Apoptotic effect of cleaved high molecular weight kininogen is regulated by extracellular matrix proteins

We previously reported that cleaved high molecular weight kininogen (HKa) and its domain 5 (D5) inhibit critical steps required for angiogenesis and in vivo neovascularization (Colman et al. 2000: Blood 95:543-550). We have further shown that D5 is able to induce apoptosis of endothelial cells, which may represent a critical part of the anti-angiogenic activity of HKa and D5 (Guo et al. 2001: Arterioscler Thromb Vasc Biol 21:1427-1433). In this study, we demonstrate that HKa- and D5-induced apoptosis is closely correlated with their anti-adhesive effect. An important new finding is that the apoptotic activity of HKa and D5 is highly regulated by their interactions with different extracellular matrix (ECM) proteins. HKa inhibited cell adhesion to vitronectin (Vn, 90%) and gelatin (Gel) (40%), but it had no apparent effect on cell adhesion to fibronectin (Fn). D5 showed a similar pattern on cell adhesion but was less potent than HKa. HKa induced apoptosis of endothelial cells grown on Vn and Gel but not cells grown on Fn which closely parallels with its anti-adhesive potency. Further results revealed that the anti-adhesive effect and the apoptotic effect of HKa are associated with its ability to inhibit phosphorylation of focal adhesion kinase (FAK) and paxillin, two important signal molecules required for cell adhesion and cell viability. We conclude that the anti-adhesive activity of HKa and D5 is responsible for their apoptotic effect and that Vn is likely an ECM component that mediates the effect of HKa and D5.     

Upregulation of Cdc2 and cyclin A during apoptosis of endothelial cells induced by cleaved high-molecular-weight kininogen

We (8) reported that the cleaved high-molecular-weight kininogen (HKa) and its domain 5 (D5) inhibited angiogenesis. Further studies (15) revealed that D5 could inhibit cell proliferation and induce apoptosis of proliferating endothelial cells, which together may represent a critical part of antiangiogenic activity of HKa and D5. In the present study, we further examined the effect of HKa on cell cycle progression and cell viability. We report that HKa induced a significant upregulation of Cdc2 and cyclin A in proliferating endothelial cells, concurrent with a marked increase of Cdc2 activity. The increased expression of Cdc2 and cyclin A by HKa was not associated with an apparent change in cell cycle profiles of basic fibroblast growth factor-stimulated proliferating cells, but closely correlated with a marked increase of apoptosis, suggesting that the elevated Cdc2 activity is involved in HKa-induced apoptosis of proliferating endothelial cells. Our results support an emerging hypothesis that Cdc2 and cyclin A are important regulators for cell cycle as well as for apoptosis.     

Two-chain high molecular weight kininogen induces endothelial cell apoptosis and inhibits angiogenesis: partial activity within domain 5.

We previously reported that the binding of two-chain high molecular weight kininogen (HKa) to endothelial cells may occur through interactions with endothelial urokinase receptors. Since the binding of urokinase to urokinase receptors activates signaling responses and may stimulate mitogenesis, we assessed the effect of HKa binding on endothelial cell proliferation. Unexpectedly, HKa inhibited proliferation in response to several growth factors, with 50% inhibition caused by approximately 10 nM HKa. This activity was Zn(2+) dependent and not shared by either single-chain high molecular weight kininogen (HK) or low molecular weight kininogen. HKa selectively inhibited the proliferation of human umbilical vein and dermal microvascular endothelial cells, but did not affect that of umbilical vein or human aortic smooth muscle cells, trophoblasts, fibroblasts, or carcinoma cells. Inhibition of endothelial proliferation by HKa was associated with endothelial cell apoptosis and unaffected by antibodies that block the binding of HK or HKa to any of their known endothelial receptors. Recombinant HK domain 5 displayed activity similar to that of HKa. In vivo, HKa inhibited neovascularization of subcutaneously implanted Matrigel plugs, as well as rat corneal angiogenesis. These results demonstrate that HKa is a novel inhibitor of angiogenesis, whose activity is dependent on the unique conformation of the two-chain molecule.     

The inhibition of tube formation in a collagen-fibrinogen, three-dimensional gel by cleaved kininogen (HKa) and HK domain 5 (D5) is dependent on Src family kinases

Cleaved high molecular weight kininogen (HKa), as well as its domain 5 (D5), inhibits migration and proliferation induced by angiogenic factors and induces apoptosis in vitro. To study its effect on tube formation we utilized a collagen-fibrinogen, three-dimensional gel, an in vitro model of angiogenesis. HKa, GST-D5 and D5 had a similar inhibitory effect of tube length by 90+/-4.5%, 86+/-5.5% and 77+/-12.9%, respectively. D5-derived synthetic peptides: G440-H455 H475-H485 and G486-K502 inhibited tube length by 51+/-3.7%, 54+/-3.8% and 77+/-1.7%, respectively. By a comparison of its inhibitory potency and its sequences, a functional sequence of HKa was defined to G486-G496. PP2, a Src family kinase inhibitor, prevented tube formation in a dose-dependent manner (100-400 nM), but PP3 at 5 microM, an inactive analogue of PP2, did not. HKa and D5 inhibited Src 416 phosphorylation by 62+/-12.3% and 83+/-6.1%, respectively. The C-terminal Src kinase (Csk) inhibits Src kinase activity. Using a siRNA to Csk, expression of Csk was down-regulated by 86+/-7.0%, which significantly increased tube length by 27+/-5.8%. The addition of HKa and D5 completely blocked this effect. We further showed that HKa inhibited Src family kinase activity by disrupting the complex of uPAR, alphavbeta3 integrin and Src. Our results indicate that the anti-angiogenic effect of HKa and D5 is mediated at least in part through Src family kinases and identify a potential novel target for therapeutic inhibition of neovascularization in cancer and inflammatory arthritis.     

Ferritin blocks inhibitory effects of two-chain high molecular weight kininogen (HKa) on adhesion and survival signaling in endothelial cells

Angiogenesis is tightly regulated through complex crosstalk between pro- and anti-angiogenic signals. High molecular weight kininogen (HK) is an endogenous protein that is proteolytically cleaved in plasma and on endothelial cell surfaces to HKa, an anti-angiogenic protein. Ferritin binds to HKa and blocks its anti-angiogenic activity. Here, we explore mechanisms underlying the cytoprotective effect of ferritin in endothelial cells exposed to HKa. We observe that ferritin promotes adhesion and survival of HKa-treated cells and restores key survival and adhesion signaling pathways mediated by Erk, Akt, FAK and paxillin. We further elucidate structural motifs of both HKa and ferritin that are required for effects on endothelial cells. We identify an histidine-glycine-lysine (HGK) -rich antiproliferative region within domain 5 of HK as the target of ferritin, and demonstrate that both ferritin subunits of the H and L type regulate HKa activity. We further demonstrate that ferritin reduces binding of HKa to endothelial cells and restores the association of uPAR with α5β1 integrin. We propose that ferritin blocks the anti-angiogenic activity of HKa by reducing binding of HKa to UPAR and interfering with anti-adhesive and anti-proliferative signaling of HKa.     

Extracellular matrix-derived peptide binds to alpha(v)beta(3) integrin and inhibits angiogenesis

Angiogenesis is associated with several pathological disorders as well as with normal physiological maintenance. Components of vascular basement membrane are speculated to regulate angiogenesis in both positive and negative manner. Recently, we reported that tumstatin (the NC1 domain of alpha 3 chain of type IV collagen) and its deletion mutant tum-5 possess anti-angiogenic activity. In the present study, we confirm that the anti-angiogenic activity of tumstatin and tum-5 is independent of disulfide bond requirement. This property of tum-5 allowed us to use overlapping synthetic peptide strategy to identify peptide sequence(s) which possess anti-angiogenic activity. Among these peptides, only the T3 peptide (69-88 amino acids) and T7 peptide (74-98 amino acids) inhibited proliferation and induced apoptosis specifically in endothelial cells. The peptides, similar to tumstatin and the tum-5 domain, bind and function via alpha(v)beta(3) in an RGD-independent manner. Restoration of a disulfide bond between two cysteines within the peptide did not alter the anti-angiogenic activity. Additionally, these studies show that tumstatin peptides can inhibit proliferation of endothelial cells in the presence of vitronectin, fibronectin, and collagen I. Anti-angiogenic effect of the peptides was further confirmed in vivo using a Matrigel plug assay in C57BL/6 mice. Collectively, these experiments suggest that the anti-angiogenic activity of tumstatin is localized to a 25-amino acid region of tumstatin and it is independent of disulfide bond linkage. Structural features and potency of the tumstatin peptide make it highly feasible as a potential anti-cancer drug.     

The inhibitory effect of HKa in endothelial cell tube formation is mediated by disrupting the uPA-uPAR complex and inhibiting its signaling and internalization

In two-dimensional (2-D) culture systems, we have previously shown that cleaved two-chain high-molecular-weight kininogen (HKa) or its domain 5 induced apoptosis by disrupting urokinase plasminogen activator (uPA) receptor (uPAR)-integrin signal complex formation. In the present study, we used a three-dimensional (3-D) collagen-fibrinogen culture system to monitor the effects of HKa on tube formation. In a 3-D system, HKa significantly inhibited tube and vacuole formation as low as 10 nM, which represents 1.5% of the physiological concentration of high-molecular-weigh kininogen (660 nM), without apparent apoptosis. However, HKa (300 nM) completely inhibited tube formation and increased apoptotic cells about 2-fold by 20-24 h of incubation. uPA-dependent ERK activation and uPAR internalization regulate cell survival and migration. In a 2-D system, we found that exogenous uPA-induced ERK phosphorylation and uPAR internalization were blocked by HKa. In a 3-D system, we found that not only uPA-uPAR association but also the activation of ERK were inhibited by HKa. HKa disrupts the uPA-uPAR complex, inhibiting the signaling pathways, and also inhibits uPAR internalization and regeneration to the cell surface, thereby interfering with uPAR-mediated cell migration, proliferation, and survival. Thus, our data suggest that the suppression of ERK activation and uPAR internalization by HKa contributes to the inhibition of tube formation. We conclude that in this 3-D collagen-fibrinogen gel, HKa modulates the multiple functions of uPAR in endothelial cell tube formation, a process that is closely related to in vivo angiogenesis.     

Identification of the anti-angiogenic site within vascular basement membrane-derived tumstatin

Components of vascular basement membrane are involved in regulating angiogenesis. Recently, tumstatin (the NC1 domain of alpha3 chain of type IV collagen) was identified as possessing anti-angiogenic activity. In the present study, the anti-angiogenic activity of tumstatin was localized to the putative 54-132-amino acid Tum-5 domain, and the activity mediated by alpha(v)beta(3) integrin interaction in an RGD-independent manner. The recombinant Tum-5 produced in Escherichia coli and Pichia Pastoris specifically inhibited proliferation and caused apoptosis of endothelial cells with no significant effect on nonendothelial cells. Tum-5 also inhibited tube formation of endothelial cells on Matrigel and induced G1 endothelial cell cycle arrest. Moreover, anti-angiogenic effect of Tum-5 was also examined in vivo using both a Matrigel plug assay in C57BL/6 mice and human prostate cancer (PC-3) xenografts in nude mice. The in vivo results demonstrate that Tum-5 at 1 mg/kg significantly inhibited growth of PC-3 tumors in association with a decrease in CD31 positive vasculature. These in vivo studies also show that, at molar equivalents, human Tum-5 is at least 10-fold more active than human endostatin. In addition, these studies for the first time suggest that through the action of endogenous inhibitors, alpha(v)beta(3) integrin may also function as a negative regulator of angiogenesis. Taken together, these findings demonstrate that Tum-5, a domain derived from tumstatin, is an effective inhibitor of tumor-associated angiogenesis and a promising candidate for the treatment of cancer.     

活化型高分子量激肽原潜在的抗肿瘤作用及其分子机制

高分子量激肽原是血浆中一种多功能的糖蛋白,与血液凝固的启动、 补体反应及炎症发生等有密切关系.新近的研究显示,活化型高分子量激肽原 具有潜在的抗肿瘤作用.本文综述活化型高分子量激肽原在细胞粘附和血管 生成中发挥的抑制作用及其活性区域,抑制细胞迁移、增殖并诱导细胞凋亡 的作用,及其在细胞表面的作用位点和分子机制.活化型高分子量激肽原作用 机制,包括抑制细胞DNA的从头合成,使细胞周期蛋白D1表达下降,以及通过 影响细胞内信号通路发挥其活性效应等.深入研究活化型高分子量激肽原在 细胞表面作用的信号转导通路可能是今后抗肿瘤研究途径之一.     

Peptides derived from type I thrombospondin repeat-containing proteins of the CCN family inhibit proliferation and migration of endothelial cells

Angiogenesis, or neovascularization, is tightly orchestrated by endogenous regulators that promote or inhibit the process. The fine-tuning of these pro- and anti-angiogenic elements (the angiogenic balance) helps establish the homeostasis in tissues, and any aberration leads to pathologic conditions. The type I thrombospondin repeats are a family of protein structural elements involved in the control of angiogenesis, and some proteins containing these repeats have been identified as negative regulators of angiogenesis. Here we identify a set of 11 novel, anti-angiogenic 18–20-amino acid peptides that are derived from proteins that belong to the CCN protein family and contain type I thrombospondin motifs. We have named these peptides spondinstatin-1, cyrostatin, connectostatin, nephroblastostatin, wispostatin-2, wispostatin-3, netrinstatin-5C, netrinstatin-5D, adamtsostatin-like-4, fibulostatin-6.1, and complestatin-C6 to reflect their origin. We have shown that these peptides inhibit proliferation and migration of human umbilical vein endothelial cells in vitro. By conducting a clustering analysis of the amino acid sequences using sequence similarity criteria and of the experimental results using a hierarchical clustering algorithm, we have demonstrated that there is an underlying correlation between the sequence and activity of the identified peptides. This combination of experimental and computational approaches introduces a novel systematic framework for studying peptide activity, identifying novel peptides with anti-angiogenic activity, and designing mimetic peptides with tailored properties.     

The anti-angiogenic His/Pro-rich fragment of histidine-rich glycoprotein binds to endothelial cell heparan sulfate in a Zn2+-dependent manner

The plasma protein histidine-rich glycoprotein (HRGP), which has been identified as an angiogenesis inhibitor, binds to heparan sulfate (HS) in a Zn(2+)-dependent manner. We wished to test whether this interaction is mechanistically important in mediation of the anti-angiogenic effect of HRGP. Inhibition of angiogenesis by HRGP is exerted through its central His/Pro-rich domain, which is proteolytically released. A 35-amino-acid residue synthetic peptide, HRGP330, derived from the His/Pro-rich domain retains the inhibitory effect on blood vessel formation in vitro and in vivo, an effect dependent on the presence of Zn(2+). We now show that HRGP330 binds heparin/HS with the same capacity as full-length HRGP, and the binding is Zn(2+)-dependent. Peptides derived from the His/Pro-rich domain of HRGP downstream of HRGP330 fail to inhibit endothelial cell migration and display a significantly reduced heparin-binding capacity. An even shorter peptide, HRGP335, covering a 26-amino-acid sequence within HRGP330 retains full heparin/HS-binding capacity. Characterization of the HS interaction shows that there is a tissue-specific HS pattern recognized by HRGP335 and that the minimal length of heparin/HS required for binding to HRGP335 is an 8-mer oligosaccharide. Saturation of the HS binding sites in HRGP330 by pre-incubation with heparin abrogates the HRGP330-induced rearrangement of endothelial cell focal adhesions, suggesting that interaction with cell surface HS is needed for HRGP330 to exert its anti-angiogenic effect.     

Novel anti-angiogenic peptides derived from ELR-containing CXC chemokines

Angiogenesis is tightly regulated by numerous endogenous pro- and anti-angiogenic proteins and peptides. Among these are the CXC chemokines, a set of multifunctional peptides. CXC chemokines containing the ELR motif act as pro-angiogenic agents by regulating both endothelial cell proliferation and migration. Here we show that a set of six 22-24-amino acid peptides derived from the pro-angiogenic ELR-containing CXC chemokines exhibit notable anti-proliferative and anti-migratory activity in vitro; we call these peptides chemokinostatins. The ability of the identified peptides to inhibit the basic components of angiogenesis even though they are derived from pro-angiogenic proteins contributes towards the understanding of the diverse role of the CXC chemokine family in angiogenesis.     

Two RGD-independent alpha vbeta 3 integrin binding sites on tumstatin regulate distinct anti-tumor properties

Vascular basement membrane is an important regulator of angiogenesis and undergoes many alterations during angiogenesis and these changes are speculated to influence neovascularization. Recently, fragments of collagen molecules have been identified to possess anti-angiogenic activity. Tumstatin (alpha3(IV)NC1 domain) is one such novel molecule with distinct anti-tumor properties and possesses an N-terminal (amino acids 54-132) anti-angiogenic and a C-terminal (amino acids 185-203) anti-tumor cell activity (Maeshima, Y., et al. 2000) J. Biol. Chem. 275, 21340-21348). Previous studies have identified the 185-203 amino acid sequence as a ligand for alpha(v)beta(3) integrin (Shahan, T. A., et al. (1999) Cancer Res. 59, 4584-4590). In the present study, we found distinct additional RGD-independent alpha(v)beta(3) integrin binding site within 54-132 amino acids of tumstatin. This site is not essential for inhibition of tumor cell proliferation but necessary for the anti-angiogenic activity. A fragment of tumstatin containing 54-132 amino acid (tum-2) binds both endothelial cells and melanoma cells but only inhibited proliferation of endothelial cells, with no effect on tumor cell proliferation. A similar experiment with fragment of tumstatin containing the 185-203 amino acid (tum-4) demonstrates that it binds both endothelial cells and melanoma cells but only inhibits the proliferation of melanoma cells. The presence of cyclic RGD peptides did not affect the alpha(v)beta(3) integrin-mediated activity of tumstatin, although significant inhibition of endothelial cell binding to vitronectin was observed. The two distinct RGD-independent binding sites on tumstatin suggest unique alpha(v)beta(3) integrin-mediated mechanisms governing the two distinct anti-tumor properties of tumstatin.     

Distinct antitumor properties of a type IV collagen domain derived from basement membrane

Vascular basement membrane is an important structural component of blood vessels. During angiogenesis this membrane undergoes many alterations and these changes are speculated to influence the formation of new capillaries. Type IV collagen is a major component of vascular basement membrane, and recently we identified a fragment of type IV collagen alpha2 chain with specific anti-angiogenic properties (Kamphaus, G. D., Colorado, P. C., Panka, D. J., Hopfer, H., Ramchandran, R., Torre, A., Maeshima, Y., Mier, J. W., Sukhatme, V. P., and Kalluri, R. (2000) J. Biol. Chem. 275, 1209-1215). In the present study we characterize two different antitumor activities associated with the noncollagenous 1 (NC1) domain of the alpha3 chain of type IV collagen. This domain was previously discovered to possess a C-terminal peptide sequence (amino acids 185-203) that inhibits melanoma cell proliferation (Han, J., Ohno, N., Pasco, S., Monboisse, J. C., Borel, J. P., and Kefalides, N. A. (1997) J. Biol. Chem. 272, 20395-20401). In the present study, we identify the anti-angiogenic capacity of this domain using several in vitro and in vivo assays. The alpha3(IV)NC1 inhibited in vivo neovascularization in matrigel plug assays and suppressed tumor growth of human renal cell carcinoma (786-O) and prostate carcinoma (PC-3) in mouse xenograft models associated with in vivo endothelial cell-specific apoptosis. The anti-angiogenic activity was localized to amino acids 54-132 using deletion mutagenesis. This anti-angiogenic region is separate from the 185-203 amino acid region responsible for the antitumor cell activity. Additionally, our experiments indicate that the antitumor cell activity is not realized until the peptide region is exposed by truncation of the alpha3(IV)NC1 domain, a requirement not essential for the anti-angiogenic activity of this domain. Collectively, these results effectively highlight the distinct and unique antitumor properties of the alpha3(IV)NC1 domain and the potential use of this molecule for inhibition of tumor growth.     

Role of the light chain of high molecular weight kininogen in adhesion, cell-associated proteolysis and angiogenesis

Cleavage of high molecular weight kininogen (HK) by plasma kallikrein results in a light chain and a heavy chain (HK). The light chain has two domains: D6, which binds (pre)kallikrein, and D5, which binds to anionic surfaces, including heparin as well as zinc. Initially, HK was thought to be important for surface-activated coagulation. HKa or D5 binds to the urokinase receptor on endothelial cells, thereby enhancing the conversion of prourokinase to urokinase by kallikrein, and, thus, cell-associated fibrinolysis. HKa or D5 is antiadhesive by competing with vitronectin binding to the urokinase receptor and/or forming a complex with vitronectin. D5 inhibits endothelial cell migration, proliferation, tube formation and angiogenesis, thus modulating inflammation and neovascularization.     

The anti-angiogenic activity of rPAI-1(23) inhibits fibroblast growth factor-2 functions

Many angiogenesis inhibitors are breakdown products of endogenous extracellular matrix proteins. Plasmin and matrix metalloproteinase-3 generate breakdown products of matrix-bound plasminogen activator inhibitor-1 (PAI-1). We produced a truncated form of PAI-1, rPAI-1(23), that possesses significant anti-angiogenic activity and stimulates high levels of apoptosis in quiescent arterial endothelial cells. Quiescent endothelial cells are less susceptible to apoptosis than angiogenic endothelial cells. The present study was designed to determine the mechanism of the rPAI-1(23) effects in bovine aortic endothelial cells. Apoptosis was measured in annexin V and caspase 3 assays. Expression of death and survival signaling molecules were examined by Western blot and kinase activity. Fibroblast growth factor 2 (FGF2) functions were analyzed in angiogenesis assays. The early response to rPAI-1(23) was an increase in annexin V-positive cells and phosphorylated (p) JNK isoform expression followed by an increase in p-Akt and p-c-Jun expression. Caspase 3 was activated at 4 h, whereas p-Akt was reduced to control levels. By 6 h of rPAI-1(23) treatment cell number was reduced by 35%, and p-c-Jun and p-JNK were degraded by proteasomes. Confocal microscopic images showed increased amounts of FGF2 in the extracellular matrix. However, rPAI-1(23) blocked FGF2 signaling through FGF receptor 1 and syndecan-4, inhibiting cell migration, tubulogenesis, and proliferation. Exogenous FGF2 stimulation could not reverse these effects. We conclude that rPAI-1(23) stimulation of apoptosis in BAEC triggers a cascade of death versus survival events that includes release of FGF2. The rPAI-1(23) anti-angiogenic activity inhibits FGF2 pro-angiogenic functions by blocking FGF2 signaling through FGF receptor 1 and syndecan-4 and downstream effectors p-Akt, p-JNK, and p-c-Jun.     

Cysteine-rich and basic domain HIV-1 Tat peptides inhibit angiogenesis and induce endothelial cell apoptosis

Previous findings suggest that both the Tat polypeptide encoded by HIV-1 and Tat-derived peptides can induce angiogenesis via activation of the KDR receptor for Vascular Endothelial Growth Factor (VEGF). We identified 20 amino acids and 12 amino acid peptides corresponding to the cysteine-rich and basic domains of HIV-1 Tat which inhibited (125)I-VEGF(165) binding to KDR and neuropilin-1 (NP-1) receptors in endothelial cells. Cysteine-rich and basic Tat peptides inhibited VEGF-induced ERK activation and mitogenesis in endothelial cells, and inhibited angiogenesis in vitro at concentrations similar to those which inhibited VEGF receptor binding. These peptides also inhibited proliferation, angiogenesis, and ERK activation induced by basic fibroblast growth factor with similar potency and efficacy. Surprisingly, we found that both cysteine-rich and basic domain Tat peptides strikingly induced apoptosis in endothelial cells, independent of their effects on VEGF and bFGF. Furthermore, we found no evidence for direct biological effects of recombinant Tat on VEGF receptor binding, ERK activation, endothelial cell survival, or mitogenesis. These findings demonstrate novel properties of Tat-derived peptides and indicate that their major effect in endothelial cells is apoptosis independent of specific inhibition of VEGF receptor activation.     

Mapping the interaction between high molecular mass kininogen and the urokinase plasminogen activator receptor

The urokinase plasminogen activator receptor (uPAR) is a multifunctional, GPI-linked receptor that modulates cell adhesion/migration and fibrinolysis. We mapped the interaction sites between soluble uPAR (suPAR) and high molecular mass kininogen (HK). Binding of biotin-HK to suPAR was inhibited by HK, 56HKa, and 46HKa with an IC50 of 60, 110, and 8 nm, respectively. We identified two suPAR-binding sites, a higher affinity site in the light chain of HK and 46HKa (His477-Gly496) and a lower affinity site within the heavy chain (Cys333-Lys345). HK predominantly bound to suPAR fragments containing domains 2 and 3 (S-D2D3). Binding of HK to domain 1 (S-D1) was also detected, and the addition of S-D1 to S-D2D3 completely inhibited biotin-HK or -46HKa binding to suPAR. Using sequential and overlapping 20-amino acid peptides prepared from suPAR, two regions for HK binding were identified. One on the carboxyl-terminal end of D2 (Leu166-Thr195) blocked HK binding to suPAR and to human umbilical vein endothelial cells (HUVEC). This site overlapped with the urokinase-binding region, and urokinase inhibited the binding of HK to suPAR. A second region on the amino-terminal portion of D3 (Gln215-Asn255) also blocked HK binding to HUVEC. Peptides that blocked HK binding to uPAR also inhibited prekallikrein activation on HUVEC. Therefore, HK interacts with suPAR at several sites. HK binds to uPAR as part of its interaction with its multiprotein receptor complex on HUVEC, and the biological functions that depend upon this binding are modulated by urokinase.     

Peptides derived from human decorin leucine-rich repeat 5 inhibit angiogenesis

Excessive angiogenesis is involved in many human diseases, and inhibiting angiogenesis is an important area of drug development. There have been conflicting reports as to whether decorin could function as an angiogenic inhibitor when used as an extracellular soluble factor. In this study, we demonstrated that not only purified decorin but also the 26-residue leucine-rich repeat 5 (LRR5) of decorin core protein functions as angiogenesis inhibitor by inhibiting both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor-induced angiogenesis. Peptide LRR5 inhibited angiogenesis through multiple mechanisms, including inhibiting VEGF-stimulated endothelial cell (EC) migration, tube formation on Matrigel, cell attachment to fibronectin, as well as induction of EC apoptosis without significantly affecting their proliferation. We further demonstrated that different subregions of LRR5 inhibited different aspects of angiogenesis, with the middle region (LRR5M, 12 residues) inhibiting endothelial cell tube formation up to 1000 times more potently than LRR5. Although the C-terminal region (LRR5C) potently inhibited VEGF-stimulated endothelial cell migration, the N-terminal region (LRR5N) is as active as LRR5 in inhibiting endothelial cell attachment to fibronectin. Although both LRR5M and LRR5N induced EC apoptosis dose-dependently similar to LRR5 through a caspase-dependent pathway, LRR5C has no such function. We further showed that the inhibition of tube formation by LRR5 and LRR5M is linked with their ability to suppress VEGF-induced focal adhesion kinase phosphorylation and the assembly of focal adhesions and actin stress fibers in ECs, but not their ability to interfere with endothelial cell attachment to the matrix. Circular dichroism studies revealed that LRR5 undergoes an inter-conversion between 3(10) helix and beta-sheet structure in solution, a characteristic potentially important for its anti-angiogenic activity. Peptide LRR5 and its derivatives are therefore novel angiogenesis inhibitors that may serve as prototypes for further development into anti-angiogenic drugs.     

Involvement of NO/cGMP signaling in the apoptotic and anti-angiogenic effects of beta-lapachone on endothelial cells in vitro

Neovascularization is an essential process in tumor development, it is conceivable that anti-angiogenic treatment may block tumor growth. In angiogenesis, nitric oxide (NO) is an important factor which mediates vascular endothelial cell growth and migration. beta-Lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho-[1,2-b]pyran-5,6-dione), a natural product extracted from the lapacho tree (Tabebuia avellanedae), has been demonstrated to possess anti-cancer and anti-viral effects. Whether beta-lapachone can induce endothelial cell death or has an anti-angiogenic effect is still an enigma. We investigated the in vitro effect of beta-lapachone on endothelial cells, including human vascular endothelial cell line, EAhy926, and human umbilical vascular endothelial cells (HUVEC). Our results revealed that (1) the intracellular cGMP levels and the mitochondria membrane potential (MMP) decreased, and calpain and caspases were activated, during beta-lapachone-induced endothelial cell death; (2) co-treatment with calpain inhibitors (ALLM or ALLN) or the intracellular calcium chelator, BAPTA, but not the general caspase inhibitor, zVAD-fmk, provided significant protection against apoptosis by preventing the beta-lapachone-induced MMP decrease and cytoplasmic calcium increase; (3) addition of NO downregulated the beta-lapachone-induced cGMP depletion and protected the cells from apoptosis by blocking the MMP decrease and the calcium increase; and (4) exogenous NO protects endothelial cells against the cell death induced by beta-lapachone, but not the anti-angiogenic effect. From all the data above, we demonstrated that NO can attenuate the apoptotic effect of beta-lapachone on human endothelial cells and suggest that beta-lapachone may have potential as an anti-angiogenic drug.