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Annals of Surgical Oncology 9:303-309 (2002)
© 2002 Society of Surgical Oncology

ORIGINAL ARTICLES

Adverse Effects of the Antiangiogenic Agent Angiostatin on the Healing of Experimental Colonic Anastomoses

Elisabeth A. te Velde, MD, Emile E. Voest, MS, PhD, Joost M. van Gorp, MD, PhD, Andrë Verheem, Jeroen Hagendoorn, Martijn F. Gebbink, PhD and Inne H. Borel Rinkes, MD, PhD

From the Department of Surgery (EATV, AV, JH, IHBR) and the Laboratory of Medical Oncology (EEV, MFG), University Medical Center Utrecht, The Netherlands; and the Department of Pathology (JMVG), Diakonessen Hospital, Utrecht, The Netherlands.

Correspondence: Address correspondence and reprint requests to: I. H. M. Borel Rinkes, MD, PhD, Department of Surgery (G04.228), University Medical Center Utrecht, PO Box 85500, 3508 GA Utrecht, The Netherlands; Fax: 31-30-2505459; E-mail: i.h.m.borelrinkes{at}chir.azu.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Antiangiogenic cancer therapy is likely to be administered long term for sustained suppression of tumor outgrowth. Surgeons will encounter more patients undergoing such therapy. Therefore, it is essential to know the effects of antiangiogenic agents on physiological angiogenesis, as occurs during the healing of colonic anastomoses.

Methods: Angiostatin was generated from human plasma and administered continuously. In 38 mice, the right colon was anastomosed after transection: group 1 (n = 13), anastomotic healing under angiostatin treatment from surgery until death (day 7); group 2 (n = 13), phosphate-buffered saline controls. For healing on discontinuation of treatment, group 3 (n = 6) received angiostatin treatment preceding surgery during 4 days; group 4 (n = 6) included controls. On day 7, all mice were inspected for signs of anastomotic leakage. Bursting pressure measurements were performed to test anastomotic strength. Neovascularization was assessed semiquantitatively by immunohistochemistry.

Results: Mice treated with angiostatin postoperatively showed significantly more signs of leakage, more adhesions, and peritonitis. One mouse died on day 5. Five mice had paralytical ileus. The bursting pressure in group 1 was 135 ± 20 mm Hg, versus 175 ± 12 mm Hg in group 2 (mean ± SEM). Significantly fewer new vessels were found surrounding the anastomosis in the treated group (6.6 ± .9) versus controls (16 ± 1.6). All controls, as well as those animals treated with angiostatin only until surgery (group 3), displayed normal healing and showed no signs of peritonitis or ileus.

Conclusions: Angiostatin impairs anastomotic healing in mice. However, on discontinuation of antiangiogenic therapy, normal anastomotic healing is promptly restored.

Key Words: Angiogenesis • Angiostatin • Colonic anastomoses • Wound healing


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Antiangiogenic therapy is a highly promising new strategy in the treatment of cancer. Because the growth and metastasizing capacity of a tumor depend on the formation of new blood vessels, many antiangiogenic agents have been developed that are directed against the endothelial cells of the tumor vasculature.1 Potential advantages of antiangiogenic therapy include that the development of drug resistance seems unlikely2 and that serious toxicity is not anticipated.3,4

Angiostatin, a fragment of plasminogen, is considered to be one of the most potent inhibitors of angiogenesis.5 Experimental models have shown that, during long-term antiangiogenic therapy with angiostatin, tumors remain in a state of dormancy.3 However, when antiangiogenic therapy is discontinued, the tumor or its metastases resume their outgrowth.2 Accordingly, angiostatin recently proved to be more potent when administered continuously, instead of twice daily.6 From a clinical point of view, antiangiogenic therapy should preferably be administered during a prolonged period of time, e.g., in an adjuvant or neoadjuvant setting, to maintain remission after surgery. As a consequence, surgeons may increasingly be confronted with patients undergoing antiangiogenic therapy. It is, therefore, of great importance to investigate any adverse effects that antiangiogenic therapy might have on physiological angiogenesis, as occurs during wound healing.

A form of wound healing that can be life threatening if impaired is the healing of intestinal anastomoses, because anastomotic leakage in the week after surgery is a disastrous event that leads to high morbidity and mortality.7 Until now, the scarce studies regarding the effects of antiangiogenic strategies on intestinal anastomotic healing have not been comprehensive and have provided contradictory results. Recent data concerning short-term administration of two mildly angiosuppressive agents suggest possible adverse effects during the early phase of wound healing.8,9 In contrast, endostatin does not seem to impair cutaneous wound healing.10 The influence of strong and continuous inhibition of angiogenesis—as would be needed for sustained tumor suppression—on intestinal healing has never been studied. Moreover, data on the temporal aspects of the relationship between angiosuppressive treatment and its discontinuation are currently lacking. Besides, the effects of angiostatin on physiological angiogenesis are unknown. Such knowledge is of the utmost importance for the design of clinical studies and administration schedules with antiangiogenic agents.

This study was undertaken to evaluate the effects of strong antiangiogenic treatment with angiostatin, with maximally suppressive conditions, on the healing of colonic anastomoses. First, we evaluated anastomotic healing under angiostatin treatment by treatment from day 0 to day 7 after surgery. In a second set of experiments, we investigated the effects of discontinuation of angiostatin therapy directly preceding colonic surgery.

Our data provide strong evidence that angiostatin impairs anastomotic healing in mice. However, on discontinuation of antiangiogenic therapy, normal anastomotic healing is promptly restored.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation and Purification of Angiostatin
Human angiostatin was generated as described by O’Reilly et al.,5 with minor modifications.6 Outdated human plasma, heated (37°C) and filtered, was diluted 1/2 with distilled water, supplemented by 3 mM of EDTA, and applied to a lysine-SepharoseTM column (Pharmacia, Uppsala, Sweden). All column purifications were performed at room temperature. After washing the column with .5 M of phosphate buffer, plasminogen was eluted with .2 M of {epsilon}-aminocaproic acid, pH 7.4. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed one band of apparent molecular weight (Mr) 92.000, corresponding to plasminogen. The eluant was dialyzed against distilled water (MWCO, 6–8000 Spectra/Por; Spectrum Laboratories, Inc., Rancho Dominguez, CA; >4 x 107 dilution; 4°C), buffered with 20 mM of Tris 7.6, and followed by proteolytic digestion with .8 U/mg plasminogen porcine pancreatic elastase (Calbiochem, San Diego, CA) (shaker overnight at 37°C; 120 rpm). The solution was applied to the same column, which had been equilibrated with a salt solution. After the column was washed, angiostatin was eluted with .2 M of {epsilon}-aminocaproic acid while the flow-through was collected and treated as recently cleaved plasminogen, to collect angiostatin fragments with low lysine affinity. The angiostatin was dialyzed against distilled water (MWCO, 6–8000 Spectra/Por; >4 x 107 dilution; 4°C) and freeze dried. After the angiostatin and its flow-through were combined, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed three distinct bands of approximately Mr 40.000, Mr 42.000, and Mr 45.000, according to the triplet described by O’Reilly et al.5

In all experiments (unless stated otherwise), the administration of angiostatin was as follows. The mice received a dorsal osmotic pump (AlzetTM pump, type 2001; Alza, Palo Alto, CA) subcutaneously (SC) for continuous administration of angiostatin in a dose of 100 mg/kg/day plus a single bolus loading dose of 2.5 mg/200 µL SC. This dose of angiostatin proved to be the optimal dose to elicit a maximal antitumor effect in the murine SC and liver metastasis models.6 All controls received an identical pump filled with phosphate-buffered saline (PBS).

The bioactivity of the angiostatin was confirmed by the mouse cornea neovascularization assay, as described elsewhere (data not shown).6 Briefly, a corneal micropocket was created by a keratotomy. A micropellet (.4 x .4 x .2 mm) containing approximately 100 ng of basic fibroblast growth factor (Life Technologies, Inc., Rockville, MD) was inserted. The mice were divided into a treated group (which received angiostatin) and a control group (which received PBS). The corneas were examined daily by use of a microscope to determine the outgrowth of newly formed vessels from the limbus toward the pellet. When the vessels in the eyes of the controls had reached the pellet, the experiment was terminated. According to the formula .2 x {pi} x maximal vessel length x clock hours, the surface area of neovascularization of both groups was determined and compared.11 At a dose of 100 mg/kg/day, continuous administration of human angiostatin consistently caused virtually complete (93%) inhibition of angiogenesis.

Animals
The animals studied were BALB/c male mice, aged 12 weeks and weighing 25 to 30 g, purchased from the General Animal Laboratory of the University Medical Center Utrecht. They were allowed food and water ad libitum. All experiments were executed according to the guidelines of the Animal Welfare Committee of the University Medical Center Utrecht.

Surgical Procedures
In all mice, the right colon was anastomosed after transection. The mice were anesthetized intraperitoneally with fentanyl citrate/fluanisone (.3 mg per mouse; Janssen-Cilag, Brussels, Belgium) and midazolam chloride (12.5 mg per mouse; Roche, Brussels, Belgium). The mice were shaved and placed on a heated surgical microscopy table. A midline laparotomy was performed under aseptic conditions. The cecum was identified and lifted from the abdominal area. The right colon was transected 1 cm distal to the cecum with microscopic scissors, taking care not to damage the mesenteric vessels. The colon was anastomosed by single-layer 8-O nonabsorbable (ProleneTM; Ethicon, Brussels, Belgium) inverted running sutures. The abdominal wall was closed by two-layer 5-O absorbable (VicrylTM, Ethicon) running sutures. Next, the osmotic pump for administration of angiostatin or its solvent was implanted SC through a 1-cm dorsolateral incision.

Study Design
For each individual experiment, groups of three or four mice underwent transection of the right colon with anastomosis. They were randomly assigned to the angiostatin-treated group or the control group.

Anastomotic Healing Under Angiostatin Treatment
The following groups were identified: group 1 (n = 13), continuous angiostatin treatment from surgery until death (day 7); and group 2 (n = 13), PBS controls. These two groups received the osmotic pump during the surgical procedure of colonic anastomosis.

Anastomotic Healing After Discontinuation of Angiostatin Treatment
In group 3 (n = 6), angiostatin was administered for 4 days before surgery. Group 4 (n = 6) included PBS controls. In the last two groups, pumps were implanted 4 days before surgery and were removed during the surgical procedure of colonic anastomosis, to determine whether any adverse effect on the healing of those anastomoses would persist on discontinuation of the angiostatin therapy.

Outcome Measures
The observers were blinded to the treatment in all outcome measures.

Clinical Performance
Two independent observers recorded the postoperative clinical condition of the mice daily, according to a predetermined clinical assessment score, as listed in Table 1. For calculation of the percentage of postoperative weight loss, body weights on day 7 after surgery were compared with those on day 0, i.e., before surgery.


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  		TABLE 1. Postoperative clinical scores
 
Macroscopic Intra-Abdominal Analysis
On day 7, the abdomen was reopened and inspected for anastomotic dehiscence, local peritonitis, ileus, and the formation of adhesions. Peritonitis was defined as macroscopic signs of inflammation, fibrin deposition, and hypervascularity. Mechanical ileus was defined as obstruction of the intestine caused by the suture, with distention proximal to the anastomosis but not distal to the anastomosis, and was considered a failure of the surgical procedure. Paralytical ileus was defined as distention of atone intestine and colon, with paling and swelling of the colonic wall, in conjunction with the absence of mechanical ileus. In addition, intestinal diameters 2 cm distal and proximal from the anastomotic line were measured with calipers to quantitatively confirm these macroscopic findings. Adhesions involving the suture line were counted, because the presence of adhesions surrounding the suture line is indicative of an inflammatory healing process, e.g., as caused by intestinal spill. All surrounding structures adhesive to the anastomosis (colon, intestine, omentum, and pelvic fatty tissue) were considered separate adhesions and were counted as such. Bacterial growth of the abdominal cavity was determined by cotton swab, seeded on agar and Schaedler plates, and incubated.

Mechanical Analysis
To test the anastomotic strength, 3 cm of colon, including the anastomotic line, was resected, while care was taken not do disturb the anastomosis and adhesions. The resected segment was ligated on both sides and, ex vivo, was connected via a cannula to a volume-directed infusion pump filled with PBS. A side arm of the cannula was connected to a pressure transducer, which in turn was connected to a recorder. The intestinal segment was gradually filled with PBS at a constant rate of 60 ml/hour while the intraluminal pressure was monitored until burst occurred, as indicated as an abrupt loss of pressure. The bursting pressure was documented, and the site of bursting was noted as being either at the anastomotic line or outside the anastomotic line.12

Immunohistochemistry
Anastomotic lines were fixed in 4% formaldehyde and embedded in paraffin. Serial sections of 4-µm thickness from each block were mounted on poly-L-lysine–coated slides, and hematoxylin and eosin–stained sections were made of every 10th section to identify the newly formed granulation tissue surrounding the suture and adjacent to the normal colon tissue (as judged by two independent observers). Generally, this granulation tissue was easily discernible from the surrounding tissue. The selected adjacent unstained slides containing granulation tissue were deparaffinized, rehydrated, and incubated with hydrogen peroxide for 10 minutes. The slides were pretreated with pepsin and preincubated with normal goat serum for 15 minutes. The tissue was incubated with a polyclonal primary antibody against factor VIII/von Willebrand factor (DAKO, Carpinteris, CA) (1:500 in PBS/bovine serum albumin) for 1 hour. Incubation with a biotinylated goat anti-polyvalent secondary antibody and large-volume streptavidin peroxidase (Lab Vision, Fremont, CA) was followed by 3,3'-diaminobenzide tetrahydrochloride, as chromogen. Sections were counterstained with hematoxylin and dehydrated. Negative controls were prepared by substituting the primary antibody for PBS and were negative in all cases.

Morphometric analysis was performed with a LEICA-Q-Prodit (Leica Microsystems BV, Rijswijk, The Netherlands). The number of factor VIII–positive newly formed vessels was counted in granulation tissue surrounding the anastomotic line. The morphometric area of the granulation tissue and the relative area of the newly formed vessels were determined in all slides (according to the following formula: area vessels/area granulation tissue).

Statistical Analysis
Independent t-tests (equal variances not assumed) were performed to determine statistical differences between the treated and nontreated groups, and Fisher’s exact test was performed for the presence of peritonitis. For the difference in clinical scores over time, repeated-measurements analysis of variance was performed. Results are presented as mean ± SEM. Data were considered significant when P < .05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Anastomotic Healing Under Angiostatin Treatment
The clinical scores showed that the postoperative recovery of the mice treated with angiostatin was worse than that of control mice (P = .001). As shown in Fig. 1, the control mice had almost completely recovered from surgery by day 7. In Table 2 the data on the clinical analysis and the intra-abdominal macroscopy are summarized. One angiostatin-treated mouse died on day 5. The clinical scores of this particular animal were 1 to 2 on days 1 to 4, indicating a particularly poor postoperative recovery. In accordance with these findings, the angiostatin-treated group on day 7 had lost more weight than the controls: 8.3% ± 2% as compared with 3.6% ± 1%.



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  		FIG. 1. Clinical appearance after colonic anastomosis on day 0. ({blacksquare}) Scores of mice treated continuously with angiostatin; ({blacktriangleup}) control group (phosphate-buffered saline).

 

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  		TABLE 2. Anastomotic healing under angiostatin treatment
 
No mechanical ileus was observed, indicating anastomotic patency. Five mice that had been treated with angiostatin developed paralytical ileus. The intestinal diameter 2 cm distal to the anastomosis was significantly wider in the angiostatin-treated group, whereas the proximal diameter did not differ between groups. Six angiostatin-treated mice had clear macroscopic signs of local peritonitis, versus none in the control group (P = .02). In three cases, this was confirmed microbiologically with objective bacterial growth. In the controls, neither peritonitis nor ileus was found. The number of adhesions was significantly higher in the angiostatin-treated group. In all mice, all the other organs had normal macroscopic appearance.

In 7 of 12 angiostatin-treated mice and in 1 of 13 controls, the rupture of the intestinal segment during measurement of the bursting pressure occurred on the anastomotic line instead of in adjacent colon tissue. As a consequence, the anastomosis was judged to be weaker than the surrounding tissue in these animals. In the controls, all but one mouse had an anastomotic line that had become at least as strong as the surrounding tissue 1 week after surgery, with bursting occurring in the normal intestinal tissue. The bursting pressure in the angiostatin-treated group was 135 ± 20 mm Hg, versus 175 ± 12 mm Hg in the controls.

On immunohistochemical analysis, 16 ± 2 newly formed vessels were counted in the granulation tissue adjacent to the suture in the controls. In contrast, the granulation tissue of the mice that were treated with angiostatin contained significantly fewer new vessels (6.6 ± 1; P < .001). In addition, the area of granulation tissue occupied by new vessels was significantly decreased in the mice that were treated with angiostatin (.5% ± .1% vs. 2.2% ± .7%; P = .005) (Fig. 2).



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  		FIG. 2. Anastomotic healing on day 7 after surgery. (A) Group 1; (B) group 2. A decreased number of newly formed vessels alongside the anastomosis in the granulation tissue were found in the angiostatin-treated mice (6.6 ± 1 vs. 16 ± 2). Also, the relative area of the newly formed vessels in the granulation tissue was less in those mice (.5% ± .1% vs. 2.2% ± .7%). Factor VIII immunohistochemistry visualized the vessels (brown staining; some representative examples indicated by arrows). S, suture; Gr, granulation tissue (delineated by arrowheads); N, normal adjacent intestinal tissue.

 
The administration of angiostatin itself, in animals without an intestinal anastomosis, did not affect mouse body weight (as shown in our previous experiments with angiostatin6), intestinal diameter, or histology (data not shown).

Anastomotic Healing on Discontinuation of Angiostatin Treatment
The clinical scores of the angiostatin-treated mice (group 3) were comparable to those of the controls (group 4) during postoperative recovery (P = .93). None of the mice died. Weight loss in theangiostatin-treated group was 2.5% ± .3% vs. 3.0% ± .4% (P = .4) (Table 3).


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  		TABLE 3. Anastomotic healing directly after discontinuation of angiostatin administration
 
None of the mice showed signs of peritonitis, ileus, or intestinal distention. The number of adhesions did not differ between the two groups (P = .08). The intestinal diameter 2 cm distal to the anastomosis was 3.1 ± .06 cm in the angiostatin-treated group, compared with 3.1 ± .05 in the control group (P = .8). Again, no macroscopic abnormalities were found in the other organs. Mechanical analysis revealed that all mice in groups 3 and 4 had bursting pressures above 130 mm Hg (control animals, 164.5 ± 7 mm Hg versus angiostatin-treated animals, 151.6 ± 6 mm Hg; not significant). Also, the anastomotic lines of these mice had become at least as strong as the adjacent intestinal tissue.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our data provide strong evidence that angiostatin impaired anastomotic healing in mice when administered continuously during the phase of postoperative repair. Until now, it has been generally assumed that antiangiogenic agents specifically affect activated, proliferating, and migrating endothelial cells and thus would lack any major side effects during treatment.3,4 In concordance with this assumption, endostatin, another strong inhibitor of angiogenesis, was recently reported to have no adverse effects on physiological angiogenesis in cutaneous wound healing,10 whereas no data exist on angiostatin in wound-healing assays. But, in contrast to the healing of cutaneous wounds, the healing of colonic anastomoses is considered more dependent on angiogenesis and less dependent on diffusion of oxygen through pre-existing vasculature.13,14 Thus, our model might be more sensitive to angiogenesis-directed interventions. This healing of colonic anastomoses could therefore serve as a model for physiologically critical angiogenesis. Putative adverse effects of antiangiogenic agents might be more apparent in this model compared with cutaneous wound healing. Indeed, in two recent studies, the short-term administration of two mildly angiosuppressive agents seemed to have adverse effects during the early phase of intestinal healing.8,9 However, the adverse effects of suramin on rats’ anastomotic healing did not include any clinical signs of dehiscence of the anastomotic sites, such as abscess formation, and were not exclusively restricted to the intestine. Even at low doses, suramin was toxic to other organs in all animals and caused intra-abdominal bleeding and splenomegaly.9 In contrast, in this study, angiostatin did not impose toxicity on any organ besides the anastomosis (data not shown).

Furthermore, the other two studies mentioned do not provide temporal data. We have examined the kinetics of the adverse effects of angiostatin on the healing of colonic anastomoses. This was undertaken by administering the agent before surgery and discontinuing the treatment on the same day as the surgical procedure. Administration of angiostatin immediately before surgery had no negative effect on the healing of the anastomoses. These results in mice suggest that surgical patients who are treated with angiostatin can safely be operated on as soon as the treatment is discontinued.

Recently, angiostatin was shown by our group to be effective against experimental colorectal liver metastases.6 Assuming that antiangiogenic treatment is used perioperatively in patients undergoing colonic resection, it should preferentially be discontinued as briefly as possible, in an attempt to keep any metastatic tumor cell deposits in a state of dormancy. This is based on the findings of recent studies that showed that, after resection of the primary tumor, the levels of circulating endogenous antiangiogenic agents produced by the primary tumor diminish, leading to accelerated metastatic outgrowth.6,15 Considering the short half-life of angiostatin (4 to 6 hours), one might speculate that brief discontinuation of angiostatin treatment and resuming its administration shortly after anastomotic healing could circumvent the risk of anastomotic leakage while still effectively suppressing metastatic outgrowth.

Although the exact working mechanism of angiostatin has not been elucidated, it was recently shown to have a direct effect on endothelial cells.16 Our immunohistochemical evaluation showed a reduction in the number of newly formed vessels in the granulation tissue surrounding the anastomotic line. On the basis of these data, we speculate that the formation of new vessels at the site of the anastomosis is impaired by angiostatin. This in turn may lead to a decrease in delivery of oxygen and nutrients to the healing colon. The adverse effects of angiostatin on physiological angiogenesis suggest mutual underlying mechanisms for tumor-induced and physiological angiogenesis. This assumption is supported by the knowledge that many other factors involved in angiogenesis, such as vascular endothelial growth factor, do indeed affect both physiological and pathologic angiogenesis.17 It could well be that the antitumor effect and the effects on physiological angiogenesis of angiostatin are both mediated through a direct effect on endothelial cells.

We conclude from these results in mice that the healing of experimental colonic anastomoses is impaired by angiostatin when it is administered during the postoperative repair. However, on discontinuation of angiostatin therapy, normal anastomotic healing is promptly restored.


   Acknowledgments
 
Supported by the Dutch Scientific Research Committee Medical Sciences (project No. 99054).

Received for publication July 18, 2001. Accepted for publication October 18, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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  4. O’Reilly MS, Boehm T, Shing Y, et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 1997; 88: 277–85.[CrossRef][Medline]
  5. O’Reilly MS, 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: 315–28.[CrossRef][Medline]
  6. Drixler TA, Borel Rinkes IHM, Ritchie ED, van Vroonhoven TJ, Gebbink MF, Voest EE. Continuous administration of angiostatin inhibits accelerated growth of colorectal liver metastases after partial hepatectomy. Cancer Res 2000; 60: 1761–5.[Abstract/Free Full Text]
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  10. Berger AC, Feldman AL, Gnant MF, et al. The angiogenesis inhibitor, endostatin, does not affect murine cutaneous wound healing. J Surg Res 2000; 91: 26–31.[CrossRef][Medline]
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  16. Claesson-Welsh L, Welsh M, Ito N, et al. Angiostatin induces endothelial cell apoptosis and activation of focal adhesion kinase independently of the integrin-binding motif RGD. Proc Natl Acad Sci U S A 1998; 95: 5579–83.[Abstract/Free Full Text]
  17. Dvorak HF, Detmar M, Claffey KP, Nagy JA, van de WL, Senger DR. Vascular permeability factor/vascular endothelial growth factor: an important mediator of angiogenesis in malignancy and inflammation. Int Arch Allergy Immunol 1995; 107: 233–5.[CrossRef][Medline]



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