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The Angiogenesis Inhibitor Vasostatin does not Impair Wound Healing at Tumor-Inhibiting Doses

2001; Elsevier BV; Volume: 117; Issue: 5 Linguagem: Inglês

10.1046/j.0022-202x.2001.01519.x

1523-1747

Bernhard Lange‐Asschenfeldt, Paula Velasco, Michael Streit, Thomas Hawighorst, Michael Detmar, Sandra E. Pike, Giovanna Tosato,

Bioactive Natural Diterpenoids Research

Inhibition of tumor angiogenesis represents a promising new approach for the treatment of human cancers. It has remained unclear, however, whether inhibition of tumor angiogenesis may also result in impaired wound healing, a process thought to be angiogenesis dependent. To determine the effects of the angiogenesis inhibitor vasostatin, a 180 amino acid calreticulin fragment, on wound healing at tumor inhibiting doses, full-thickness wounds were generated on the back of nude mice that were also injected intradermally with CA46 Burkitt lymphoma cells. Mice were treated with daily injections of vasostatin or vehicle control at a site between the wounds and the transplanted tumor cells over 14 d. Vasostatin potently inhibited tumor growth and significantly reduced tumor angiogenesis, as measured by computer-assisted image analysis of CD31-stained tumor sections. Moreover, vasostatin treatment resulted in an increased fraction of mature tumor-associated blood vessels. In contrast, no impairment of wound healing was observed in vasostatin-treated mice, despite a significantly reduced vascularity of the wound granulation tissue. Our results reveal a different sensitivity of malignant tumor growth and physiologic wound healing to inhibition of angiogenesis, and they suggest that therapeutic inhibition of tumor angiogenesis may be achieved without impairment of tissue repair. Inhibition of tumor angiogenesis represents a promising new approach for the treatment of human cancers. It has remained unclear, however, whether inhibition of tumor angiogenesis may also result in impaired wound healing, a process thought to be angiogenesis dependent. To determine the effects of the angiogenesis inhibitor vasostatin, a 180 amino acid calreticulin fragment, on wound healing at tumor inhibiting doses, full-thickness wounds were generated on the back of nude mice that were also injected intradermally with CA46 Burkitt lymphoma cells. Mice were treated with daily injections of vasostatin or vehicle control at a site between the wounds and the transplanted tumor cells over 14 d. Vasostatin potently inhibited tumor growth and significantly reduced tumor angiogenesis, as measured by computer-assisted image analysis of CD31-stained tumor sections. Moreover, vasostatin treatment resulted in an increased fraction of mature tumor-associated blood vessels. In contrast, no impairment of wound healing was observed in vasostatin-treated mice, despite a significantly reduced vascularity of the wound granulation tissue. Our results reveal a different sensitivity of malignant tumor growth and physiologic wound healing to inhibition of angiogenesis, and they suggest that therapeutic inhibition of tumor angiogenesis may be achieved without impairment of tissue repair. The majority of current cancer treatments, including polychemotherapy and radiation therapy, are associated with serious adverse effects such as myelodepression, impaired tissue repair, and development of tumor resistance to therapy. The inhibition of tumor angiogenesis represents a promising new approach for the treatment of human cancers, leading to tumor starvation and to repression of metastatic spread (Hanahan and Folkman, 1996Hanahan D. Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (5847) Google Scholar). Therapeutic inhibition of tumor angiogenesis appears to be associated with a relatively low rate of toxic adverse effects (Boehm et al., 1997Boehm T. Folkman J. Browder T. O'Reilly M.S. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance.Nature. 1997; 390: 404-407Crossref PubMed Scopus (1579) Google Scholar). It has remained unclear, however, whether antiangiogenic therapy at tumor-relevant doses may also affect other angiogenesis-dependent processes such as tissue repair. Cutaneous wound healing has been thought to be critically dependent on the induction of angiogenesis. Skin wounds are characterized by the formation of a richly vascularized granulation tissue that supports the nutritional needs of rapidly proliferating and migrating epidermal keratinocytes, fibroblasts, and leukocytes (Dvorak, 1986Dvorak H.F. Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing.N Engl J Med. 1986; 315: 1650-1659Crossref PubMed Scopus (3230) Google Scholar;Singer and Clark, 1999Singer A.J. Clark R.A. Cutaneous wound healing.N Engl J Med. 1999; 341: 738-746Crossref PubMed Scopus (4298) Google Scholar). Naturally occurring angiogenesis inhibitors include thrombospondin-1 (Iruela-Arispe et al., 1991Iruela-Arispe L. Bornstein P. Sage H. Thrombospondin exerts an antiangiogenic effect on cord formation by endothelial cells in vitro.Proc Natl Acad Sci USA. 1991; 88: 5026-5030Crossref PubMed Scopus (230) Google Scholar), thrombospondin-2 (Volpert et al., 1995Volpert O.V. Tolsma S.S. Pellerin S. Feige J.J. Chen H. Mosher D.F. Bouck N. Inhibition of angiogenesis by thrombospondin-2.Biochem Biophys Res Commun. 1995; 217: 326-332https://doi.org/10.1006/bbrc.1995.2780Crossref PubMed Scopus (188) Google Scholar), angiostatin, a fragment of plasminogen (O'Reilly et al., 1994O'Reilly M.S. Holmgren L. Shing Y. et al.Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma.Cell. 1994; 79: 315-328Abstract Full Text PDF PubMed Scopus (3081) Google Scholar), and endostatin, a fragment of collagen type XVIII (O'Reilly et al., 1997O'Reilly M.S. Boehm T. Shing Y. et al.Endostatin: an endogenous inhibitor of angiogenesis and tumor growth.Cell. 1997; 88: 277-285Abstract Full Text Full Text PDF PubMed Scopus (4098) Google Scholar). Vasostatin is a 180 amino acid NH2-terminal fragment of human calreticulin (Pike et al., 1998Pike S.E. Yao L. Jones K.D. et al.Vasostatin, a calreticulin fragment, inhibits angiogenesis and suppresses tumor growth.J Exp Med. 1998; 188: 2349-2356Crossref PubMed Scopus (268) Google Scholar). Calreticulin is found in the sarcoplasmic reticulum of skeletal muscle cells and is involved in the regulation of calcium storage (Michalak et al., 1992Michalak M. Milner R.E. Burns K. Opas M. Calreticulin.Biochem J. 1992; 285: 681-692Crossref PubMed Scopus (396) Google Scholar). Both vasostatin and calreticulin inhibit endothelial proliferation in vitro and suppress tumor growth and angiogenesis in vivo (Tosato et al., 1994Tosato G. Sgadari C. Taga K. et al.Regression of experimental Burkitt's lymphoma induced by Epstein-Barr virus-immortalized human B cells.Blood. 1994; 83: 776-784PubMed Google Scholar;Pike et al., 1998Pike S.E. Yao L. Jones K.D. et al.Vasostatin, a calreticulin fragment, inhibits angiogenesis and suppresses tumor growth.J Exp Med. 1998; 188: 2349-2356Crossref PubMed Scopus (268) Google Scholar;Pike et al., 1999Pike S.E. Yao L. Setsuda J. et al.Calreticulin and calreticulin fragments are endothelial cell inhibitors that suppress tumor growth.Blood. 1999; 94: 2461-2468Crossref PubMed Google Scholar). To investigate whether angiogenesis inhibition at tumor-inhibiting doses might impair cutaneous wound healing, we evaluated full-thickness wounds in tumor-bearing mice treated with the endogenous angiogenesis inhibitor vasostatin. We implanted CA46 Burkitt lymphoma cells into the back skin of nude mice and also generated full-thickness skin wounds on the contralateral side in the same animals. Mice were treated with vasostatin or vehicle control, and tumor growth and wound closure were closely evaluated. We demonstrate that vasostatin potently inhibited tumor growth and angiogenesis of CA46 Burkitt lymphomas without any detectable effect on the rate of wound closure, despite significantly reduced vascularity of the wound granulation tissue. Our results reveal a different sensitivity of malignant tumor growth versus physiologic tissue repair to inhibition of angiogenesis, and they suggest that the therapeutic inhibition of angiogenesis at tumor-inhibiting doses is not necessarily associated with impaired tissue repair. Human CA46 Burkitt lymphoma cells (Sgadari et al., 1996Sgadari C. Angiolillo A.L. Cherney B.W. et al.Interferon-inducible protein-10 identified as a mediator of tumor necrosis in vivo.Proc Natl Acad Sci USA. 1996; 93: 13791-13796Crossref PubMed Scopus (196) Google Scholar) were cultured in RPMI 1640 medium supplemented with 12% fetal bovine serum, 2 mmol per l L-glutamine, and 5 µg per ml gentamicin (all purchased from Life Technologies, Rockville, MD). 2 × 107 cells in 100 µl phosphate-buffered saline were injected intradermally into the right side of the back of 8-wk-old female Swiss nu/nu mice. Tumor sizes were determined by measuring the largest and the smallest tumor diameter, using a digital caliper (Streit et al., 1999Streit M. Riccardi L. Velasco P. Brown L.F. Hawighorst T. Bornstein P. Detmar M. Thrombospondin-2: a potent endogenous inhibitor of tumor growth and angiogenesis.Proc Natl Acad Sci USA. 1999; 96: 14888-14893https://doi.org/10.1073/pnas.96.26.14888Crossref PubMed Scopus (246) Google Scholar). Tumor volumes were calculated using the formula: volume = 4/3 × π × (½ smaller diameter)2 × ½ larger diameter. Tumors were harvested from at least three animals for each treatment group and for each time point and were embedded in OCT compound (Sakura, Torrance, CA) and snap frozen in liquid nitrogen. Six hours after the first injection of either vasostatin or buffer (30 h after tumor injection into the contralateral side), mice were anesthetized with a single intraperitoneal injection of avertin (0.5 µg per 10 g body weight 2,2,2-tribromoethanol in 2.5% tert-amyl alcohol; Sigma). A full-thickness wound was created on the left side of the back of each mouse, using a 6 mm biopsy punch (Acuderm, Ft. Lauderdale, FL). Wound closure was monitored daily for 14 d in at least 10 mice for each time point by covering each wound with a transparent plastic sheet and marking the wound areas on the plastic sheet. Wound area measurements were performed on scanned transparencies, using the IP-LAB software (Scanalytics, Fairfax, VA) as described previously (Streit et al., 2000Streit M. Velasco P. Riccardi L. et al.Thrombospondin-1 suppresses wound healing and granulation tissue formation in the skin of transgenic mice.EMBO J. 2000; 19: 3272-3282https://doi.org/10.1093/emboj/19.13.3272Crossref PubMed Scopus (172) Google Scholar). Wound closure was expressed as the percentage of the initial wound area. Wounds were harvested from at least three animals per treatment and time point on days 3, 6, and 14. An area of 7–8 mm in diameter, including the complete epithelial margin of the wounds and the muscle fascia, was excised and cut in half. Wound specimens were either frozen in OCT compound or fixed for 48 h in 4% paraformaldehyde in phosphate-buffered saline and then embedded in paraffin. All experiments were repeated at least twice. One day after intradermal injection of tumor cells, 100 µg vasostatin (n = 16) or vehicle control (n = 16; 0.9% saline, 50 mg per ml human albumin, 5 mg per ml mannitol) were injected subcutaneously in close vicinity to the site of tumor cell inoculation. Subcutaneous injections were continued daily in between the tumors and the wounds over 2 wk. Recombinant vasostatin was produced as recently described (Pike et al., 1998Pike S.E. Yao L. Jones K.D. et al.Vasostatin, a calreticulin fragment, inhibits angiogenesis and suppresses tumor growth.J Exp Med. 1998; 188: 2349-2356Crossref PubMed Scopus (268) Google Scholar). The purified protein was tested for endotoxin by the Limulus Amebocyte Lysate kinetic-QCL assay (Bio Whittaker, Walkersville, MD) and was found to contain less than 5 EU per 10 µg protein. Immunohistochemistry was performed on 6 µm frozen or paraffin sections of wounds and tumors as described previously (Detmar et al., 1997Detmar M. Brown L.F. Berse B. Jackman R.W. Elicker B.M. Dvorak H.F. Claffey K.P. Hypoxia regulates the expression of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) and its receptors in human skin.J Invest Dermatol. 1997; 108: 263-268Crossref PubMed Scopus (225) Google Scholar). Vessels were stained with a monoclonal antibody against the endothelial junction molecule CD31 (Albelda et al., 1991Albelda S.M. Muller W.A. Buck C.A. Newman P.J. Molecular and cellular properties of PECAM-1 (endoCAM/CD31): a novel vascular cell-cell adhesion molecule.J Cell Biol. 1991; 114: 1059-1068Crossref PubMed Scopus (591) Google Scholar) (dilution 1:50; Pharmingen, San Diego, CA). Perivascular smooth muscle cells and pericytes were stained with an antihuman α-smooth muscle actin (α-SMA) antibody (dilution 1:100; clone 14A, Dako, Denmark). Masson's trichrome staining was performed on paraffin sections according to standard techniques (Prophet et al., 1992Prophet E. Mills B. Arrington J. Sobin L. Laboratory Methods in Histotechnology. American Registry of Pathology, Washington, DC1992: 1-236–237Google Scholar). The maturation status of blood vessels was assessed by double immunofluorescence staining for CD31 and α-SMA, using secondary antibodies labeled with either Texas Red or fluorescein isothiocyanate (1:50; Jackson ImmunoResearch Laboratory, West Grove, PA) as described previously (Benjamin et al., 1998Benjamin L.E. Hemo I. Keshet E. A plasticity window for blood vessel remodelling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF.Development. 1998; 125: 1591-1598Crossref PubMed Google Scholar). The vessel maturation index was calculated as the percentage of CD31-positive vessels that were associated with α-SMA-positive periendothelial cells. A total of at least 400 blood vessels were evaluated in each treatment group. Nuclear staining was performed with 20 µg per ml Hoechst bisbenzimide (Sigma). Endothelial cell proliferation was studied by intraperitoneal injection of mice with 5-bromo-deoxyuridine (BrdU; 250 mg per kg of body weight; Sigma) 2 h prior to sacrifice, followed by double immunofluorescence staining with anti-BrdU (Pharmingen) and anti-CD31 antibodies. Sections were mounted in Mowiol (Calbiochem, La Jolla, CA) and fluorescence and conventional light microscopy were performed using a Nikon E-600 microscope (Nikon, Melville, NY). Wound and tumor sections were obtained from three mice of each experimental group at 3, 6, and 14 d after wounding. Six micron frozen sections were stained with a monoclonal rat antimouse CD31 antibody and were analyzed using a Nikon E-600 microscope. Digital images were captured with a Spot digital camera (Diagnostic Instruments, Sterling Heights, MI), and morphometric analyses were performed using the IP-LAB software. Three different fields per section were examined at 60× magnification and the number of vessels per mm2, the average vessel size, the vessel size distribution, and the percentage of tumor area or granulation tissue area covered by blood vessels were determined as described previously (Streit et al., 1999Streit M. Riccardi L. Velasco P. Brown L.F. Hawighorst T. Bornstein P. Detmar M. Thrombospondin-2: a potent endogenous inhibitor of tumor growth and angiogenesis.Proc Natl Acad Sci USA. 1999; 96: 14888-14893https://doi.org/10.1073/pnas.96.26.14888Crossref PubMed Scopus (246) Google Scholar). Furthermore, combined CD31 and BrdU immunofluorescence stains of wound and tumor sections, obtained at 14 d after initiation of treatment, were analyzed using a Nikon E-600 microscope. Digital images were captured with a Spot digital camera and were examined at 10× magnification. The number of proliferating endothelial cells was determined by counting the number of BrdU- and CD31-positive cells at 60× magnification as described previously (Streit et al., 2000Streit M. Velasco P. Riccardi L. et al.Thrombospondin-1 suppresses wound healing and granulation tissue formation in the skin of transgenic mice.EMBO J. 2000; 19: 3272-3282https://doi.org/10.1093/emboj/19.13.3272Crossref PubMed Scopus (172) Google Scholar). The two-sided unpaired Student's t test was used for statistical analyses. Fourteen days after intradermal injection of CA46 Burkitt lymphoma cells, detectable tumor formation was found in 81% of the mice that were treated with vehicle control but only in 44% of vasostatin-treated mice. Treatment with vasostatin potently inhibited tumor growth with a reduction of the average tumor size by 79% (12.6 ± 6.3 mm3) compared with control-treated tumors (60.2 ± 18.4 mm3) at 6 d after tumor cell injection. Fourteen days after tumor cell injection, tumor growth was significantly inhibited (p ≤ 0.01) by more than 90% in the vasostatin-treated group (40.5 ± 23.2 mm3) compared with the control group (455.4 ± 120.4 mm3) Figure 1. Histologic analysis of viable tumor areas did not reveal major differences of the tumor architecture or cellular morphology between control and vasostatin-treated tumors Figure 2a, b. Staining for the endothelial junction molecule CD31, however, demonstrated rarefication of tumor-associated blood vessels after vasostatin treatment Figure 2c, d.Figure 2Vasostatin inhibits tumor angiogenesis. Comparable tumor architecture and cellular morphology in the viable areas of control (A) and vasostatin-treated (B) CA46 Burkitt lymphomas 14 d after intradermal injection. Hematoxylin and eosin stain. Immunostaining with an anti-CD31 antibody demonstrated rarefication of tumor blood vessels in vasostatin-treated tumors (D), compared with control tumors (C). Double immunofluorescence staining for CD31 (green) and α-SMA (red) demonstrates increased numbers of blood vessels associated with α-SMA positive mural cells (yellow, arrowheads) in vasostatin-treated tumors (F) compared to control tumors (E). Scale bars: 250 µm.View Large Image Figure ViewerDownload (PPT) To investigate the effects of vasostatin on the maturation status of the tumor vasculature, we simultaneously visualized endothelial cells and mural cells (smooth muscle cells and pericytes) by differential immunofluorescence staining for CD31 and α-SMA (Benjamin et al., 1999Benjamin L.E. Golijanin D. Itin A. Pode D. Keshet E. Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal.J Clin Invest. 1999; 103: 159-165Crossref PubMed Scopus (1017) Google Scholar). We found an increased number of vessels associated with α-SMA-positive cells in vasostatin-treated tumors Figure 2f compared with control-treated tumors Figure 2e. Quantitative evaluation revealed that the vessel maturation index, defined as the percentage of CD31-positive vessels associated with α-SMA-positive periendothelial cells, was significantly higher in vasostatin-treated tumors (83.5% ± 7.1%) than in control tumors (55.2% ± 11.6%; p < 0.01). Computer-assisted morphometric analyses of CD31-stained tumor sections revealed a significantly decreased vascular density in vasostatin-treated tumors with a more than 30% reduction in vessel numbers on day 6 (p < 0.01) and a more than 40% reduction at day 14 after tumor transplantation (p < 0.001) Figure 3a. In contrast, the average size of tumor vessels was unaffected by vasostatin treatment Figure 3b and no major changes in the size distribution of tumor-associated blood vessels were found Figure 3d. Overall, the relative tumor area covered by blood vessels was decreased by more than 30% on day 6 and by more than 40% (p < 0.001) on day 14 in vasostatin-treated tumors Figure 3c. Excisional full-thickness wounds were created on the backs of tumor-bearing 8-wk-old mice at 6 h after the first injection of vasostatin or vehicle control. Twenty-four hours post wounding, wounds were covered with a dry scab that remained adherent for 5–7 d in all mice. After 3 d, the wound areas were reduced to approximately 50% of the original size Figure 4. Daily wound area measurements did not reveal any significant difference in the rate of wound closure between vasostatin-treated mice (58.1% ± 11% of the original wound area at day 3, 44.1% ± 12% at day 6, and 3.6% ± 1.1% at day 14) or control-treated mice (54.8% ± 12.9% at day 3, 49.0% ± 9.7% at day 6, and 4.1% ± 2.1% at day 14) Figure 4 and complete wound closure occurred within 14 d in both groups. Comparable results were obtained in three independent experiments. Invasion of granulation tissue from the wound margins into the wound bed was already observed 3 d after injury and on day 6 the wound bed was completely filled with granulation tissue in both control and vasostatin-treated mice Figure 5a, b. After 14 d, both control-treated wounds and vasostatin-treated wounds Figure 5c, d were completely covered by a multilayered neo-epidermis.Figure 5Reduced vascularity of granulation tissue in vasostatin-treatment mice. Comparable formation of granulation tissue in vasostatin-treated (B) full-thickness wounds at 6 d after wounding, compared to control-treated wounds (A). Hematoxylin and eosin stain. Control-treated wounds (C) and vasostatin-treated wounds (D) show typical organization and maturation of granulation tissue 14 d after wounding. Hematoxylin and eosin stains. CD31 immunostains depict diminished vascularization in the granulation tissue of vasostatin-treated wounds (F) compared to control-treated wounds (E) at 14 d post wounding. Pronounced inhibition of vascular endothelial cell proliferation and neovascularization in granulation tissue of vasostatin-treated wounds (H) at 14 d post wounding compared with numerous proliferating endothelial cells in control-treated wounds (G) at 14 d post wounding. Immunofluorescence staining demonstrates CD31-stained blood vessels (red), BrdU-labeled proliferating nonendothelial cells (green), and CD31/BrdU double-stained proliferating vascular endothelial cells (yellow, arrowheads). Scale bars: 250 µm.View Large Image Figure ViewerDownload (PPT) Vasostatin treatment resulted in a significant reduction of the vessel density in the granulation tissue compared to control-treated wounds Figure 5e, f. Furthermore, treatment with vasostatin led to a 48.1% reduction of the number of proliferating endothelial cells in tumors and to a 52.4% inhibition of proliferating endothelial cells in wounds at day 14 Figure 5g, h. We observed a more than 20% decrease of vessel number on days 3 (p < 0.01) and 14 (p < 0.001) and a more than 15% reduction after 6 d (p < 0.05) Figure 6a. The average vessel size was slightly smaller in the granulation tissue of vasostatin-treated mice Figure 6b, and no major changes in the size distribution of blood vessels were found Figure 6d. The relative area of granulation tissue that was covered by blood vessels, however, reflecting changes in both vessel number and size, was significantly reduced following vasostatin treatment Figure 6c, with a more than 35% reduction on days 3 (p < 0.01) and 6 (p < 0.05) and a more than 25% reduction on day 14 (p < 0.05). In order to grow beyond a minimal size and to metastasize, tumors need to induce the growth of new blood vessels (angiogenesis), providing a lifeline for tumor sustenance and waste disposal (Hanahan and Folkman, 1996Hanahan D. Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.Cell. 1996; 86: 353-364Abstract Full Text Full Text PDF PubMed Scopus (5847) Google Scholar). Inhibition of tumor angiogenesis represents a new approach for the treatment of cancers and might potentially avoid the development of tumor cell resistance and the occurrence of some of the adverse effects observed with polychemotherapy (Boehm et al., 1997Boehm T. Folkman J. Browder T. O'Reilly M.S. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance.Nature. 1997; 390: 404-407Crossref PubMed Scopus (1579) Google Scholar). Because antiangiogenic therapies for the treatment of human cancers will most probably be administered over prolonged time periods, it is of considerable clinical importance to evaluate whether long-term inhibition of angiogenesis may also inhibit other physiologic or pathologic processes that are thought to be angiogenesis dependent. In particular, it remains to be established whether systemic inhibition of angiogenesis might result in impaired tissue repair as it occurs in ischemic heart disease or in cutaneous wound healing. Although a small number of studies have aimed at characterizing the effects of distinct antiangiogenic regimens on wound healing (Klein et al., 1999Klein S.A. Bond S.J. Gupta S.C. Yacoub O.A. Anderson G.L. Angiogenesis inhibitor TNP-470 inhibits murine cutaneous wound healing.J Surg Res. 1999; 82: 268-274Abstract Full Text PDF PubMed Scopus (51) Google Scholar;Berger et al., 2000Berger A.C. Feldman A.L. Gnant M.F. et al.The angiogenesis inhibitor, endostatin, does not affect murine cutaneous wound healing.J Surg Res. 2000; 91: 26-31Abstract Full Text PDF PubMed Scopus (106) Google Scholar;Bloch et al., 2000Bloch W. Huggel K. Sasaki T. et al.The angiogenesis inhibitor endostatin impairs blood vessel maturation during wound healing.FASEB J. 2000; 14: 2373-2376Crossref PubMed Scopus (146) Google Scholar;Streit et al., 2000Streit M. Velasco P. Riccardi L. et al.Thrombospondin-1 suppresses wound healing and granulation tissue formation in the skin of transgenic mice.EMBO J. 2000; 19: 3272-3282https://doi.org/10.1093/emboj/19.13.3272Crossref PubMed Scopus (172) Google Scholar;Wood et al., 2000Wood J.M. Bold G. Buchdunger E. et al.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), the results have been controversial. In particular, it has remained unclear whether some of the observed inhibition of wound healing might have been the result of effects of angiogenesis inhibitors on nonendothelial cells such as fibroblasts, leading to delayed granulation tissue formation (Streit et al., 2000Streit M. Velasco P. Riccardi L. et al.Thrombospondin-1 suppresses wound healing and granulation tissue formation in the skin of transgenic mice.EMBO J. 2000; 19: 3272-3282https://doi.org/10.1093/emboj/19.13.3272Crossref PubMed Scopus (172) Google Scholar) or to inhibiting effects on the activity of matrix metalloproteinases (Klein et al., 1999Klein S.A. Bond S.J. Gupta S.C. Yacoub O.A. Anderson G.L. Angiogenesis inhibitor TNP-470 inhibits murine cutaneous wound healing.J Surg Res. 1999; 82: 268-274Abstract Full Text PDF PubMed Scopus (51) Google Scholar;Streit et al., 2000Streit M. Velasco P. Riccardi L. et al.Thrombospondin-1 suppresses wound healing and granulation tissue formation in the skin of transgenic mice.EMBO J. 2000; 19: 3272-3282https://doi.org/10.1093/emboj/19.13.3272Crossref PubMed Scopus (172) Google Scholar). Moreover, conflicting results have been reported regarding the effects of the angiogenesis inhibitor endostatin on the vascularity of wound granulation tissue (Berger et al., 2000Berger A.C. Feldman A.L. Gnant M.F. et al.The angiogenesis inhibitor, endostatin, does not affect murine cutaneous wound healing.J Surg Res. 2000; 91: 26-31Abstract Full Text PDF PubMed Scopus (106) Google Scholar;Bloch et al., 2000Bloch W. Huggel K. Sasaki T. et al.The angiogenesis inhibitor endostatin impairs blood vessel maturation during wound healing.FASEB J. 2000; 14: 2373-2376Crossref PubMed Scopus (146) Google Scholar), and the criteria for the evaluation of wound healing have not been well defined. Importantly, none of these previous studies directly investigated the effects of angiogenesis inhibition on wound healing at tumor-inhibiting doses within the same animals. In this study, we examined the effects of the angiogenesis inhibitor vasostatin on wound healing in tumor-bearing mice. Vasostatin treatment potently reduced the tumor-forming efficiency of CA46 Burkitt lymphoma cells and significantly inhibited malignant tumor growth. These data are in accordance with the previously reported antitumoral activity of vasostatin (Pike et al., 1998Pike S.E. Yao L. Jones K.D. et al.Vasostatin, a calreticulin fragment, inhibits angiogenesis and suppresses tumor growth.J Exp Med. 1998; 188: 2349-2356Crossref PubMed Scopus (268) Google Scholar;Yao et al., 2000Yao L. Pike S.E. Setsuda J. et al.Effective targeting of tumor vasculature by the angiogenesis inhibitors vasostatin and interleukin-12.Blood. 2000; 96: 1900-1905PubMed Google Scholar) and they confirm that the doses used in our experiments were indeed tumor suppressive. Vasostatin treatment also resulted in a pronounced reduction of the vascular density of tumors whereas the average size of tumor-associated blood vessels was only slightly reduced. It is of interest that we found a significantly increased percentage of α-SMA-positive tumor vessels after vasostatin treatment, indicating that vasostatin induced a higher degree of vessel maturation, similar to that found in slowly developing tissues or under physiologic conditions in normal skin (Benjamin et al., 1998Benjamin L.E. Hemo I. Keshet E. A plasticity window for blood vessel remodelling is defined by pericyte coverage of the preformed endothelial network and is regulated by PDGF-B and VEGF.Development. 1998; 125: 1591-1598Crossref PubMed Google Scholar). Pericytes are closely associated with endothelial cells in the microvasculature (Beck and D'Amore, 1997Beck L. D'Amore P.A. Vascular development: cellular and molecular regulation.FASEB J. 1997; 11: 365-373Crossref PubMed Scopus (433) Google Scholar) and it has been suggested that blood vessels with pericyte coverage have a diminished ability to sprout and to form new capillaries (Darland and D'Amore, 1999Darland D.C. D'Amore P.A. Blood vessel maturation: vascular development comes of age.J Clin Invest. 1999; 103: 157-158Crossref PubMed Scopus (252) Google Scholar). The reduced vascular density observed in tumors after vasostatin treatment reflects these blood vessels' reduced ability to form vascular sprouts as a result of an increase in their maturation (Pettersson et al., 2000Pettersson A. Nagy J.A. Brown L.F. et al.Heterogeneity of the angiogenic response induced in different normal adult tissues by vascular permeability factor/vascular endothelial growth factor.Lab Invest. 2000; 80: 99-115Crossref PubMed Scopus (351) Google Scholar). Importantly, treatment with tumor-inhibiting doses of vasostatin (100 µg per d) did not result in delayed closure of full-thickness skin wounds in tumor-bearing mice. The model of full-thickness wounds induced in mouse skin represents a well-established experimental system for studies of wound healing and has revealed important insights into the distinct roles of a number of growth factors in tissue repair (Werner et al., 1992Werner S. Peters K.G. Longaker M.T. Fuller-Pace F. Banda M.J. Williams L.T. 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Therefore, our findings indicate that inhibition of wound angiogenesis does not necessarily result in impaired wound healing. Taken together, our results suggest that malignant tumor growth is more sensitive to angiogenesis inhibition than physiologic tissue repair. This may be explained, in part, by our findings that tumor tissue was much less vascularized than wound granulation tissue. In fact, control-treated wounds showed a 3-fold higher vascular density than control-treated CA46 Burkitt lymphomas in this study. Although it remains to be established whether these findings apply to the majority of malignant tumors, our previous studies in transplanted SCC-13 or A431 squamous cell carcinomas also detected vascular densities that were markedly lower than those found here in wound granulation tissue (Streit et al., 1999Streit M. Riccardi L. Velasco P. Brown L.F. Hawighorst T. Bornstein P. Detmar M. Thrombospondin-2: a potent endogenous inhibitor of tumor growth and angiogenesis.Proc Natl Acad Sci USA. 1999; 96: 14888-14893https://doi.org/10.1073/pnas.96.26.14888Crossref PubMed Scopus (246) Google Scholar;Detmar et al., 2000Detmar M. Velasco P. Richard L. et al.Expression of vascular endothelial growth factor induces an invasive phenotype in human squamous cell carcinomas.Am J Pathol. 2000; 156: 159-167Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Our results demonstrate that angiogenesis in wound repair, a process essential to survival, is more redundant than angiogenesis in tumors, which appear to be critically dependent on their blood supply. These findings may have implications for the development of antiangiogenic cancer therapies and indicate that different thresholds may apply to efficient angiogenesis inhibition in tumors and in nonmalignant tissues (Eberhard et al., 2000Eberhard A. Kahlert S. Goede V. Hemmerlein B. Plate K.H. Augustin H.G. Heterogeneity of angiogenesis and blood vessel maturation in human tumors: implications for antiangiogenic tumor therapies.Cancer Res. 2000; 60: 1388-1393PubMed Google Scholar). In summary, we show that the angiogenesis inhibitor vasostatin potently inhibited the tumor growth and angiogenesis of CA46 Burkitt lymphomas transplanted into nude mice without major impairment of wound healing. Our results suggest a different sensitivity of tumors and wounds to inhibition of angiogenesis, most probably related to the more pronounced and more redundant vascularization of the wound granulation tissue. They also indicate that efficient antiangiogenic therapy of malignant tumors can be achieved without impairment of tissue repair. Supported by National Institutes of Health/National Cancer Institute Grants CA69184 and CA86410 (M.D.), American Cancer Society Research Project Grant 99-23901 (M.D.), Deutsche Forschungsgemeinschaft (B.L.-A.; T.H.), the Dermatology Foundation (M.S.), and the Cutaneous Biology Research Center through the Massachusetts General Hospital/Shiseido Co. Ltd. Agreement (to M.D.).