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VEGF-A Induces Angiogenesis by Perturbing the Cathepsin-Cysteine

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VEGF-A Induces Angiogenesis by Perturbing the Cathepsin-Cysteine Published OnlineFirst May 12, 2009; DOI: 10.1158/0008-5472.CAN-08-4539 Published Online First on May 12, 2009 as 10.1158/0008-5472.CAN-08-4539 Research Article VEGF-A Induces Angiogenesis by Perturbing the Cathepsin-Cysteine Protease Inhibitor Balance in Venules, Causing Basement Membrane Degradation and Mother Vessel Formation Sung-Hee Chang,1 Keizo Kanasaki,2 Vasilena Gocheva,4 Galia Blum,5 Jay Harper,3 Marsha A. Moses,3 Shou-Ching Shih,1 Janice A. Nagy,1 Johanna Joyce,4 Matthew Bogyo,5 Raghu Kalluri,2 and Harold F. Dvorak1 Departments of 1Pathology and 2Medicine, and the Center for Vascular Biology Research, Beth Israel Deaconess Medical Center and Harvard Medical School, and 3Departments of Surgery, Children’s Hospital and Harvard Medical School, Boston, Massachusetts; 4Cancer Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, New York; and 5Department of Pathology, Stanford University, Stanford, California Abstract to form in many transplantable mouse tumor models are mother Tumors initiate angiogenesis primarily by secreting vascular vessels (MV), a blood vessel type that is also common in many endothelial growth factor (VEGF-A164). The first new vessels autochthonous human tumors (2, 3, 6–8). MV are greatly enlarged, to form are greatly enlarged, pericyte-poor sinusoids, called thin-walled, hyperpermeable, pericyte-depleted sinusoids that form mother vessels (MV), that originate from preexisting venules. from preexisting venules. The dramatic enlargement of venules We postulated that the venular enlargement necessary to form leading to MV formation would seem to require proteolytic MV would require a selective degradation of their basement degradation of their basement membranes. Vascular basement membranes, rigid structures that resist vascular expansion. To membranes are primarily composed of laminins and type IV collagen (9–11). They are rigid, noncompliant (nonelastic) struc- identify the specific proteases responsible for MV formation, f we induced angiogenesis in mouse tissues with an adenoviral tures and allow only an increase of 30% in cross-sectional area in vector expressing VEGF-A164 (Ad-VEGF-A164) or with VEGF-A– response to increased internal pressure (12); by contrast, MV commonly have cross-sectional areas that are 4 to 5 times those of secreting TA3/St mammary tumors. We found that MV formation resulted from greatly increased activity of cathe- the venules from which they arise (2, 3, 7, 8). psins (B>S>L) in venules transitioning into MV, as well as from The specific proteases responsible for generating MV have not a reciprocal decrease in the expression of several cysteine been identified. Tumors are complex entities in which many protease inhibitors (CPI), stefin A and cystatins B and C, by different proteases participate in a wide range of simultaneous these same venules. Using a fluorescence probe that selectively processes that include tumor, stromal, inflammatory and vascular binds cellular sites of cathepsin protease activity in vivo,we cell proliferation, and migration. Several different classes of showed that increased cathepsin activity was localized proteases have been identified in tumors including matrix metal- exclusively to perivenular cells, not to venule endothelial loproteases (MMP) and serine and cysteine proteases (13–16). Of cells. CPI strikingly inhibited angiogenesis in the Matrigel these, MMPs have received the most attention (15, 17, 18). assay, and Ad-VEGF-A164–induced angiogenesis was reduced However, in recent years, cysteine proteases, and particularly by f50% in cathepsin B–null mice. Thus, VEGF-A, whether cathepsins B, L, Sand H, have been implicated in tumor cell expressed by interstitial cells infected with an adenoviral invasion, metastasis and, more recently, in tumor angiogenesis vector or by tumor cells, upsets the normal cathepsin-CPI (13, 19–26). Cathepsins are members of the papain subfamily of balance in nearby venules, leading to degradation of their cysteine proteases (13); they are found in lysosomes and have basement membranes, an important first step in angiogenesis. traditionally been associated with intracellular functions (27, 28). [Cancer Res 2009;69(10):4537–44] More recent data indicate that cathepsins are secreted, can function extracellularly to degrade matrix proteins, and have significant roles in tumor angiogenesis (13, 20–24, 29). Introduction Endogenous inhibitors of cathepsins, members of the cysteine protease inhibitor (CPI) family, have also been implicated in tumor To grow beyond minimal size, tumors must induce a new progression. CPI are small, 11- to 13-kDa proteins that include vascular supply (1). They do so by overexpressing growth factors, stefin A and cystatins B and C (27). RIP-Tag 2 tumors grow faster in particularly vascular endothelial growth factor/vascular permeabil- cystatin C null than in wild-type mice (30), and changes in CPI have ity factor (VEGF-A) and its 164 (mouse)/165 (human) isoform been reported in several different tumors (31–35). (2–4). However, unlike the angiogenesis of normal development, The goal of the present investigation was to identify the specific the new blood vessels that tumors induce are highly abnormal and proteases and protease inhibitors that participate in MV formation, differ strikingly from the microvessels of normal tissues with as well as the cell types that make them. To avoid the complexities respect to both structure and function (2, 3, 5). The first new vessels of the tumor environment, in which many cell types, proteases, and protease inhibitors participate, we made use of an adenoviral 164 164 Requests for reprints: Harold F. Dvorak, Department of Pathology, Beth Israel vector that expresses VEGF-A (Ad-VEGF-A ); when injected Deaconess Medical Center, 330 Brookline Avenue, RN 227C, Boston, MA 02215. Phone: into mouse tissues, Ad-VEGF-A164 induces an angiogenic response 617-667-0654; Fax: 617-667-2913; E-mail: [email protected]. I2009 American Association for Cancer Research. that closely mimics that induced by malignant tumors (2, 3, 7, 36). doi:10.1158/0008-5472.CAN-08-4539 We report here that increased expression of several cathepsins www.aacrjournals.org 4537 Cancer Res 2009; 69: (10). May 15, 2009 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2009 American Association for Cancer Research. Published OnlineFirst May 12, 2009; DOI: 10.1158/0008-5472.CAN-08-4539 Cancer Research Figure 1. Degradation of vascular basement membranes in MV induced by Ad-VEGF-A164 or by TA3/St tumors. A, confocal microscopy of ear whole mounts from normal control (ctl.) mice and from mice whose ears had been injected 3 d earlier with Ad-VEGF-A164. Immediately before sacrifice, mice were injected i.v. with FITC-lectin (green). Ears were then immunostained with antibodies against laminin or collagen IV and visualized with Texas red–conjugated secondary antibodies. Note extensive loss of red staining (laminin or collagen) in MV compared with control venules. Scale bar, 50 Am. B, immunohistochemistry for laminin and collagen IV in MV of TA3/St tumors harvested 4 d after implantation. Note patchily reduced MV staining (red arrowheads), compared with strong, continuous basement membrane staining of normal venules (insets). Scale bar, 50 Am. C, immunoblot with an antibody against laminin h1 chain performed on extracts of normal ears (time 0) and on ears harvested at 1 to 21 d after Ad-VEGF-A164 injection. Note increasing low molecular weight laminin h1 chain fragments (bracket) at 1 to 5 d. In contrast, on days 7 and 10, there is increased expression of intact laminin h1 chain (arrow), as well as high molecular weight fragments. D, glomeruloid microvascular proliferations in ears 7 d after Ad-VEGF-A164 injection show extensive new laminin deposition (brown stain). Scale bar, 50 Am. (B>S>L), accompanied by a reciprocal decrease in the expression of (R&D Systems), cathepsin B (Neuromics), or CD31 (BD Biosciences). Images their inhibitors, members of the CPI family, is responsible for the were obtained from two to five different ears or tumors at each time point. vascular basement membrane degradation that allows MV to form. For confocal microscopy, mice were injected i.v. with FITC-lectin (Vector Laboratories). After perfusion, ears were removed, split into dorsal and Furthermore, increased cathepsin and reduced CPI expression was ventral halves, exposed to primary antibodies, and incubated with Texas localized selectively to venules that were transitioning into MV. We red–conjugated anti-rabbit immunoglobulin (Invitrogen). Images were substantiated these results with a VEGF-A–expressing tumor. obtained from three or more ears at each time point. For imaging cathepsin Together, these findings implicate venule-associated cathepsins B/S/L activity, 25 nmol of GB123 was injected i.v. 8 h later, FITC-Lectin was and their CPI inhibitors in the earliest stage of tumor angiogenesis. injected i.v., mice were sacrificed, and tissues were visualized in a LSM 510 Subsequently, this degradative process was reversed as MV evolved Meta confocal microscope (39). into daughter vessels, a process accompanied by extensive new Protein extraction and Western blot analysis. Ear sites were rapidly vascular basement membrane synthesis. frozen in liquid nitrogen, ground with a mortar and pestle, and lysed on ice in 1 AL sample buffer/mg ear tissue: 62.5 Amol/L Tris, 250 mmol/L HEPES, 0.5% NP40, 5% 2-mercaptoethanol, 2% SDS, 5% sucrose, and 0.005% Materials and Methods bromophenol blue. Immunoblotting was performed with a specific antibody h Animals, adenoviral vectors, and tumors. Ad-VEGF-A164 and Ad-LacZ against laminin 1 chain (CHEMICON). Experiments were repeated four and their injection into athymic nude mice (National Cancer Institute) were times. described previously (7). A/J mice (Jackson Laboratory) and the TA3/St Isolation of RNA, quantitative reverse transcription-PCR. Total RNA tumor were described previously (37). Cathepsin B–null mice on the FVB was isolated with the Qiagen RNeasy Mini kit and reverse transcribed using TaqMan Reverse Transcription Reagents (Applied Biosystems).
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