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Regulation of neovascularization by human neutrophil peptides (-defensins): a link between inflammation and angiogenesis

TRIANTAFYLLOS CHAVAKIS^*,,1, DOUGLAS B. CINES, JOONG-SUP RHEE^*, OLIN D. LIANG^*, UWE SCHUBERT^*, HANS-PETER HAMMES, ABD AL-ROOF HIGAZI, PETER P. NAWROTH, KLAUS T. PREISSNER^* and KHALIL BDEIR

^* Institute for Biochemistry, Justus-Liebig-Universität, Giessen,
Department of Internal Medicine I, University of Heidelberg, Heidelberg, Germany;
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA; and
Department of Internal Medicine, University Clinic Mannheim, Germany

¹ Correspondence: Department of Medicine I, University Heidelberg, Bergheimer Strasse 58, Heidelberg D-69115, Germany. Email: triantafyllos.chavakis{at}med.uni-heidelberg.de

SPECIFIC AIMS

Angiogenesis is a complex biological process that involves several interrelated reactions, such as endothelial cell adhesion, migration, proliferation, and differentiation. It is orchestrated by several growth factors and components of the extracellular matrix, including fibronectin and its receptor, integrin 5ß1. There is emerging evidence that inflammatory cells, particularly neutrophils, regulate endothelial cell functions in angiogenesis. We asked whether human neutrophil peptides (HNP) (also known as -defensins), abundant in human neutrophils and secreted upon neutrophil activation, can influence angiogenesis. We investigated mechanisms that may provide a link between inflammation and neovascularization.

PRINCIPAL FINDINGS

1. Inhibition of endothelial cell adhesion and migration by HNPs
Extracellular matrix (ECM) -associated proteins such as fibronectin (FN), vitronectin (VN), fibrinogen (FBG), and collagen are deposited into an adhesive fibrillar network and control endothelial cell adhesion and migration. In earlier studies we observed that HNPs (a family of four, closely related antimicrobial peptides, which are abundant in neutrophil granules) can accumulate in the vessel wall by binding to ECM-associated FN. We therefore investigated potential involvement of HNPs in endothelial cell adhesion and migration. HNPs specifically blocked 5ß1-integrin-dependent endothelial cell adhesion to FN whereas adhesion to FBG, VN, or collagen was not affected. HNPs specifically inhibited migration of HUVEC toward fibronectin under basal culture conditions and under stimulation by VEGF, whereas migration toward collagen, VN, or FBG was not affected (Fig. 1 ). The antiadhesive and antimigratory effect of HNPs was dose dependent, reaching a maximum inhibition at an HNP concentration of 10 µM, well within the plasma concentration attained during systemic infection. The far more cationic ß-defensin, HBD-2, did not inhibit endothelial cell adhesion or migration at the same concentrations.

View larger version (19K): [in this window] [in a new window]   Figure 1. Effect of HNPs on endothelial cell migration. Migration of HUVEC toward collagen (CN), vitronectin (VN), fibrinogen (FBG), and fibronectin (FN) is shown alone (–) or after addition of VEGF and in the absence (filled bars) or presence of HNPs (open bars) (5 µM). Cell migration is expressed as % of control, represented as cell migration in the absence of any stimulus or competitor. Data are mean ± SD (n=3) of a typical experiment; similar results were obtained in 3 separate experiments.

2.Effect of HNPs on interaction between FN and 5ß1-integrin
We studied the mechanism by which HNPs inhibit FN-5ß1-integrin-dependent adhesion and migration. ¹²⁵I- HNPs specifically bound to immobilized 5ß1-integrin in a dose-dependent and saturable manner with an estimated K_d of 7.0 ± 1.0 µM. Binding of HNPs to 5ß1 was not inhibited by RGDS and was only partially inhibited by EDTA. Thus, HNPs do not bind exclusively to the known RGD-inhibitable ligand binding site for FN on activated 5ß1, and the integrin does not have to be in active conformation to bind HNPs. Binding of HNPs to 5ß1 was inhibited by heparin.

We then investigated the effect of HNPs on FN-5ß1 interaction. Contrary to our expectations, HNPs caused a dose-dependent increase in FN binding to 5ß1. HNPs profoundly altered the way in which FN bound to 5ß1. Binding of FN to 5ß1-integrin in the absence of HNPs was inhibited by RGDS, whereas binding of FN to 5ß1 in the presence of HNPs was not RGD dependent. Although heparin had no effect on binding of FN to 5ß1 in the absence of HNPs, heparin abolished FN-5ß1 binding in the presence of 5 µM HNPs. These data indicate that binding of HNPs to 5ß1 impedes binding of FN to the ligand binding site on activated integrin while also promoting its association with the integrin through an RGD-independent process that does not contribute to cell adhesion.

3. Inhibition of endothelial proliferation by HNPs
HNPs inhibited HUVEC proliferation in a dose-dependent manner in the absence or presence of known activators of endothelial proliferation, such as VEGF or sphingosine-1-phosphate (Fig. 2 ). HNP1-3 induced apoptosis of HUVEC in a dose-dependent manner. In contrast, HBD-2 had no antiproliferative or proapoptotic effect on HUVEC. The antiproliferative effect of HNPs was slightly more potent when endothelial cells were cultivated on FN compared with the other two substrates.

View larger version (14K): [in this window] [in a new window]   Figure 2. Effect of HNPs on endothelial cell proliferation. HUVEC were incubated with 10 ng/ml VEGF (open bars) or 1 µM sphingosine-1-phosphate (filled bars) in the absence (–) or presence of increasing concentrations of HNPs. Proliferation of HUVEC is expressed as % of control, defined as cell proliferation in the absence of any stimulus or competitor. Data are mean ± SD (n=3) of a typical experiment; similar results were obtained in 3 separate experiments.

4. Inhibition of capillary sprout formation and angiogenesis in chicken chorioallantoic membrane (CAM) assay by HNPs
We next examined the role of HNPs in a more complex in vitro assay that simulates angiogenesis. Capillary-like tube formation in three-dimensional fibrin gels was inhibited by blockade of either v- or ß1-integrin and HNPs reduced the number of capillary-like tubes to the same extent as antibody against ß1-integrin. Extent of inhibition was dose dependent and mirrored observed in vitro activity. Almost complete inhibition of tube formation by HNPs was observed at concentrations >5 µM. HBD-2 (10 µM) had no effect on capillary tube formation.

Antiangiogenic activity of HNPs in vivo was investigated using CAM assay. On day 13, there were few if any capillaries present in the center of the methylcellulose disc exposed to HNPs; preexisting small vessels in CAMs were fragmented whereas flanking larger vessels were unaffected. HNPs inhibited angiogenesis under control conditions and under stimulation with basic fibroblast growth factor.

CONCLUSIONS AND SIGNIFICANCE

Emerging evidence points to the likelihood that inflammatory changes within the vessel wall regulate neovascularization associated with wound healing, tumor growth, and chronic inflammatory diseases. We report here that HNPs, which are abundant in neutrophil granules and secreted in high local concentrations when these cells are activated, inhibit endothelial cell adhesion and migration (primarily by interfering with the FN-5ß1-integrin system), block endothelial cell proliferation, and abrogate capillary tube formation and angiogenesis.

HNPs specifically inhibited 5ß1-mediated endothelial cell adhesion to end migration toward FN, whereas adhesion and migration in response to other ECM proteins such as VN, FBG, or collagen was unaffected. The antiadhesive and antimigratory effect of HNPs cannot be attributed solely to a nonspecific effect of the cationic charge or hydrophobic properties of peptides because little difference was seen between the potency of purified HNP-1 and HNP-3 and the more highly cationic and hydrophobic ß-defensin HBD-2 had no effect on either process.

We found that HNPs bind specifically to 5ß1-integrin. Binding was only partially inhibited by EDTA, suggesting that integrin activation is not required. HNP binding to 5ß1 also was not blocked by RGD, indicating that defensin does not behave as a classical integrin ligand. This is consistent with our finding that HNPs, contrary to our initial hypothesis, did not inhibit FN-5ß1 interaction. HNPs caused a dose-dependent enhancement of FN binding to 5ß1. Moreover, HNPs transformed FN binding to 5ß1 from a divalent cation and RGD-dependent, heparin-insensitive process associated with cell adhesion to one that is heparin dependent and RGD-insensitive, similar to the effect of these peptides on binding of Lp(a) to FN.

Vascular effects of HNPs extend beyond inhibition of endothelial cell adhesion and migration, although other pathways appear to be operative. HNPs 1) completely blocked VEGF- and sphingosine-1-phosphate-induced proliferation of endothelial cells and induced apoptosis; 2) inhibited endothelial proliferation whether cells were plated on FN, VN, or gelatin, indicating that their antiproliferative and proapoptotic effect does not require interference with 5ß1-FN; 3) inhibited capillary-like tube formation in 3-dimensional fibrin-matrices to the same extent as antibody to ß1-integrin; 4) inhibited neovascularization in CAM assay in vivo. The more highly cationic ß-defensin, HBD-2, had no significant effect on capillary sprout formation, excluding a nonspecific effect based on charge density or hydrophobicity.

There are several physiological implications of the observed antiangiogenic function of HNPs. First, one mechanism by which the innate immune system copes with invasive microbes is to invest them in fibrin, which deprives them of nutrition. HNPs may contribute to this by inhibiting plasminogen activation and the formation of new vessels required to supply oxygen and nutrients to rapidly dividing organisms. This initial walling off may be followed by a period of enhanced neovascularization during the healing phase that may be mediated in part by other antimicrobial peptides (e.g., cathelicidin peptide LL-37, which is generated by several leukocytes and stimulates endothelial cell proliferation and neovascularization via mechanism involving G-protein-coupled formyl peptide receptor-like 1). Local balance between pro- and antiangiogenic peptides may serve a regulatory function that contributes to host survival and tissue repair. Second, antiangiogenic properties of HNPs may extend to several pathophysiological processes. For example, besides impairing vascular metabolism of Lp(a) and LDL and inhibiting fibrinolysis, HNPs may impede development of a functional vasa vasorum in atherosclerotic vessels. The hypothesis that HNPs are endogenous modulators of plaque stability and neoangiogenesis merits further study. The presence of HNPs in human tumors may serve a salutary function, helping to control tumor angiogenesis and thereby tumor growth.

HNP1-3 thus may provide a platform for developing a novel class of antiangiogenesis compounds in cancer and other conditions, e.g., diverse retinopathies associated with exuberant and pathological vascular growth. Studies are under way in human HNP-1-expressing mice to test these concepts.

View larger version (43K): [in this window] [in a new window]   Figure 3. Model showing potential mechanisms underlying inhibition of angiogenesis by HNPs.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1009fje; doi: 10.1096/fj.03-1009fje

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