultrathin sections (approximately 100 nm) of a nascent pillar (A, section 3; B, section 14; C, section 20; D, section 22; E, section 36). The pillar was present in 22 sections. The collagen bundle is densely packed with fibers and oriented parallel to the axis of the pillar (B–D). A–E: The collagen bundle does not extend into the connective tissue (up). E: In contrast, the collagen bundle is still visible (arrow) and runs with a sharp change in direction along the circumference of the vessel (down). This suggests that the putative direction of the collagen bundle transport is from right to left. Apparently, the bundle just reached the left side of the vessel. In A, an arrow points at a small process of the EC, indicating the location of the pillar. The body parts of the ECs in contact with the collagen bundle show homogeneous staining because of the high microfilament content (arrowheads in B). Scale bar = 2 μm. F–H: High-power micrographs showing details of the pillar region (F, section 1; G, section 14; G inset, section 10; H, section 36). F represents the first section that suggests the presence of a pillar. The small cellular process (arrow) is densely packed within a meshwork of microfilaments. Microfilaments are also present under the plasma membrane in the left part of the cell. The large arrowhead points at an area containing intermediate filaments, whereas small arrowheads point at microtubules. Scale bar = 0.5 μm (F). G: Details of B. The cell body above the collagen bundle contains many microfilaments but no other organelles. The high electron density of the plasma membrane is cut at a low angle. Adhesion spots are also visible (small arrowheads). A basement membrane is lacking in the area where the EC faces the collagen bundle (small arrows). However, it is present in other areas of the vessel wall (large arrow). Inset: An adhesion area where the plasma membrane is cut at a low angle. The adhesion spots are situated exactly and regularly above the collagen fibers (small arrowheads). Scale bar = 0.5 μm (G and inset). H: The collagen bundle (cut perpendicularly) of the pillar extends outside into the connective tissue and runs around the circumference of the vessel. Although adhesion spots are not visible, a basement membrane is not present at the area where the EC faces the collage bundle. Many microfilaments are present in this body part (arrowheads) of the EC. An intact basement membrane is present under the other parts of the EC (arrows). Scale bar = 1 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1Collagen Bundle Diameters in the Peritumoral Connective Tissue and in Transcapillary PillarsValueDiameter (μm)Collagen bundles within the peritumoral connective tissue (n = 260)Collagen bundles within the pillars (n = 55)Pillars (including endothelium) (n = 55)Mean ± SD1.61 ± 0.91.72 ± 1.0⁎The diameters of the collagen bundles of the connective tissue and the pillars did not differ significantly (P = 0.4).2.5 ± 1.1Range0.3–5.90.4–5.00.8–5.8 The diameters of the collagen bundles of the connective tissue and the pillars did not differ significantly (P = 0.4). Open table in a new tab Figure 4Immunofluorescent labeling of pillars. A–D: Vessel with pillar (arrow) stained for CD31 (A, red), vinculin (B, green), and collagen I (C, blue). The pictures show horizontal views of 35 optical sections. CD31 and vinculin are present mainly on the two sides of the pillar, whereas collagen is positioned in the middle. D shows the merged picture. Inset: One optical section showing that vinculin is present in small spots along the periphery of the pillar (green). There is a high density of adhesions in the connective tissue. Black areas (except for the vessel lumen) correspond to tumor tissue. Scale bar = 20 μm (A–D). E and F: Vessel with pillar (arrow) stained for CD31 (green) and phalloidin (red). Phalloidin staining, representing filamentous actin, colocalizes with CD31 staining at the sides of the pillar. Myofibroblasts outside of the vessel are also stained by phalloidin–tetra rhodamine isothiocyanate. Scale bar = 20 μm (E and F).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The part of the cell body of the ECs that formed the pillars frequently contained a high density of microfilaments, excluding all other cellular organelles (Figure 3, G and H). These microfilaments were generally not in a parallel arrangement; rather, they formed a mesh. The presence of polymerized actin within the ECs of the pillars was also confirmed by phalloidin staining (" @default.
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• W2019200520 title "A New Mechanism for Pillar Formation during Tumor-Induced Intussusceptive Angiogenesis: Inverse Sprouting" @default.
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