Impaired Angiogenesis, Delayed Wound Healing

[CANCER RESEARCH 64, 4699 – 4702, July 15, 2004]

Advances in Brief

Impaired Angiogenesis, Delayed Wound Healing and Retarded Tumor Growth in Perlecan Heparan Sulfate-Deficient Mice Zhongjun Zhou,1,2 Jianming Wang,2,4 Renhai Cao,3 Hiroyuki Morita,2 Raija Soininen,2,5 Kui Ming Chan,1 Baohua Liu,1 Yihai Cao,3 and Karl Tryggvason2 1

Department of Biochemistry, Faculty of Medicine, University of Hong Kong, Hong Kong; 2Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, and Microbiology and Tumorbiology Center, Karolinska Institute, Stockholm, Sweden; 4Institute of Molecular Medicine, Department of Medicine, University of California at San Diego, La Jolla, California; and 5Department of Medical Biochemistry and Molecular Biology, University of Oulu, Oulu, Finland 3

Abstract Perlecan, a modular proteoglycan carrying primary heparan sulfate (HS) side chains, is a major component of blood vessel basement membranes. It sequesters growth factors such as fibroblast growth factor 2 (FGF-2) and regulates the ligand-receptor interactions on the cell surface, and thus it has been implicated in the control of angiogenesis. Both stimulatory and inhibitory effects of perlecan on FGF-2 signaling have been reported. To understand the in vivo function of HS carried by perlecan, the perlecan gene heparan sulfate proteoglycan 2 (Hspg2) was mutated in mouse by gene targeting. The HS at the NH2 terminus of perlecan was removed while the core protein remained intact. Perlecan HS-deficient (Hspg2⌬3/⌬3) mice survived embryonic development and were apparently healthy as adults. However, mutant mice exhibited significantly delayed wound healing, retarded FGF-2-induced tumor growth, and defective angiogenesis. In the mouse corneal angiogenesis model, FGF-2-induced neovascularization was significantly impaired in Hspg2⌬3/⌬3 mutant mice. Our results suggest that HS in perlecan positively regulates the angiogenesis in vivo.

Introduction Basic fibroblast growth factor (FGF-2) is a member of a large family of growth factors that regulate the growth and differentiation of a number of cell types. FGF-2 is a key regulator of angiogenesis and is involved in a number of pathophysiological processes such as wound repair and tumor growth (1). The binding of FGF-2 to its receptors requires the participation of heparan sulfate (HS) or heparin (1). HS proteoglycans (HSPGs) are a group of modular proteins that carry HS side chains. According to their relationship with cells, HSPGs are divided into two major groups, membrane-associated HSPGs (syndecans 1– 4 and glypican 1– 6) and extracellular HSPGs (perlecan and agrin; Ref. 2). Studies of perlecan expression in mice revealed its early expression in the heart primordium and large blood vessels and subsequently in a number of mesenchymal tissues (3). The fact that expression of perlecan is always prominent in the endothelial cell basement membranes of all vascularized organs (4) indicates its crucial role in vasculogenesis and angiogenesis. Perlecan is not only a structural protein, but is also able to bind a number of growth factors and modulate their activities (4, 5). The expression pattern of perlecan parallels the distribution of FGF-2 in various basement membranes, suggesting its involvement in FGF-2 signaling (4). Although many

studies demonstrated that the perlecan is a key regulator in ligandreceptor binding, the in vivo role for HS in perlecan molecules during angiogenesis has not yet been studied. The mouse Hspg2 gene encodes the large perlecan core protein. Hspg2 null mice were embryonic lethal, due to the disruption of basement membrane structure (5). To study the potential in vivo role of perlecan HS side chains, a 45-bp in-frame deletion was made to the Hspg2 gene to remove all three HS chain attachment sites from the domain I, resulting in the HS chains deficiency in a specific HSPGperlecan HS deficiency. Hspg2⌬3/⌬3 mice survived embryonic development. Adult mice appear healthy and fertile and are grossly indistinguishable from their littermate controls. However, a careful examination showed that Hspg2⌬3/⌬3 mice had defects in lens development (6). The Hspg2⌬3/⌬3 mice provided the unique model to study the in vivo roles for specific HSPG HS chains in FGF-2 signaling and angiogenesis. The purpose of this study was to address the following questions: (a) Is angiogenesis in physiological and pathological conditions affected in Hspg2⌬3/⌬3 mice? (b) What is the role for perlecan HS in FGF-2-induced angiogenesis? Here, we show that deficiency in perlecan HS does not affect the structure or composition of the blood vessel basement membranes. We found that perlecan HS promoted angiogenesis in vivo, because the removal of perlecan HS side chains led to impaired FGF-2-mediated angiogenesis. Materials and Methods

Animals. The generation and characterization of Hspg2⌬3/⌬3 mice has been described elsewhere (6). In brief, exon 3 of mouse perlecan gene, Hspg2, which is 45 bp in size, was removed in-frame. After homologous recombination in embryonic stem cells, the mutated allele was transmitted through the germ line. Heterozygous mutant mice were back-crossed to C57BL/6 mice for seven generations. Homozygous mutants (Hspg2⌬3/⌬3) were obtained through mating between heterozygous animals. All animal experiments have been approved by the Stockholm Animal Ethics Committee. Skin Wound-Healing Assay. The skin wound-healing assay was performed essentially as described previously (7). Two 8-mm full-thickness punch wounds were created on the dorsal surface of each of the 6-week-old mice with a dermal biopsy punch (Miltex GmbH, Ludwigshafen, Germany). Twenty-four h after the surgery, the wounds were recorded as initial condition. The wounds were then recorded daily on transparent parafilm for 9 days. All recordings were scanned, and the wound areas were calculated with Scion Image software. Tumor Experiments. K1000, the NIH3T3 cells stably transfected with FGF-2 cDNA with a signal peptide (8), were maintained in DMEM supplied Received 3/5/04; revised 5/4/04; accepted 5/28/04. Grant support: University of Hong Kong Basic Research Seeds Fund, Swedish with 10% fetal bovine serum and 100 IU/ml penicillin-streptomycin (Life Cancer Foundation, Novo-Nordisk Foundation, the Swedish Medical Council, and EU Technologies, Inc.). Before inoculation, cells were trypsinized, washed, and Grant QLGI-CT-01131. J. Wang was a recipient of a postdoctoral fellowship from suspended in PBS. Cells of 5 ⫻ 106 were inoculated s.c. on the backs of Karolinska Institute. newborn mice. Tumors were dissected and weighed at designated days after The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with the inoculation. 18 U.S.C. Section 1734 solely to indicate this fact. Cornea Micropocket Assay. The corneal micropocket assay was perRequests for reprints: Zhongjun Zhou, Department of Biochemistry, Faculty of formed as described previously (9). In brief, a corneal micropocket was created Medicine, University of Hong Kong, 21 Sassoon Road, Hong Kong. Phone: 852on one eye of each of the 6-week-old Hspg2⌬3/⌬3 mice and controls. A 28199542; Fax: 852-28551254; E-mail: [email protected]. 4699

PERLECAN AND ANGIOGENESIS

micropellet containing 80 ng of human recombinant FGF-2 (Invitrogen) was implanted into each pocket. Corneal neovascularization was examined 5 days after the operation. Immunohistochemistry. Immunohistochemical staining was performed on cryostat sections. Fresh tumor samples and skin wound samples were collected and snap-frozen in OCT compound (Sakura, Finetek, the Netherlands). Cryostat sections (10 ␮m thick) were fixed in 1% paraformaldehyde and stained with biotin antimouse CD31 (PharMingen) or rat monoclonal antibodies against perlecan (NeoMarkers), nidogen (NeoMarkers), laminin ␣4 [a kind gift from Rupert Timpl (Max-Planck Institute for Biochemistry, Martinsried, Germany)], and collagen IV ␣1 chain. Measurement of Blood Vessel Density. To measure the blood vessel density, tumors of comparable size from control and Hspg2⌬3/⌬3 mice were used. Tumors from control mice were dissected 7 days after the inoculation, and tumors from Hspg2⌬3/⌬3 mice were dissected 10 days after the inoculation. Cryostat sections (10 ␮m thick) were immunostained with CD31 antibody. Five microscopic fields were randomly selected from each tumor section and recorded with a CCD camera. In wound-healing experiments, skin wound samples were harvested at day 5, and the sections were counterstained with hematoxyline after CD31 immunostaining. Two or three microscopic fields of granulation tissue were chosen from each section. The number of blood vessels was counted in sections of three animals from each group, and the density was calculated using Scion Image software. Cell Surface Binding of 125I-FGF-2. Binding of 125I-FGF-2 to murine embryonic fibroblasts (MEF) cells was determined as described previously (10), with the following modifications. MEF cells were seeded at a density of 2 ⫻ 105/well in 48-well plates and cultured until confluence in full medium. After washing with 1⫻ PBS, cells were then incubated for an additional 24 h in serum-free DMEM for conditioned medium collection. A binding solution was prepared by mixing DMEM medium with various concentrations of 125 I-FGF-2 and cooling to 4°C for 10 min. Confluent MEF cells were washed three times with ice-cold DMEM. The binding solution was added to the cells and incubated at 4°C for 2.5 h. The binding medium was then removed, and the cells were gently washed three times with ice-cold DMEM. The FGF-2 bound to cell surface was extracted with ice-cold 2 M NaCl/20 mM sodium acetate (pH 4.0) for 15 min, and the salt extracts were counted in a ␥ counter. Nonspecific binding was considered as the value obtained in the presence of a 100-fold excess of nonradioactive FGF-2. The value was then normalized by the cell number.

Statistics. Data were analyzed with the two-tailed Student’s t test.

Results Delayed Wound Healing in Hspg2⌬3/⌬3 Mice. Angiogenesis is an essential step in the wound-healing process. New blood vessels are required for the supply of oxygen and nutrients, for wound debris removal, and for granulation formation. FGF-2-mediated angiogenesis plays critical role in wound healing (1). To investigate the role of perlecan HS in physiological angiogenesis, we examined wound repair in mice. Six-week-old Hspg2⌬3/⌬3 mice and their sex- and agematched wild-type control mice from the same background were used in this study. Two full skin thickness punch wounds were created on the backs of these mice (Fig. 1A). Twenty-four h later, the wound area was first recorded and regarded as the initial wound. The wound area was then recorded daily, and the healing process was quantified as the percentage of the initial wound area. The wound-healing process was significantly delayed in mutant mice (Fig. 1, A and B), with significant differences found between days 3 and 9 (Fig. 1B). From day 3 after surgery, the formation of granulation at the edge of the wound bed was observed both in wild-type and in Hspg2⌬3/⌬3 mice. However, the extent of development of granulation tissue was less in Hspg2⌬3/⌬3 mice and appeared to be poorly vascularized (Fig. 1C). To quantify the vascularity, wound tissue was sampled at day 5 after the surgery, and sections were immunostained with the endothelial marker CD31. The number of blood vessels was counted. We found that blood vessel density in granulation tissue of Hspg2⌬3/⌬3 mice was significantly lower (182 ⫾ 21/mm2, mean ⫾ SE) in comparison with that in wild-type controls (287 ⫾ 25/mm2, mean ⫾ SE, P ⬍ 0.001; Fig. 1D). No obvious difference in the gross vascular structure was observed between mutants and controls. Impaired FGF-2-Induced Tumor Angiogenesis and Tumor Growth in Hspg2⌬3/⌬3 Mice. Tumor growth is also angiogenesis dependent. We therefore studied tumor growth and tumor angiogenesis in Hspg2⌬3/⌬3 mice to further address the role of perlecan HS in angiogenesis. Basic FGF-transformed cells K1000 were used in this

Fig. 1. Delayed wound healing, reduced granulation tissue formation, and vascular density in Hspg2⌬3/⌬3 mice. Two punch wounds were created on the back of each Hspg2⌬3/⌬3 and control mouse. A, skin wounds in Hspg2⌬3/⌬3 and sex- and age-matched control mice 4 days after surgery, showing the difference in wound sizes. B, quantification of the wound-healing process. Wound areas were measure from days 2 to 9 in seven Hspg2⌬3/⌬3 and nine control mice (mean ⫾ SD). The wound size at 24 h after surgery was set as initial wound (100%). ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.01; ⴱⴱⴱ, P ⬍ 0.001. C, CD31 immunostaining, showing granulation tissue formation in the wound bed 5 days after surgery. D, quantification of blood vessel density in granulation tissue. The number of blood vessels was counted in three Hspg2⌬3/⌬3 and three control mice (mean ⫾ SE); ⴱⴱ, P ⬍ 0.01.

4700


Fig. 2. Tumor growth in newborn Hspg2⌬3/⌬3 and control mice. A, tumor weight from 21 Hspg2⌬3/⌬3 and 37 littermate control mice (mean ⫾ SE); ⴱⴱ, P ⬍ 0.01. B, quantification of vascular density in K1000 tumors from three Hspg2⌬3/⌬3 and three littermate controls (mean ⫾ SE); ⴱ, P ⬍ 0.05. CD31 immunostaining of K1000 tumor sections showing the vascular density in tumors of similar size from a Hspg2⌬3/⌬3 mouse at day 10 (C) and a littermate control at day 7 (D). Immunostaining of tumor sections with antibodies against perlecan (E and F), laminin ␣4 (G and H), and collagen IV ␣1 chain (I and J). Perlecan is prominent in endothelial basement membranes. Vascular morphology and the intensity of staining were comparable between Hspg2⌬3/⌬3 mice and controls. A reduction in vascular density was found in Hspg2⌬3/⌬3 mice.

experiment. K1000 cells of 5 ⫻ 106 were inoculated s.c. on the backs of newborn Hspg2⌬3/⌬3 and control mice. Ten days after inoculation, tumors were dissected and weighed. Eight litters of newborn mice, amounting to 21 mutant and 37 control individuals, were used in this study. The tumor mass from Hspg2⌬3/⌬3 mice weighed 0.47 ⫾ 0.10 g (mean ⫾ SE) and was significantly less than that of their littermate controls (0.79 ⫾ 0.17 g, mean ⫾ SE, P ⬍ 0.01; Fig. 2A). To elucidate the retarded tumor growth in Hspg2⌬3/⌬3 mice, we evaluated blood vessel formation in tumors. Tumors from control mice were sampled at day 7, and tumors from mutant mice were sampled at day 10, so the tumors from controls and mutants were comparable in size. Tumor sections were stained with CD31 (Fig. 2, C and D), and the numbers of blood vessels were counted in three mutants and three controls, using Scion Image software. Tumors from mutant mice showed a

significantly reduced blood vessel density of 98 ⫾ 14/mm2, compared with 142 ⫾ 30/mm2 in controls (mean ⫾ SE, P ⬍ 0.05; Fig. 2, B–D). Perlecan HS Depletion Did Not Alter the Integrity of the Blood Vessel Basement Membranes. The integrity of the blood vessel basement membranes may also affect angiogenesis (11). We therefore carefully examined the morphology of blood vessels in tumor tissues and in granulation tissues. No blood vessel deformities, i.e., dilation, constriction, or signs of blood cell leakage, were observed in tumors from Hspg2⌬3/⌬3 mice. To examine whether the loss of perlecan HS altered the composition of major basement membrane components in blood vessels, we immunostained tumor sections with antibodies against perlecan (Fig. 2, E and F), laminin ␣4 (Fig. 2, G and H), collagen IV (Fig. 2, I and J), and nidogen (data not shown). No obvious differences in signal intensities or staining patterns were observed between Hspg2⌬3/⌬3 and control mice. Impaired FGF-2-Induced Corneal Angiogenesis in Hspg2⌬3/⌬3 Mice. To further investigate the impaired angiogenesis in Hspg2⌬3/⌬3 mice, we used the corneal micropocket assay to study FGF-2-induced angiogenesis in vivo. An FGF-2-containing pellet was implanted into the cornea of each 6-week-old mouse. Three animals of each genotype group (Hspg2⌬3/⌬3, heterozygous, and control) were used in these experiments. Five days after pellet implantation, blood vessel formation was evident in all mice. No differences were found between wild-type and heterozygous mice. However, all Hspg2⌬3/⌬3 mice showed a severely impaired angiogenesis (Fig. 3). Reduced FGF-2 Binding to Perlecan HS-Deficient Cells. To understand the mechanism of the impaired angiogenesis observed in Hspg2⌬3/⌬3 mice, we isolated MEFs from Hspg2⌬3/⌬3 mice and wild-type mice. The role of perlecan HS in the ligand-receptor interaction was investigated in vitro. 125I-FGF-2 of different concentrations was incubated for 2.5 h at 4°C with wild-type MEFs and Hspg2⌬3/⌬3 MEFs. After washing, the cell surface-bound 125I-FGF-2 was extracted and measured by ␥ counter. At the range of 5–20 ng/ml, wild-type MEFs exhibited a significant higher binding efficiency than Hspg2⌬3/⌬3 cells (P ⬍ 0.05), suggesting that in wild-type cells, the remaining wild-type perlecan HS on cell surface facilitates the binding of growth factor to cell surface (Fig. 4). These results indicated that perlecan with HS, but not the perlecan core protein, could facilitate the growth factor-receptor binding and promote cellular response. Loss of HS side chains in the perlecan protein compromised the ligand-receptor interaction in Hspg2⌬3/⌬3 cells. Discussion Genetic approaches have been applied to understand the in vivo roles of HSPGs. Mouse models lacking functional HS chains have been generated through the disruption of enzymes involved in HS biosynthesis (12). Diverse phenotypes were observed in these animal models with limited consistency. Knockout mice of several membrane-associated HSPGs have also been produced. These

Fig. 3. Corneal micropocket assay. Impaired FGF-2-induced angiogenesis in a Hspg2⌬3/⌬3 mouse compared with that in a wild-type control mouse at day 5 after growth factor implantation.

4701


of perlecan HS in the extracellular matrix is to bind FGF-2 and other growth factors, protect them from proteolytic degradation, and store the ligands in a dormant state. Perlecan on the cell surface may directly or indirectly present the growth factors to their receptor and facilitate the growth factor signaling. Perlecan without its HS chains failed to trap growth factors at the extracellular matrix of endothelial basement membranes and was unable to protect or present growth factors efficiently to the cells surface or higher affinity receptors. Therefore, impaired angiogenesis in the mutant mice was expected. Our results also indicate that the other membrane-associated HSPGs may also be important in the presentation of growth factors to their receptors. Lack of perlecan HS side chains did not result in severe developmental defects in neuronal and vascular tissues or total loss of mitotic response to FGF-2, indicating the functional redundancy in the regulation of growth factors receptors binding by HSPGs. Acknowledgments Fig. 4. Effects of perlecan HS on FGF-2 binding. MEF cell surface-bound 125I-FGF-2 increased along with the increase in the concentration of 125I-FGF-2. A significantly higher mount of 125I-FGF-2 bound to wild-type MEFs than to Hspg2⌬3/⌬3 MEFs (ⴱ, P ⬍ 0.05; ⴱⴱ, P ⬍ 0.01).

We thank Maria Laisi and Dadi Niu for excellent technical support. We also thank Neil Portwood for proofreading of the manuscript.

References knockout mice provide valuable models to understand the function of a specific HSPG in signaling (2). A complete knockout of perlecan core protein caused embryonic lethality due to the rupture of basement membranes in the heart and abnormal cartilage development, suggesting a critical role for this protein in the maintenance of cartilage and basement membrane integrity (6, 13). Because the signaling functions of HSPGs are mediated by their HS chains (14), mice lacking HS side chains while maintaining the structural role of perlecan core protein provide an unique model to study the signaling function of HS chains of a specific HSPG in vivo. We have shown in this study that perlecan HS played an important role in the regulation of wound healing and tumor growth. Loss of perlecan HS resulted in impaired angiogenesis, delayed wound healing and retarded tumor growth. Our data suggest that perlecan regulates FGF-2 signaling and angiogenesis through its HS side chains. To our knowledge, this is the first animal model demonstrating the in vivo function of HS side chains in a specific HSPG. A number of studies have been carried out to understand the biochemical and biological roles of perlecan in growth factor signaling (14 –17). Several functions of perlecan HS have been proposed based on in vitro studies: (a) Perlecan HS sequesters and traps growth factors, protecting them from proteolytic degradation; and (b) perlecan HS binds growth factors by acting as a low-affinity receptor and mediates high-affinity growth factor-receptor binding. Data from our in vitro binding support the notion that although a proportion of perlecan is secreted into the culture medium (17), a significant amount of perlecan attached to cell surfaces (18, 19). Cell surface perlecan participated directly or indirectly in the presentation of growth factors to its high-affinity receptor. Both the ligand trapping and presenting functions of perlecan seemed to be carried out through its HS side chains. Based on our and other’s results, it is likely that the major function

1. Nugent MA, Iozzo RV. Fibroblast growth factor-2. Int J Biochem Cell Biol 2000; 32:115–20. 2. Perrimon N, Bernfield M. Specificities of heparan sulphate proteoglycans in developmental processes. Nature 2000;404:725– 8. 3. Handler M, Yurchenco PD, Iozzo RV. Developmental expression of perlecan during murine embryogenesis. Dev Dyn 1997;210:130 – 45. 4. Iozzo RV. Matrix proteoglycans: from molecular design to cellular function. Annu Rev Biochem 1998;67:609 –52. 5. Costell M, Gustafsson E, Aszodi A, et al. Perlecan maintains the integrity of cartilage and some basement membranes. J Cell Biol 1999;147:1109 –22. 6. Rossi M, Morita H, Sormunen R, et al. Heparan sulfate chains of perlecan are indispensable in the lens capsule but not in the kidney. EMBO J 2003;22:236 – 45. 7. Malinda KM, Sidhu GS, Mani H, et al. Thymosin ␤4 accelerates wound healing. J Invest Dermatol 1999;113:364 – 8. 8. Soutter AD, Nguyen M, Watanabe H, Folkman J. Basic fibroblast growth factor secreted by an animal tumor is detectable in urine. Cancer Res 1993;53:5297–9. 9. Zhou Z, Apte SS, Soininen R, et al. Impaired endochondral ossification and angiogenesis in mice deficient in membrane-type matrix metalloproteinase I. Proc Natl Acad Sci USA 2000;97:4052–7. 10. Yayon A, Klagsbrun M, Esko JD, Leder P, Ornitz DM. Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell 1991;64:841– 8. 11. Thyboll J, Kortesmaa J, Cao R, et al. Deletion of the laminin ␣4 chain leads to impaired microvessel maturation. Mol Cell Biol 2002;22:1194 –202. 12. Forsberg E, Kjellen L. Heparan sulfate: lessons from knockout mice. J Clin Investig 2001;108:175– 80. 13. Arikawa-Hirasawa E, Watanabe H, Takami H, Hassell JR, Yamada Y. Perlecan is essential for cartilage and cephalic development. Nat Genet 1999;23:354 – 8. 14. Aviezer D, Hecht D, Safran M, Eisinger M, David G, Yayon A. Perlecan, basal lamina proteoglycan, promotes basic fibroblast growth factor-receptor binding, mitogenesis, and angiogenesis. Cell 1994;79:1005–13. 15. Nugent MA, Karnovsky MJ, Edelman ER. Vascular cell-derived heparan sulfate shows coupled inhibition of basic fibroblast growth factor binding and mitogenesis in vascular smooth muscle cells. Circ Res 1993;73:1051– 60. 16. Koyama N, Kinsella MG, Wight TN, Hedin U, Clowes AW. Heparan sulfate proteoglycans mediate a potent inhibitory signal for migration of vascular smooth muscle cells. Circ Res 1998;83:305–13. 17. Forsten KE, Courant NA, Nugent MA. Endothelial proteoglycans inhibit bFGF binding and mitogenesis. J Cell Physiol 1997;172:209 –20. 18. Hayashi K, Madri JA, Yurchenco PD. Endothelial cells interact with the core protein of basement membrane perlecan through ␤ 1 and ␤ 3 integrins: an adhesion modulated by glycosaminoglycan. J Cell Biol 1992;119:945–59. 19. Tran PK, Tran-Lundmark K, Soininen R, et al. Increased intimal hyperplasia and smooth muscle cell proliferation in transgenic mice with heparan sulfate-deficient perlecan. Circ Res 2004;94:550 – 8.

4702