Domain 5 of high molecular weight kininogen (kininostatin) down-regulates endothelial cell proliferation and migration and inhibits angiogenesis

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 543-550 ◽  
Author(s):  
Robert W. Colman ◽  
Bradford A. Jameson ◽  
Yingzhang Lin ◽  
Donald Johnson ◽  
Shaker A. Mousa
Keyword(s):  
Molecular Weight ◽  
Cell Proliferation ◽  
Cell Migration ◽  
Endothelial Cell ◽  
High Molecular Weight ◽  
Endothelial Cell Migration ◽  
Endothelial Cell Proliferation ◽  
High Molecular Weight Kininogen ◽  
Cell Binding

We have demonstrated that high molecular weight kininogen (HK) binds specifically on endothelial cells to domain 2/3 of the urokinase receptor (uPAR). Inhibition by vitronectin suggests that kallikrein-cleaved HK (HKa) is antiadhesive. Plasma kallikrein bound to HK cleaves prourokinase to urokinase, initiating cell-associated fibrinolysis. We postulated that HK cell binding domains would inhibit angiogenesis. We found that recombinant domain 5 (D5) inhibited endothelial cell migration toward vitronectin 85% at 0.27 μM with an IC50 (concentration to yield 50% inhibition) = 0.12 μM. A D5 peptide, G486-K502, showed an IC50 = 0.2 μM, but a 25-mer peptide from a D3 cell binding domain only inhibited migration 10% at 139 μM (IC50 > 50 μM). D6 exhibited weaker inhibitory activity (IC50 = 0.50 μM). D5 also potently inhibited endothelial cell proliferation with an IC50 = 30 nM, while D3 and D6 were inactive. Using deletion mutants of D5, we localized the smallest region for full activity to H441-D474. To further map the active region, we created a molecular homology model of D5 and designed a series of peptides displaying surface loops. Peptide 440-455 was the most potent (IC50 = 100 nM) in inhibiting proliferation but did not inhibit migration. D5 inhibited angiogenesis stimulated by fibroblast growth factor FGF2 (97%) in a chicken chorioallantoic membrane assay at 270 nM, and peptide 400-455 was also inhibitory (79%). HK D5 (for which we suggest the designation, “kininostatin”) is a potent inhibitor of endothelial cell migration and proliferation in vitro and of angiogenesis in vivo.

Domain 5 of cleaved high molecular weight kininogen inhibits endothelial cell migration through Akt

2005 ◽  
Vol 94 (09) ◽  
pp. 606-614 ◽  
Author(s):  
Vaibhav Katkade ◽  
Abigail A. Soyombo ◽  
Irma Isordia-Salas ◽  
Harlan N. Bradford ◽  
John P. Gaughan ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Signaling Pathways ◽  
High Molecular Weight ◽  
Focal Adhesions ◽  
Akt Signaling ◽  
Pi3 Kinase ◽  
Endothelial Cell Migration ◽  
High Molecular Weight Kininogen

SummaryDomain 5 (D5) of cleaved high molecular weight kininogen (HKa) inhibits angiogenesis in vivo and endothelial cell migration in vitro, but the cell signaling pathways involved in HKa and D5 inhibition of endothelial cell migration are incompletely delineated. This study examines the mechanism of HKa and D5 inhibition of two potent stimulators of endothelial cell migration, sphingosine 1-phosphate (S1P) and vascular endothelial growth factor (VEGF), that act through the PI3-kinase-Akt signaling pathway. HKa and D5 inhibit bovine pulmonary artery endothelial cell (BPAE) or human umbilical vein endothelial cell chemotaxis in the modified-Boyden chamber in response to VEGF or S1P. The inhibition of migration by HKa is reversed by antibodies to urokinase-type plasminogen activator receptor. Both HKa and D5 decrease the speed of BPAE cell migration and alter the morphology in live, time-lapse microscopy after stimulation with S1P or VEGF. HKa and D5 reduce the localization of paxillin to the focal adhesions after S1P and VEGF stimulation. To better understand the intracellular signaling pathways, we examined the effect of HKa on the phosphorylation of Akt and its downstream effector, GSK-3α.HKa and D5 inhibit phosphorylation of Akt and GSK-3α after stimulation withVEGF and S1P. Inhibitors of Akt and PI3-kinase, the upstream activator of Akt, block endothelial cell migration and disrupt paxillin localization to the focal adhesions after stimulation with VEGF and S1P. Therefore we suggest that HKa through its D5 domain alters PI3-kinase- Akt signaling to inhibit endothelial cell migration through alterations in the focal adhesions.

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.

JS-K, a Novel Nitric Oxide (NO) Generator, Shows Potent Anti-Angiogenic Activity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3414-3414 ◽  
Author(s):  
Paul J. Shami ◽  
Gurmeet Kaur ◽  
Jagadambal Thillainathan ◽  
Lee Jia ◽  
Joseph E. Saavedra ◽  
...  
Keyword(s):  
Cell Migration ◽  
Endothelial Cell ◽  
Cell Growth ◽  
Cancer Therapeutics ◽  
Myelogenous Leukemia ◽  
Endothelial Cell Migration ◽  
In Vitro Assays ◽  
Endothelial Cell Proliferation

Abstract NO induces differentiation and apoptosis in Acute Myelogenous Leukemia (AML) cells. Glutathione S-Transferases (GST) play an important role in multidrug resistance and are upregulated in 90% of AML cells. We have designed a novel prodrug class that releases NO on metabolism by GST. O2-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K, a member of this class) has potent antileukemic activity in vitro and in vivo (Molecular Cancer Therapeutics 2:409-417,2003). The purpose of this study was to determine the effect of JS-K on angiogenesis. The anti-angiogenic properties of JS-K were tested in 3 different in vitro assays: proliferation, cord formation (reflecting new vessel formation) and migration using Human Umbilical Vein Endothelial Cells (HUVEC). JS-K inhibited the proliferation of HUVEC’s with a 50% inhibitory concentration (IC50) of 0.432, 0.466, and 0.505 μM at 24, 48, and 72 hours, respectively. At concentrations of 1 μM or above, HUVEC proliferation was totally inhibited. In the cord formation assay, treatment with JS-K lad to a decrease in both the number of cord junctions and cord length with an IC50 of 0.637 and 0.696 μM, respectively. At a concentration of 1 μM, JS-K inhibited cord formation completely. JS-K inhibited cell migration at 5 hours using 10 ng/mL VEGF as a chemoattractant. At that time point, migration inhibition occurred at JS-K concentrations that did not affect cell growth with an IC50 of 0.493 μM. We conclude that JS-K is a potent inhibitor of 3 important elements of angiogenesis, namely endothelial cell proliferation, cord formation, and endothelial cell migration. These experiments identify a new mechanism by which JS-K and similar compounds may inhibit leukemia and solid tumor cell growth in vivo. Determining whether the anti-angiogenic effects of JS-K are NO-dependent will require further studies. (NO1-CO-12400).

ADAMTS13 Promotes Angiogenesis and Modulates VEGF-Mediated Angiogenic Activities

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1145-1145
Author(s):  
Manfai Lee ◽  
Jonathan Baza ◽  
George M. Rodgers
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Endothelial Cell Migration ◽  
Molar Ratio ◽  
Assay Method ◽  
Boyden Chamber ◽  
Dependent Manner

Abstract Abstract 1145 Severe plasma ADAMTS13 deficiency results in the clinical disorder thrombotic thrombocytopenic purpura. However, other potential pathophysiological roles of ADAMTS13 in endothelial cell biology remain unexplored. To assess the possible role of ADAMTS13 in angiogenesis, cell proliferation and migration of human umbilical vein endothelial cells (HUVEC) were studied in vitro. ADAMTS13 was found to be a highly potent chemoattractant, and additionally was capable of neutralizing VEGF activity in two angiogenesis assays-cell proliferation and cell migration. In the Boyden chamber cell migration assay, treatment of endothelial cells with exogenous recombinant ADAMTS13 promoted cell migration in a dose-dependent manner, with 1 ng/mL increasing cell migration across a gelatinized polycarbonate membrane by 14-fold. In the same model, 5 ng/mL VEGF165 (molar ratio of ADAMTS13:VEGF165 = 1/19) only increased cell migration by 7 fold. A steady decrease in endothelial cell migration was observed when the concentration of ADAMTS13 exceeded 1 ng/mL (Figure 1). Coincubation of 30 ng/mL ADAMTS13 with 6.16 ng/mL VEGF165 (molar ratio of ADAMTS13/VEGF165 = 1.3/1) inhibited endothelial cell migration by 45% compared to VEGF alone (Figure 2). A second model using an in vitro scratch-wound assay confirmed the Boyden chamber data. Substitution of ADAMTS13 with ADAM17, an analog of ADAMTS13 without the thrombospondin domain reversed the inhibition of VEGF-mediated cell migration, suggesting that the thrombospondin domain of ADAMTS13 is responsible for the inhibitory interaction with VEGF165. This finding was in agreement with our previously published co-immunoprecipitation assay data (Blood 2010, 116, 4307). Similar patterns of inhibition were observed with VEGF121 and VEGF189, indicating that other isoforms of VEGF may interact with the TSP domain of ADAMTS13. Using a manual proliferation assay method, HUVEC treated with 30 ng/mL ADAMTS13 and 6.16 ng/mL VEGF165 proliferated 40% slower than the control treated with VEGF alone. Combined with our findings on the inhibition of endothelial cell-tube formation in a Matrigel assay with ADAMTS13 and VEGF165 previously reported, our cumulative data suggest that 1) ADAMTS13 promotes angiogenesis by increasing cell migration and 2) ADAMTS13 can modulate VEGF-mediated angiogenic activities. Disclosures: No relevant conflicts of interest to declare.

Covalently grafted VEGF165 in hydrogel models upregulates the cellular pathways associated with angiogenesis

2011 ◽  
Vol 301 (5) ◽  
pp. C1086-C1092 ◽  
Author(s):  
A. M. Porter ◽  
C. M. Klinge ◽  
A. S. Gobin
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Umbilical Vein ◽  
Biological Response ◽  
Biomedical Science ◽  
Endothelial Cell Proliferation ◽  
Peg Hydrogels

Angiogenesis is an important biological response known to be involved in many physiological and pathophysiological situations. Cellular responses involved in the formation of new blood vessels, such as increases in endothelial cell proliferation, cell migration, and the survival of apoptosis-inducing events, have been associated with vascular endothelial growth factor isoform 165 (VEGF165). Current research in the areas of bioengineering and biomedical science has focused on developing polyethylene glycol (PEG)-based systems capable of initiating and sustaining angiogenesis in vitro. However, a thorough understanding of how endothelial cells respond at the molecular level to VEGF165 incorporated into these systems has not yet been established in the literature. The goal of the current study was to compare the upregulation of key intracellular proteins involved in angiogenesis in human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC) seeded on PEG hydrogels containing grafted VEGF165 and adhesion peptides Arg-Gly-Asp-Ser (RGDS). Our data suggest that the covalent incorporation of VEGF165 into PEG hydrogels encourages the upregulation of signaling proteins responsible for increases in endothelial cell proliferation, cell migration, and the survival after apoptosis-inducing events.

scholarly journals Distinct Vegfa isoforms control endothelial cell proliferation through PI3 kinase signalling mediated regulation of cdkn1a/p21

2020 ◽  
Author(s):  
Martin Lange ◽  
Elvin Leonard ◽  
Nils Ohnesorge ◽  
Dennis Hoffmann ◽  
Susana F. Rocha ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Proliferation ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Pi3 Kinase ◽  
Endothelial Cell Migration ◽  
Endothelial Cell Proliferation ◽  
Downstream Signaling ◽  
Matrix Bound ◽  
Cell Migration And Proliferation

SUMMARYThe formation of appropriately patterned blood vessel networks requires endothelial cell migration and proliferation. Signaling through the Vascular Endothelial Growth Factor A (VEGFA) pathway is instrumental in coordinating these processes. mRNA splicing generates short (diffusible) and long (extracellular matrix bound) Vegfa isoforms. The differences between these isoforms in controlling cellular functions are not understood. In zebrafish, vegfaa generates short and long isoforms, while vegfab only generates long isoforms. We found that mutations in vegfaa affected endothelial cell migration and proliferation. Surprisingly, mutations in vegfab specifically reduced endothelial cell proliferation. Analysis of downstream signaling revealed no change in MAPK (ERK) activation, while inhibiting PI3 kinase signaling phenocopied vegfab mutants. The cell cycle inhibitor cdkn1a/p21 was upregulated in vegfab deficient embryos. Accordingly, reducing cdkn1a/p21 restored endothelial cell proliferation. Together, these results suggest that extracellular matrix bound Vegfa acts through PI3K signaling to specifically control endothelial cell proliferation during angiogenesis independently of MAPK (ERK) regulation.

Prolylcarboxypeptidase Promotes Endothelial Cell Proliferation and Vascular Repair

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1142-1142 ◽  
Author(s):  
Gregory N Adams ◽  
Gretchen LaRusch ◽  
Alvin H. Schmaier
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Wound Repair ◽  
Endothelial Cell Migration ◽  
Endothelial Cell Proliferation ◽  
Vascular Health ◽  
Transfected Cells ◽  
Vessel Growth

Abstract Abstract 1142 Background. The S28 serine protease, prolylcarboxypeptidase (PRCP) degrades bradykinin, angiotensin II, alpha melanocyte stimulating hormone and actives plasma prekallikrein. Additionally our studies indicate that PRCP depletions in vivo and in cultured cells are associated with increased reactive oxygen species (ROS) and loss of constitutive anticoagulant function of endothelium (Blood 2011; 117:3929). PRCP-depleted mice are prothrombotic and hypertensive. We observed that PRCP-depleted cells in culture have reduced growth. We posited that PRCP promotes vascular health by influencing cell proliferation, angiogenesis, and wound repair. Methods and Results. Initial investigations determined that PRCP influences vascular endothelial cell proliferation. Bovine aortic endothelial cells (BAEC) were depleted of PRCP by siRNA knockdown resulting in 5% residual mRNA. After transfecting equal numbers of BAEC, at 24 h, the PRCP siRNA transfected cells have reduced proliferation, −18±3 change in cells/high power field (HPF) (mean±SEM), compared to the sham transfected cells, +23±8 cells/HPF, p<0.05. Additionally, PRCP siRNA-treated BAEC demonstrate less proliferation as measured by the MTS assay (Promega) (0.23±0.01 OD490 nm in PRCP-depleted cells vs 0.31±0.01 OD490 nm in sham transfected cells, p<0.02). Alternatively, when BAEC are transfected with full-length PRCP cDNA, at 24 h there is increased proliferation, +58±9 cells/HPF, vs +31±2 of sham-transfected cells, p<0.05. On a BAEC scratch assay, the degree of endothelial cell migration at 5 h in PRCP siRNA-knocked down cells is only 69% of that seen with sham-transfected cells (38±4% scratch coverage in PRCP knockdown BAEC vs 55±5% in sham knockdowns). These combined studies indicate that the content of PRCP in endothelial cells directly correlates with the degree of cell migration and proliferation. Studies next determined the influence of PRCP on angiogenesis. PRCP-depleted mice (PRCPgt/gt) have reduced new vessel growth into sub-cutaneous matrigel plugs containing FGF and VEGF. Matrigel plugs from the PRCP gt/gt mice show 3.5±0.5 Hgb mg/dL/mg-matrigel vs 6.7±1.2 Hgb mg/dL/mg matrigel in plugs in littermate wild type (WT) mice (p<0.03). When sections from the matrigel plugs are stained for the vascular marker CD31, the percent area of new vessels in the PRCPgt/gt (5.5±0.9%), as determined by ImageJ analysis, is significantly less (p<0.04) than that seen (11.9±2.1%) in WT plugs. These data indicate that host PRCP levels influence induced angiogenesis in the whole animal. Additional studies examined if PRCPgt/gt have reduced wound repair angiogenesis. Punch biopsies (5 mm) were performed on PRCPgt/gt. At day 7, no wound healed in PRCPgt/gt but 5/10 wounds healed in WT. The mean size of the PRCPgt/gt wounds is 5.5±1.1 mm2 vs 1.4±0.7 mm2 for WT, p<0.05. Also, at day 7, the wounds of PRCPgt/gt have 11.6±1.0 % area of CD31 stained vessels vs 15.0±1.0 % area of CD31 stained vessels in control wounds, p<0.03. Since there is no difference in the number of vessels in unwounded skin biopsies in PRCPgt/gt vs WT, the reduced vessel growth and delayed wound closure indicates that PRCPgt/gt mice have reduced repair angiogenesis. Conclusions. These combined studies indicate that PRCP levels in endothelial cells influence cell proliferation and growth. In the whole animal this cell biology observation translates into less induced and wound repair angiogenesis. Since PRCP-depleted endothelial cells and vessels from PRCPgt/gt have increased ROS with loss of anticoagulant properties and PRCPgt/gt have higher thrombosis risk, the finding that PRCP also influences endothelial cell growth and angiogenesis suggests that PRCP promotes vascular health and injury repair. Disclosures: No relevant conflicts of interest to declare.

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.

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