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

2000 ◽  
Vol 14 (15) ◽  
pp. 2589-2600 ◽  
Author(s):  
Jing‐Chuan Zhang ◽  
Kevin Claffey ◽  
Ramasamy Sakthivel ◽  
Zbigniev Darzynkiewicz ◽  
David Elliot Shaw ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Endothelial Cell ◽  
High Molecular Weight ◽  
Cell Apoptosis ◽  
High Molecular Weight Kininogen ◽  
Endothelial Cell Apoptosis ◽  
Partial Activity

Induction of Endothelial Cell Apoptosis by Cleaved High Molecular Weight Kininogen Is Mediated Through a Lck and p53-Dependent Pathway

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3305-3305
Author(s):  
Venkaiah Betapudi ◽  
Keith R. McCrae
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
High Molecular Weight ◽  
Cell Apoptosis ◽  
Negative Regulator ◽  
Dependent Manner ◽  
High Molecular Weight Kininogen ◽  
Endothelial Cell Apoptosis ◽  
Single Chain

Abstract Abstract 3305 Background and objective: High molecular weight kininogen (HK) is an abundant plasma protein that serves as an important component of the intrinsic pathway of coagulation. HK normally circulates as in the single chain form, but may be cleaved by plasma kallikrein to release the nonapeptide bradykinin, resulting in the formation cleaved high molecular weight kininogen (HKa) that consists of a heavy and light chain linked by a single disulfide bond. Conformational changes occurring after kallikrein cleavage result in increased exposure of histidine and glycine-rich regions with kininogen domain 5 that impart HKa with unique properties, including the ability to inhibit angiogenesis by causing selective apoptosis of proliferating endothelial cells. However, the receptors that mediate the antiangiogenic activity of HKa remain controversial, and the signaling pathways that lead to apoptosis have not been defined. Previous studies suggested possible involvement of SRC family kinases (SFK) in this process, and the purpose of this work was to further define the activation of SFKs and their downstream targets during HKa-induced endothelial cell apoptosis. Results: We first assessed the activation of SFKs in proliferating endothelial cells stimulated with bFGF before and after incubation with HKa (6–20 nM). SFKs are maintained in an inactive state through tyrosine phosphorylation of their C-terminal region mediated by the negative regulator C-terminal Src kinase (Csk). Exposure of endothelial cells to HKa caused downregulation of Csk in a dose-dependent manner within 60 minutes. In parallel, we observed a significant increase in expression of the proapoptotic SFK Lck in endothelial cells exposed to HKa, though expression of other SFKs including Lyn, Fyn, Src, Hck and Blk were not significantly altered. Increased expression of Lck was associated with activation of p53 and increased expression of the pro-apoptotic Bcl-2 family members Bax and Bak. Endothelial cell lysates prepared within 60 minutes of exposure to HKa demonstrated significant increases in the activity of caspases 3 and 7, as well as depletion of DNA fragmentation factors (DFF) 45 and 35, which cleave and inactivate DFF40, a major endonuclease involved in apoptosis. In parallel studies, endothelial cells depleted of Lck by treatment with Lck siRNA displayed loss of p53 phosphorylation, caspase 3 and 7 activity, and expression of Bax and Bad with no effects on the expression of Bad and Bid. Conclusion: These findings demonstrate a critical role for Csk in regulation of SFK activation and endothelial homeostasis, and demonstrate that downregulation of Csk by HKa leads to activation of a Lck-dependent, p53-mediated apoptotic pathway. Increasing the expression of Lck may represent a novel mechanism for regulation of aberrant angiogenesis. Disclosures: No relevant conflicts of interest to declare.

Apoptosis of Proliferating Endothelial Cells Induced by Cleaved High Molecular Weight Kininogen (HKa) Involves an Oxidation-Dependent Pathway.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3926-3926
Author(s):  
Danyu Sun ◽  
Keith McCrae
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
High Molecular Weight ◽  
Cell Apoptosis ◽  
Type I ◽  
High Molecular Weight Kininogen ◽  
Endothelial Cell Apoptosis ◽  
Cells Cultured

High molecular weight kininogen (HK) is a single-chain glycoprotein that plays a central role in contact activation. We previously reported that the cleaved form of high molecular weight kininogen (HKa), which lacks bradykinin, induces apoptosis of proliferating endothelial cells and inhibits angiogenesis in vivo. This activity involves binding of HKa to tropomyosin exposed on the endothelial cell surface. We have also demonstrated that endothelial cells cultured on types I or IV collagen are endowed with relative resistance to the apoptotic effects of HKa. We hypothesized that one mechanism that might account for this specific effect could be the ability of collagen to inhibit hydroxyl radical induced apoptosis through scavenging of hydroxyl radicals and inhibition of cellular lipid peroxidation. To address this issue, we assessed the ability of HKa to inhibit endothelial cell proliferation in the absence or presence of glutathione (GSH), an intracellular thiol antioxidant which scavenges reactive oxygen species (ROS) and prevents ROS-induced cell damage. The results of these experiments demonstrated that GSH blocked the ability of HKa to inhibit endothelial proliferation and induce endothelial cell apoptosis on all ECM proteins tested, including gelatin, laminin, vitronectin, fibronectin, and collagens I and IV. Similar results were obtained when N-acetylcysteine (NAC), a cell membrane permeable GSH precursor, was employed. However, neither GSH nor NAC protected endothelial cells from apoptosis induced by 2-methoxyestradiol, which also induces selective apoptosis of proliferating endothelial cells, suggesting that the ability of GSH to block HKa-induced endothelial cell apoptosis is specific. To further examine the role of ROS in HKa induced endothelial cell apoptosis, we measured the cellular GSH level and content of lipid peroxidation products MDA and 4-HNE following exposure to HKa. These studies demonstrated that exposure of endothelial cells cultured on gelatin to HKa led to a rapid fall in intracellular GSH, accompanied by a three fold increase in MDA and 4-HNE. In contrast, little change in the levels of oxidation products were observed when cells were cultured on collagen type I or IV. These results demonstrate that the ability of HKa to induce apoptosis of proliferating endothelial cells is associated with the production of reactive oxygen species (ROS) and a change in intracellular redox status. To further test this conclusion, we examined another redox sensitive signaling regulator, thioredoxin (TRX), which normally occurs primarily in the cytoplasm but translocates to the nucleus in response to oxidative stress. In response to HKa, thioredoxin quickly translocated from the cytoplasm to the nucleus of endothelial cells cultured on gelatin, though nuclear translocation of thioredoxin did not occur when cells were cultured on type I collagen. These results suggest that nuclear accumulation of TRX may be intimately involved in HKa induced endothelial cell apoptosis by sensing oxidative stress. In conclusion, our data suggests that HKa induces endothelial cell apoptosis through an oxidation dependent pathway, which is co-regulated through additional signals emanating from the extracellular matrix. Ongoing studies are focused on defining the mechanisms by which ROS are generated, and the role of HKa-tropomyosin interactions in stimulating this oxidant pathway.

Inhibition of angiogenesis by two-chain high molecular weight kininogen (HKa) and kininogen-derived polypeptides

2002 ◽  
Vol 80 (2) ◽  
pp. 85-90 ◽  
Author(s):  
Jing-Chuan Zhang ◽  
Xiaoping Qi ◽  
Jose' Juarez ◽  
Marian Plunkett ◽  
Fernando Donaté ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
High Molecular Weight ◽  
Cell Apoptosis ◽  
Endothelial Cell Proliferation ◽  
Dependent Manner ◽  
High Molecular Weight Kininogen ◽  
Endothelial Cell Apoptosis ◽  
Domain 3

We recently reported that the two-chain form of human high molecular weight kininogen (HKa) inhibits angiogenesis by inducing endothelial cell apoptosis (Zhang et al. 2000). This property appears to be primarily conferred by HKa domain 5 (HKa D5). In this manuscript, we further characterize the activity of these polypeptides toward proliferating endothelial cells, as well as their in vivo anti-angiogenic activity in the chick chorioallantoic membrane (CAM). We also demonstrate that short peptides derived from endothelial cell binding regions in HKa domains 3 and 5 inhibit endothelial cell proliferation and induce endothelial cell apoptosis. Like HKa and HKa D5, peptides derived from the latter domain induce endothelial cell apoptosis in a Zn2+-dependent manner, while those derived from domain 3 function independently of Zn2+. The implications of these findings to the regulation of angiogenesis and development of anti-angiogenic therapeutics are discussed.Key words: angiogenesis, kininogen, endothelial cells, apoptosis, peptides.

scholarly journals Endothelial-cell apoptosis induced by cleaved high-molecular-weight kininogen (HKa) is matrix dependent and requires the generation of reactive oxygen species

Blood ◽  
2006 ◽  
Vol 107 (12) ◽  
pp. 4714-4720 ◽  
Author(s):  
Danyu Sun ◽  
Keith R. McCrae
Keyword(s):  
Reactive Oxygen Species ◽  
Molecular Weight ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
High Molecular Weight ◽  
Cell Apoptosis ◽  
Oxygen Species ◽  
High Molecular Weight Kininogen ◽  
Endothelial Cell Apoptosis ◽  
Cells Cultured

AbstractHigh–molecular-weight kininogen (HK) is an abundant plasma protein that plays a central role in activation of the kallikrein-kinin system. Cleavage of HK by plasma kallikrein results in release of the nonapeptide bradykinin (BK), leaving behind cleaved high–molecular-weight kininogen (HKa). Previous studies have demonstrated that HKa induces apoptosis of proliferating endothelial cells and inhibits angiogenesis in vivo, activities mediated primarily through its domain 5. However, the mechanisms by which these effects occur are not well understood. Here, we demonstrate that HKa induces apoptosis of endothelial cells cultured on gelatin, vitronectin, fibronectin, or laminin but not collagen type I or IV. The ability of HKa to induce endothelial-cell apoptosis is dependent on the generation of intracellular reactive oxygen species and associated with depletion of glutathione and peroxidation of endothelial-cell lipids, effects that occur only in cells cultured on matrix proteins permissive for HKa-induced apoptosis. Finally, the ability of HKa to induce endothelial-cell apoptosis is blocked by the addition of reduced glutathione or N-acetylcysteine. These studies demonstrate a unique role for oxidant stress in mediating the activity of an antiangiogenic polypeptide and highlight the importance of the extracellular matrix in regulating endothelial-cell survival.

Definition of the Apoptotic Pathway Induced in Proliferating Endothelial Cells by Cleaved High Molecular Weight Kininogen.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3937-3937
Author(s):  
Danyu Sun ◽  
Keith R. McCrae
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cytochrome C ◽  
Broad Spectrum ◽  
Cell Apoptosis ◽  
High Molecular Weight Kininogen ◽  
Endothelial Cell Apoptosis ◽  
Apoptotic Pathway ◽  
Induced Apoptosis

Abstract Previous studies from our laboratory and others have demonstrated that cleaved high molecular weight kininogen (HKa) induces selective apoptosis of proliferating endothelial cells and inhibits angiogenesis. Recently we reported that the induction of endothelial cell apoptosis by HKa was matrix dependent and requires the generation of reactive oxygen species. To further define the mechanisms of HKa-induced endothelial cell apoptosis, we have investigated the pathway through which apoptosis occurs. HKa-induced endothelial cell apoptosis was caspase-dependent, as demonstrated by the cleavage of pro-caspase 3 and the inhibition of apoptosis by the broad spectrum caspase inhibitor z-VAD-fmk. Activation of the intrinsic pathway of apoptosis was also confirmed by demonstrating cleavage of pro-caspase 9 as well as the release of cytochrome C from mitochondria. These results are consistent with our previous work, as apoptosis induced through oxidative stress is a well known activator of the intrinsic apoptotic pathway. Given the resistance to HKa-induced endothelial cell apoptosis conferred by culture of endothelial cells on collagen I or IV, we questioned whether MMPs may be involved in this process. Interestingly, we observed that the broad spectrum MMP inhibitor GM6001 completely protected endothelial cells from HKa-induced apoptosis. Moreover, we observed that HKa-induced apoptosis was associated with PECAM-1 cleavage within 1 hour of exposure, and that inhibition of apoptosis by either GM6001 or z-VAD-fmk also inhibited the cleavage of PECAM-1. These findings are likely to be relevant to the apoptotic pathway, as intact PECAM-1 has been previously reported to inhibit cytochrome c release after exposure of cells to cytotoxic stimuli, while a cleaved cytoplasmic domain of PECAM-1 enhances the susceptibility of cells to apoptosis. In summary, our studies demonstrate that the antiangiogenic activity of HKa may be critically dependent on MMP activation, which in turn leads to ROS generation and caspase activation. The subsequent cleavage of PECAM-1, presumably by caspases, may be another important event in HKa-induced apoptosis.

Interaction of high molecular weight kininogen binding proteins on endothelial cells

2004 ◽  
Vol 91 (01) ◽  
pp. 61-70 ◽  
Author(s):  
Baby Tholanikunnel ◽  
Berhane Ghebrehiwet ◽  
Allen Kaplan ◽  
Kusumam Joseph
Keyword(s):  
Molecular Weight ◽  
Endothelial Cell ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Surface Proteins ◽  
Factor Xii ◽  
High Molecular Weight Kininogen ◽  
Dot Blot ◽  
Cell Plasma ◽  
Molecular Weight Peak

SummaryCell surface proteins reported to participate in the binding and activation of the plasma kinin-forming cascade includes gC1qR, cytokeratin 1 and u-PAR. Each of these proteins binds high molecular weight kininogen (HK) as well as Factor XII. The studies on the interaction of these proteins, using dot-blot analysis, revealed that cytokeratin 1 binds to both gC1qR and u-PAR while gC1qR and u-PAR do not bind to each other. The binding properties of these proteins were further analyzed by gel filtration. When biotinylated cytokeratin 1 was incubated with either gC1qR or u-PAR and gel filtered, a new, higher molecular weight peak containing biotin was observed indicating complex formation. The protein shift was also similar to the biotin shift. Further, immunoprecipitation of solubilized endothelial cell plasma membrane proteins with anti-gC1qR recovered both gC1qR and cytokeratin 1, but not u-PAR. Immunoprecipitation with anti-u-PAR recovered only u-PAR and cytokeratin 1. By competitive ELISA, gC1qR inhibits u-PAR from binding to cytokeratin 1; u-PAR inhibits gC1qR binding to a lesser extent and requires a 10-fold molar excess. Our data suggest that formation of HK (and Factor XII) binding sites along endothelial cell membranes consists of bimolecular complexes of gC1qR-cytokeratin 1 and u-PAR-cytokeratin 1, with gC1qR binding being favored.

scholarly journals Interaction of high-molecular-weight kininogen with endothelial cell binding proteins suPAR, gC1qR and cytokeratin 1 determined by Surface Plasmon Resonance (BiaCore)

2011 ◽  
Vol 105 (06) ◽  
pp. 1053-1059 ◽  
Author(s):  
Berhane Ghebrehiwet ◽  
Kusumam Joseph ◽  
Alice Kao ◽  
Khalil Bdeir ◽  
Douglas Cines ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Surface Plasmon Resonance ◽  
Endothelial Cell ◽  
High Molecular Weight ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Binding Proteins ◽  
Binding Protein ◽  
High Molecular Weight Kininogen ◽  
Cell Binding

SummaryThe physiologic activation of the plasma kallikrein-kinin system requires the assembly of its constituents on a cell membrane. High-molecular-weight kininogen (HK) and cleaved HK (HKa) both interact with at least three endothelial cell binding proteins: urokinase plasminogen activator receptor (uPAR), globular C1q receptor (gC1qR,) and cytokeratin 1 (CK1). The affinity of HK and HKa for endothelial cells are KD=7–52 nM. The contribution of each protein is unknown. We examined the direct binding of HK and HKa to the soluble extracellular form of uPAR (suPAR), gC1qR and CK1 using surface plasmon resonance. Each binding protein linked to a CM-5 chip and the association, dissociation and KD (equilibrium constant) were measured. The interaction of HK and HKa with each binding protein was zinc-dependent. The affinity for HK and HKa was gC1qR>CK1>suPAR, indicating that gC1qR is dominant for binding. The affinity for HKa compared to HK was the same for gC1qR, 2.6-fold tighter for CK1 but 53-fold tighter for suPAR. Complex between binding proteins was only observed between gC1qR and CK1 indicating that a binary CK1-gC1qR complex can form independently of kininogen. Although suPAR has the weakest affinity of the three binding proteins, it is the only one that distinguished between HK and HKa. This finding indicates that uPAR may be a key membrane binding protein for differential binding and signalling between the cleaved and uncleaved forms of kininogen. The role of CK1 and gC1qR may be to initially bind HK to the membrane surface before productive cleavage to HKa.

CULTURED HUMAN ENDOTHELIAL CELLS BIND AND INTERNALIZE HIGH MOLECULAR WEIGHT KININOGEN

1987 ◽  
Author(s):  
Freek van Iwaarden ◽  
G Philip ◽  
de Groot ◽  
Bonno N Bouma
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Surface ◽  
High Molecular Weight ◽  
Binding Sites ◽  
High Molecular Weight Kininogen ◽  
Human Endothelial Cells ◽  
Endothelial Cell Surface ◽  
Coagulation Pathway

The presence of High Molecular Weight kininogen (HMWK) was demonstrated in cultured human endothelial cells (EC) by immunofluorescence techniques. Using an enzyme linked immunosorbent assay a concentration of 58 ng HMWK/10 cells was determined. Immunoprecipitation studies performed with lysed metabolically labelled endothelial cells and mono-specific antisera directed against HMWK suggested that HMWK is not synthesized by the endothelial cells. Endothelial cells cultured in the presence of HMWK-depleted serum did not contain HMWK. This, suggests that endothelial cells can internalize HMWK. Using 125I-HMWK it was demonstrated that cultured endothelial cells bind HMWK in a time-dependent, specific and saturable.way. The cells were found to internalize 125I-HMWK, since I-HMWK was detected in solubilized endothelial cells after the cell bound 125I-HMWK had been eluted with dextran sulphate.The binding of I-HMWK required the presence of zinc ions. Optimal binding of 125I-HMWK was observed at 50 μM Zn++ . Calcium ions inhibited the Zn++ dependent binding of 125I-HMWK |25EC. In the presence of 3 mM CaCl2 the total binding of 125I-HMWK was significantly decreased, and a .concentration of 200 μM Zn++ was Required for the binding of 125I-HMWK to thecells. Higher,. Ca concentrations did not further decrease the binding of 125I-HMWK. Analysis of tl^e binding data by the ligand computer program indicated 3.2 x 10 binding sites per cell for HMWK with a Kd of 35 nM at 50 μM ZnCl2 and 1 mM CaCl2. Specify binding of HMWK did also occur at physiological plasma Zn++ concentrations. Half maximal binding was observed at HMWK concentrations of ± 105 nM at 10 μM ZnCl2 and 45 nM at 25 μM ZnCl2. The HMWK binding sites were saturatecT at HMWK concentrations of 130 nM with 1.6 x 10 molecules of HMWK bound per cell and at 80 nM with 2.8 x 10 molecules of HMWK bound per cell at 10 and 25 pM ZnCl2 respectively. These results suggest that at physiological zinc, calcium and HMWK concentrations the HMWK binding sites on the endothelial cell are saturated. The presence of HMWK on the endothelial cell surface may play a role in the initiation of the intrinsic coagulation pathway. M ZnCl2 and 45 nM at 25 μM ZnCl2. The HMWK binding sites were saturatecT at HMWK concentrations of 130 nM with 1.6 x 10 molecules of HMWK bound per cell and at 80 nM with 2.8 x 10 molecules of HMWK bound per cell at 10 and 25 μM ZnCl2 respectively. These results suggest that at physiological zinc, calcium and HMWK concentrations the HMWK binding sites on the endothelial cell are saturated. The presence of HMWK on the endothelial cell surface may play a role in the initiation of the intrinsic coagulation pathway. M ZnCl2 and 45 nM at 25 μM ZnCl2. The HMWK binding sites were saturatecT at HMWK concentrations of 130 nM with 1.6 x 10 molecules of HMWK bound per cell and at 80 nM with 2.8 x 10 molecules of HMWK bound per cell at 10 and 25 μM ZnCl2 respectively. These results suggest that at physiological zinc, calcium and HMWK concentrations the HMWK binding sites on the endothelial cell are saturated. The presence of HMWK on the endothelial cell surface may play a role in the initiation of the intrinsic coagulation pathway. M ZnCl2 and 45 nM at 25 μM ZnCl2. The HMWK binding sites were saturatecT at HMWK concentrations of 130 nM with 1.6 x 10 molecules of HMWK bound per cell and at 80 nM with 2.8 x 10 molecules of HMWK bound per cell at 10 and 25 μM ZnCl2 respectively. These results suggest that at physiological zinc, calcium and HMWK concentrations the HMWK binding sites on the endothelial cell are saturated. The presence of HMWK on the endothelial cell surface may play a role in the initiation of the intrinsic coagulation pathway.M ZnCl2 and 45 nM at 25 μM ZnCl2. The HMWK binding sites were saturatecT at HMWK concentrations of 130 nM with 1.6 x 16 molecules of HMWK bound per cell and at 80 nM with 2.8 x 106 molecules of HMWK bound per cell at 10 and 25 μM ZnCl2 respectively. These results suggest that at physiological zinc, calcium and HMWK concentrations the HMWK binding sites on the endothelial cell are saturated. The presence of HMWK on the endothelial cell surface may play a role in the initiation of the intrinsic coagulation pathway.

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