Plasma Kallikrein-kinin System Research Articles

Kallikrein-kinin in stem cell therapy.

The tissue kallikrein-kinin system exerts a wide spectrum of biological activities in the cardiovascular, renal and central nervous systems. Tissue kallikrein-kinin modulates the proliferation, viability, mobility and functional activity of certain stem cell populations, namely mesenchymal stem cells (MSCs), endothelial progenitor cells (EPCs), mononuclear cell subsets and neural stem cells. Stimulation of these stem cells by tissue kallikrein-kinin may lead to protection against renal, cardiovascular and neural damage by inhibiting apoptosis, inflammation, fibrosis and oxidative stress and promoting neovascularization. Moreover, MSCs and EPCs genetically modified with tissue kallikrein are resistant to hypoxia- and oxidative stress-induced apoptosis, and offer enhanced protective actions in animal models of heart and kidney injury and hindlimb ischemia. In addition, activation of the plasma kallikrein-kinin system promotes EPC recruitment to the inflamed synovium of arthritic rats. Conversely, cleaved high molecular weight kininogen, a product of plasma kallikrein, reduces the viability and vasculogenic activity of EPCs. Therefore, kallikrein-kinin provides a new approach in enhancing the efficacy of stem cell therapy for human diseases.

Lack of Plasma Kallikrein-Kinin System Cascade in Teleosts

The kallikrein-kinin system (KKS) consists of two major cascades in mammals: “plasma KKS” consisting of high molecular-weight (HMW) kininogen (KNG), plasma kallikrein (KLKB1), and bradykinin (BK); and “tissue KKS” consisting of low molecular-weight (LMW) KNG, tissue kallikreins (KLKs), and [Lys0]-BK. Some components of the KKS have been identified in the fishes, but systematic analyses have not been performed, thus this study aims to define the KKS components in teleosts and pave a way for future physiological and evolutionary studies. Through a combination of genomics, molecular, and biochemical methods, we showed that the entire plasma KKS cascade is absent in teleosts. Instead of two KNGs as found in mammals, a single molecular weight KNG was found in various teleosts, which is homologous to the mammalian LMW KNG. Results of molecular phylogenetic and synteny analyses indicated that the all current teleost genomes lack KLKB1, and its unique protein structure, four apple domains and one trypsin domain, could not be identified in any genome or nucleotide databases. We identified some KLK-like proteins in teleost genomes by synteny and conserved domain analyses, which could be the orthologs of tetrapod KLKs. A radioimmunoassay system was established to measure the teleost BK and we found that [Arg0]-BK is the major circulating form instead of BK, which supports that the teleost KKS is similar to the mammalian tissue KKS. Coincidently, coelacanths are the earliest vertebrate that possess both HMW KNG and KLKB1, which implies that the plasma KKS could have evolved in the early lobe-finned fish and descended to the tetrapod lineage. The co-evolution of HMW KNG and KLKB1 in lobe-finned fish and early tetrapods may mark the emergence of the plasma KKS and a contact activation system in blood coagulation, while teleosts may have retained a single KKS cascade.

Discovery and Characterization Of a Highly Specific Antibody Inhibitor Of Plasma Kallikrein

The plasma-kallikrein kinin (contact) system contributes to the physiological and pathophysiological reactions of vascular biology. Activation of this pathway causes the release of the potent nonapeptide vasodilator bradykinin following proteolytic cleavage of high-molecular weight kininogen (HMWK) by the serine protease plasma kallikrein (pKal). Normal vascular homeostasis requires regulation of pKal activity by interactions with the C1-inhibitor (C1-INH). This is most apparent in individuals with hereditary angioedema (HAE), a disease characterized by a genetic deficiency in C1-INH that results in persistent pKal activity and consequent bradykinin release. These events can ultimately manifest as unpredictable and potentially fatal attacks of subcutaneous and mucosal edema. Inhibition of pKal proteolytic activity has proven to be a viable therapeutic option for HAE, however there remains an unmet medical need for a long-lasting prophylactic treatment for this disease. Given the potential for target specificity and long serum half-life with antibody therapeutics, we used phage display to select a fully human antibody inhibitor (DX-2930) specific for pKal. In vitro enzyme inhibition and affinity assays demonstrate that DX-2930 is a potent antibody inhibitor of pKal (Ki = 125 pM) that binds the active form of pKal, but not the proenzyme form (prekallikrein) or any other serine protease tested. DX-2930 binding consequently prohibits pKal from cleaving bradykinin out of HMWK and thereby prevents the activation of the bradykinin receptor B2. A 2.1Å resolution X-ray crystallographic structure of pKal complexed to a DX-2930 Fab construct supports these findings, demonstrating that the pKal proteolytic active site is intimately bound - and thereby occluded - by the Fab. This structural analysis provides both a rationale for the potency and specificity of DX-2930, and demonstrates the utility of using antibodies to specifically target an antigen among a family of related proteins (e.g. serine proteases). To further address the functional activity of DX-2930, we demonstrate that subcutaneous dosing of DX-2930 effectively reduces carrageenan-induced paw edema in vivo in rats when injected 24 hours prior to challenge. Combined with our finding that DX-2930 has a prolonged serum residence time in cynomolgus monkeys (t1/2 = 301 hours, SC), the data presented here demonstrates the potential of DX-2930 for the prophylactic inhibition of pKal-mediated diseases, such as HAE. Disclosures:Kenniston:Dyax Corp: Employment. Sexton:Dyax Corp: Employment. Martik:Dyax Corp: Employment, former employee of Dyax Corp Other. Faucette:Dyax Corp: Employment. Viswanathan:Dyax Corp: Employment. Kastrapeli:Dyax: Employment. Kopacz:Dyax Corp: Employment. Conley:Dyax Corp: Employment. Lindberg:Dyax Corp: Employment. Cosic:Dyax Corp: Employment. Comeau:Dyax Corp: Employment. Mason:Dyax Corp: Employment. DiLeo:Dyax Corp: Employment. Chen:Dyax Corp: Employment. Ladner:Dyax Corp: Employment. Edwards:Emerald Biostructures: Employment. TenHoor:Dyax Corp: Employment. Nixon:Dyax Corp: Employment. Adelman:Dyax Corp: Employment.

Endothelial Cell Permeability during Hantavirus Infection Involves Factor XII-Dependent Increased Activation of the Kallikrein-Kinin System

Hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) are diseases caused by hantavirus infections and are characterized by vascular leakage due to alterations of the endothelial barrier. Hantavirus-infected endothelial cells (EC) display no overt cytopathology; consequently, pathogenesis models have focused either on the influx of immune cells and release of cytokines or on increased degradation of the adherens junction protein, vascular endothelial (VE)-cadherin, due to hantavirus-mediated hypersensitization of EC to vascular endothelial growth factor (VEGF). To examine endothelial leakage in a relevant in vitro system, we co-cultured endothelial and vascular smooth muscle cells (vSMC) to generate capillary blood vessel-like structures. In contrast to results obtained in monolayers of cultured EC, we found that despite viral replication in both cell types as well as the presence of VEGF, infected in vitro vessels neither lost integrity nor displayed evidence of VE-cadherin degradation. Here, we present evidence for a novel mechanism of hantavirus-induced vascular leakage involving activation of the plasma kallikrein-kinin system (KKS). We show that incubation of factor XII (FXII), prekallikrein (PK), and high molecular weight kininogen (HK) plasma proteins with hantavirus-infected EC results in increased cleavage of HK, higher enzymatic activities of FXIIa/kallikrein (KAL) and increased liberation of bradykinin (BK). Measuring cell permeability in real-time using electric cell-substrate impedance sensing (ECIS), we identified dramatic increases in endothelial cell permeability after KKS activation and liberation of BK. Furthermore, the alterations in permeability could be prevented using inhibitors that directly block BK binding, the activity of FXIIa, or the activity of KAL. Lastly, FXII binding and autoactivation is increased on the surface of hantavirus-infected EC. These data are the first to demonstrate KKS activation during hantavirus infection and could have profound implications for treatment of hantavirus infections.

Both plasma kininogen and bradykinin receptors are required for collagen antibody-induced arthritis (P4079)

Abstract Although experimental and clinical studies have suggested that plasma kallikrein-kinin system (KKS) is involved in arthritis, their role in the disease pathogenesis has never been studied using gene deficient mouse model. High molecular weight kininogen (HK) is a major component of KKS, the activation of which induces HK cleavage to generate proinflammatory bradykinin (BK). In mice, kininogen-1 (Kng1) gene in liver drives production of HK in plasma. We generated a Kng1-/- mouse strain whose liver Kng1 mRNA and plasma HK protein were absent. To induce collagen antibody-induced arthritis (CAIA), three groups of mice (WT, Kng1-/-, and B2RB1R-/- that lack double BK receptors) were injected I.P. with anti-type II collagen antibody cocktail (3 mg) on day 0 and LPS (50 μg) on day 3. WT mice started to develop joint swelling and erythema on day 5, and the increase in ankle joint diameter became marked on day 6 (1.32±0.13 mm) and peaked on day 8 (1.41±0.17 mm). As detected by ELISA, plasma levels of IL-1β were increased on day 8 than day 0 (p<0.01). Real time PCR indicated that IL-1β mRNA in monocytes of WT mice was increased by 3 fold. In contrast, either Kng1-/- or B2RB1R-/- mice did not develop marked joint swelling, their plasma IL-1β and monocytic IL-1β mRNA levels were not significantly increased. Our results demonstrate an essential role of HK in the pathogenesis of arthritis, BK serving as a key mediator. Blockade of HK cleavage has a potential for anti-arthritic treatment.

Activation of plasma kallikrein‐kinin system mediates synovial recruitment of endothelial progenitor cells in arthritis

OBJECTIVEThis study aimed at determining whether activation of the plasma kallikrein‐kinin system (KKS) mediates synovial recruitment of endothelial progenitor cells (EPCs) in arthritis.METHODSEPCs were isolated from Lewis rat bone marrow, and expression of progenitor cell‐lineage markers and functional properties were characterized. EPCs were injected intravenously into Lewis rats with arthritis, and their recruitment and formation of de novo blood vessels in inflamed synovium were evaluated. The role of plasma KKS was examined using plasma kallikrein inhibitors. A transendothelial migration assay was used to determine the role of bradykinin and its receptor in EPC mobilization.RESULTSEPCs from Lewis rats exhibited a strong capacity to form tubes and vacuoles and expressed increased levels of bradykinin type 2 receptor (B2R) and progenitor cell markers CD34 and Sca‐1. In Lewis rats with arthritis, EPCs were recruited into inflamed synovium at the acute phase of disease and formed de novo blood vessels. Inhibition of plasma kallikrein significantly suppressed synovial recruitment of EPCs and hyperproliferation of synovial cells. Bradykinin stimulated transendothelial migration of EPCs in a concentration‐dependent manner. This was mediated by B2R, as demonstrated by the finding that knockdown of B2R with silencing RNA completely blocked bradykinin‐stimulated transendothelial migration. Moreover, bradykinin selectively up‐regulated expression of the homing receptor CXCR4 in EPCs.CONCLUSIONThese observations demonstrate a novel role of plasma KKS activation in the synovial recruitment of EPCs in arthritis, acting via kallikrein activation and B2R‐dependent mechanisms. B2R might be involved in the mobilization of EPCs via up‐regulation of CXCR4.

Plasma kallikrein-kinin system and diabetic retinopathy

Diabetic retinopathy (DR) occurs, to some extent, in most people with at least 20 years' duration of diabetes mellitus. The progression of DR to its sight-threatening stages is usually associated with the worsening of underlying retinal vascular dysfunction and disease. The plasma kallikrein-kinin system (KKS) is activated during vascular injury, where it mediates important functions in innate inflammation, blood flow, and coagulation. Recent findings from human vitreous proteomics and experimental studies on diabetic animal models have implicated the KKS in contributing to DR. Vitreous fluid from people with advanced stages of DR contains increased levels of plasma KKS components, including plasma kallikrein (PK), coagulation factor XII, and high-molecular-weight kininogen. Both bradykinin B1 and B2 receptor isoforms (B1R and B2R, respectively) are expressed in human retina, and retinal B1R levels are increased in diabetic rodents. The activation of the intraocular KKS induces retinal vascular permeability, vasodilation, and retinal thickening, and these responses are exacerbated in diabetic rats. Preclinical studies have shown that the administration of PK inhibitors and B1R antagonists to diabetic rats ameliorates retinal vascular hyperpermeability and inflammation. These findings suggest that components of plasma KKS are potential therapeutic targets for diabetic macular edema.

Cleaved High Molecular Weight Kininogen Induces Endothelial Progenitor Cell Senescence by Stimulation of JNK/FOXO4/MnSOD Pathway.

Abstract 2181 Objectives:High molecular weight kininogen (HK) is a major component of plasma kallikrein-kinin system (KKS), and is cleaved to its active form (HKa) upon the activation of this system. Although the KKS activation is widely involved in a variety of pathological settings, the activities of its activation product HKa remain largely unknown. Recently we have reported that HKa accelerates the onset of endothelial progenitor cell (EPC) senescence by induction of reactive oxygen species (Arterioscler Thromb Vasc Biol. 2011;31:883), whereas the mechanism is not clear. In this study, we investigated whether HKa induces EPC senescence via stimulation of c-Jun N-terminal kinases (JNK)-related pathway. Methods:Human EPCs were treated with HKa in different concentrations for 72 hours, which were followed by the measurement of phospharylation of JNK at Thr183/tyr185 and transcription factor FOXO4 at Thr451, as well as expression of Mn-superoxide dismutase (MnSOD) at protein and mRNA level. To narrow down the functional domain of HKa, recombinant proteins of human HK heavy chain (HC, 19–380aa) and light chain (LC, 390–644aa) were generated for determining which domain(s) mediates the effect of HKa. Results:In a concentration-dependent manner, HKa treatment induced phosphorylation of JNK at Thr183/Tyr185, and its downstream phosphorylation of transcription factor FOXO4 at Thr451. Concomitantly, HKa upregulated the expression of MnSOD at protein and mRNA levels as measured by Western blot and real time RT-PCR. However, it did not affect the expression of catalase. Similar to HKa, the heavy chain (HC), but not the light chain, increased the percentage of senescent EPCs (63%±6.6 for HC v.s. 77.5%±6.02 for HKa). Moreover, HC at 100 nM increased FOXO4 phosphorylation at Thr451 and the expression of MnSOD in EPCs. Besides, both of HKa and its HC stimulated the production of intracellular H2O2. Conclusion:These above results demonstrate that HKa accelerates the onset of EPC senescence by stimulating JNK/FOXO4/MnSOD pathway, and its effect is mediated by the HC. Disclosures:No relevant conflicts of interest to declare.

Role of plasma kallikrein–kinin system activation in synovial recruitment of endothelial progenitor cells in experimental arthritis

To examine whether activation of the plasma kallikrein-kinin system (KKS) mediates synovial recruitment of endothelial progenitor cells (EPCs) in arthritis. EPCs were isolated from Lewis rat bone marrow, and expression of progenitor cell-lineage markers and functional properties were characterized. EPCs were injected intravenously into Lewis rats with arthritis, and their recruitment and formation of de novo blood vessels in inflamed synovium were evaluated. The role of plasma KKS was examined using a plasma kallikrein inhibitor (EPI-KAL2) and an antikallikrein antibody (13G11). A transendothelial migration assay was used to determine the role of bradykinin and its receptor in EPC mobilization. EPCs from Lewis rats exhibited a strong capacity to form tubes and vacuoles and expressed increased levels of bradykinin type 2 receptor (B2R) and progenitor cell markers CD34 and Sca-1. In Lewis rats with arthritis, EPCs were recruited into inflamed synovium at the acute phase of disease and formed de novo blood vessels. Inhibition of plasma kallikrein by EPI-KAL2 and 13G11 significantly suppressed synovial recruitment of EPCs and hyperproliferation of synovial cells. Bradykinin stimulated transendothelial migration of EPCs in a concentration-dependent manner. This was mediated by B2R, as demonstrated by the finding that knockdown of B2R with silencing RNA completely blocked bradykinin-stimulated transendothelial migration. Moreover, bradykinin selectively up-regulated expression of the homing receptor CXCR4 in EPCs. These observations demonstrate a novel role of plasma KKS activation in the synovial recruitment of EPCs in arthritis, acting via kallikrein activation and B2R-dependent mechanisms. B2R might be involved in the mobilization of EPCs via up-regulation of CXCR4.

Hereditary angioedema: Classification, pathogenesis, and diagnosis

Hereditary angioedema (HAE) is a rare autosomal dominant genetic disorder associated with a deficiency in C1 inhibitor. More than 200 mutations in this gene, located on chromosome 11, have been identified. Although HAE is often inherited, 20-25% of cases are from new spontaneous mutations and they have no family history of swelling. Decreased C1 inhibitor activity leads to inappropriate activation of multiple pathways, including the complement and contact systems and the fibrinolysis and coagulation systems. Reduced C1 inhibitor activity results in increased activation of plasma kallikrein-kinin system proteases and increased bradykinin levels. Bradykinin is felt to be the main mediator of symptoms in HAE. Patients with HAE have recurrent episodes of swelling of the extremities, abdomen, face, and upper airway. Angioedema involving the gastrointestinal tract can lead to intestinal wall edema, which results in abdominal pain, nausea, vomiting, and diarrhea. Laryngeal swelling is life-threatening and may lead to asphyxia. Common triggers of an attack include trauma, stress, infection, menstruation, oral contraceptives, hormone replacement therapy, and angiotensin-converting enzyme inhibitors. Laboratory testing including C4, C1 inhibitor level, and function is needed to confirm or rule out the diagnosis of HAE. The treatment of HAE has improved significantly in recent years with the availability of several safe and effective therapies. Several consensus guidelines have been created to further assist in the management of HAE patients. This review will provide an update on the classification, pathophysiology, clinical presentation, and diagnosis of HAE.

Heparin affects the interaction of kininogen on endothelial cells

In the plasma kallikrein-kinin system, it has been shown that when plasma prekallikrein (PK) and high molecular weight kininogen (HK) assemble on endothelial cells, plasma kallikrein (huPK) becomes available to cleave HK, releasing bradykinin, a potent mediator of the inflammatory response. Because the formation of soluble glycosaminoglycans occurs concomitantly during the inflammatory processes, the effect of these polysaccharides on the interaction of HK on the cell surface or extracellular matrix (ECM) of two endothelial cell lines (ECV304 and RAEC) was investigated. In the presence of Zn+2, HK binding to the surface or ECM of RAEC was abolished by heparin; reduced by heparan sulfate, keratan sulfate, chondroitin 4-sulfate or dermatan sulfate; and not affected by chondroitin 6-sulfate. By contrast, only heparin reduced HK binding to the ECV304 cell surface or ECM. Using heparin-correlated molecules such as low molecular weight dextran sulfate, low molecular weight heparin and N-desulfated heparin, we suggest that these effects were mainly dependent on the charge density and on the N-sulfated glucosamine present in heparin. Surprisingly, PK binding to cell- or ECM-bound-HK and PK activation was not modified by heparin. However, the hydrolysis of HK by huPK, releasing BK in the fluid phase, was augmented by this glycosaminoglycan in the presence of Zn2+. Thus, a functional dichotomy exists in which soluble glycosaminoglycans may possibly either increase or decrease the formation of BK. In conclusion, glycosaminoglycans that accumulated in inflammatory fluids or used as a therapeutic drug (e.g., heparin) could act as pro- or anti-inflammatory mediators depending on different factors within the cell environment.

Mesothelial cells activate the plasma kallikrein-kinin system during pleural inflammation

Abstract Pleural inflammation underlies the formation of most exudative pleural effusions and the plasma kallikrein-kinin system (KKS) is known to contribute. Mesothelial cells are the predominant cell type in the pleural cavity, but their potential role in plasma KKS activation and BK production has not been studied. Bradykinin concentrations were higher in pleural fluids than the corresponding serum samples in patients with a variety of diseases. Bradykinin concentrations did not correlate with disease diagnosis, but were elevated in exudative effusions. It was demonstrated, using a range of primary and transformed mesothelial and mesothelioma cell lines, that cells assembled high molecular weight kininogen and plasma prekallikrein to liberate bradykinin, a process inhibited by novobiocin, a heat shock protein 90 (HSP90) inhibitor, cysteine, bradykinin and protamine sulphate. Of the common plasma prekallikrein activators, mesothelial cells expressed HSP90, but not prolylcarboxypeptidase or Factor XII. Calcium mobilisation was induced in some mesothelium-derived cell lines by bradykinin. Des-Arg(9)-bradykinin was inactive, indicating that mesothelial cells are responsive to bradykinin, mediated via the bradykinin receptor subtype 2. In summary, pleural mesothelial cells support the assembly and activation of the plasma KKS by a mechanism dependent on HSP90, and may contribute to KKS-mediated inflammation in pleural disease.

Cleaved High-Molecular-Weight Kininogen Accelerates the Onset of Endothelial Progenitor Cell Senescence by Induction of Reactive Oxygen Species

Cleaved high-molecular-weight kininogen (HKa), an activation product of the plasma kallikrein-kinin system, inhibits endothelial cell functions. We questioned whether HKa affects the function of endothelial progenitor cells (EPCs) and accelerates their senescence. Treatment with HKa for 2 weeks markedly inhibited the formation of large colonies and proliferation of EPCs on collagen surfaces, whereas HKa did not affect collagen-mediated EPC adhesion and survival. Concomitantly, treated EPCs displayed flattened and giant cell morphological changes and formation of intracellular vacuoles. As determined by acidic β-galactosidase staining, HKa increased senescent EPCs by 2- and >3-fold after culture for 1 and 2 weeks, respectively. In addition, HKa suppressed the telomerase activity of EPCs. HKa concentration-dependently increased the generation of intracellular reactive oxygen species (ROS) and markedly upregulated p38 kinase phosphorylation and prosenescence molecule p16(INK4a) expression. SB203580, a p38 inhibitor, attenuated the level of HKa-enhanced p16(INK4a) expression. Either quenching of ROS or inhibition of p38 kinase prevented HKa-induced EPC senescence. HKa accelerates the onset of EPC senescence by activating the ROS-p38 kinase-p16(INK4a) signaling cascade. This novel activity of HKa points out the likelihood of HKa serving as an endogenous inducer of EPC senescence.

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

The 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.

Assessment of activation of the plasma kallikrein-kinin system in frontal and temporal cortex in Alzheimer's disease and vascular dementia

Decreased cerebral blood flow and blood-brain barrier disruption are features of Alzheimer's disease (AD). The plasma kallikrein-kinin system modulates cerebrovascular tone through release of vasoactive bradykinin (BK). Cerebroventricular infusion of Aβ1–40 enhances BK release, suggesting that the activity of this system may be elevated in AD. We investigated the profile of the activating protease of this system, plasma kallikrein (PK), in frontal and temporal brain tissue from postmortem confirmed cases of AD, vascular dementia (VaD), and controls. Measurements of neuron specific enolase messenger ribonucleic acid (mRNA) and protein were used to adjust for neuronal loss. Adjusted PK mRNA was significantly increased in the frontal cortex in AD, and the frontal and temporal cortex in VaD. Similar trends were seen for PK protein level in AD and VaD. PK activity was significantly increased in the frontal and temporal cortex in AD. Increased PK activity in AD is likely to contribute to increased BK release and may thereby influence cerebral blood flow and vascular permeability.

Activation of the plasma kallikrein-kinin system on human lung epithelial cells

Activation of the tissue kallikrein-kinin system (KKS) plays a major inflammatory role in the lung, but the contribution of the plasma KKS remains unclear. Plasma KKS involves assembly and activation of high molecular weight kininogen (HK) and plasma prekallikrein (PPK) on cell surfaces, resulting in the liberation of the inflammatory peptide, bradykinin (BK), from HK by plasma kallikrein (PK). To this end, we determined the possible contribution of plasma KKS in BK formation using airway epithelium. The HK binding proteins, urokinase plasminogen activator receptor, cytokeratin 1 and gC1qR, were expressed on transformed A549 and BEAS-2B cell lines, as well as on normal lung tissue, but Mac-1 was absent. A549 cells bound FITC-labelled HK, which was only partially inhibited by a combination of antibodies to the HK binding proteins. HK-PPK complex activation on the transformed epithelial cell lines, as well as primary epithelial cells, resulted in PK formation and liberation of BK. HK-PPK activation was inhibited by cysteine, BK and protamine, and by novobiocin, a heat shock protein 90 (HSP90) inhibitor. In summary, lung epithelial cells support the assembly and activation of the plasma KKS by a mechanism dependent on HSP90, and could contribute to KKS-mediated inflammation in lung disease.

Cleaved high molecular weight kininogen accelerates endothelial progenitor cell senescence via induction of reactive oxygen species

High‐molecular‐weight kininogen (HK) is a component of the plasma kallikrein‐kinin system (KKS) and plays a central role in the activation of this system. Upon cleavage by plasma kellikrein, HK is converted into cleaved form of HK (HKa), which gains a function of antiadhesion and antiangiogenesis. In this study, we further investigated whether HKa regulates the functions of endothelial progenitor cells (EPCs). Our observations demonstrate that HKa at 50 mM markedly inhibited colony formation as well as proliferation of EPCs as assessed by BrdU incorporation assay. This effect did not result from its antiadhesive activity and induction of apoptosis, because HKa failed to inhibit EPC adhesion to collagen surfaces and did not increase the percentage of apoptotic cells. In the absence of HKa, EPCs became slightly senescent as determined by acidic ¦Â‐galactosidase staining. HKa, however, significantly accelerated the onset of EPC senescence. Since reduced form of glutathione, an ROS scavenger, prevented the increase in acidic ¦Â‐galactosidase‐positive cells, HKa acceleration of EPC senescence is through ROS production. Taken together, the present study demonstrates a novel role for HKa in inhibition of EPC function, and suggests the likelihood for HKa serving as endogenous inducer of EPC dysfunction by accelerating the onset of EPC senescence.

The plasma kallikrein-kinin system in patients with spontaneous pneumothorax during rapid reexpansion

The plasma kallikrein-kinin system in patients with spontaneous pneumothorax during rapid reexpansion

Cleaved high molecular weight kininogen inhibits tube formation of endothelial progenitor cells via suppression of matrix metalloproteinase 2.

Endothelial progenitor cells (EPCs) contribute to postnatal neovascularization, thus promoting wide interest in their therapeutic potential in vascular injury and prevention of their dysfunction in cardiovascular diseases. Cleaved high molecular weight kininogen (HKa), an activation product of the plasma kallikrein-kinin system (KKS), inhibits the functions of differentiated endothelial cells including in vitro and in vivo angiogenesis. In this study, our results provided the first evidence that HKa is able to target EPCs and inhibits their tube forming capacity. We determined the effect of HKa on EPCs using a three-dimensional vasculogenesis assay. Upon stimulation with vascular endothelial growth factor (VEGF) alone, EPCs formed vacuoles and tubes, and differentiated into capillary-like networks. As detected by gelatinolytic activity assay, VEGF stimulated secretion and activation of matrix metallopeptidase 2 (MMP-2), but not MMP-9, in the conditioned medium of 3D culture of EPCs. Specific inhibition or gene ablation of MMP-2, but not MMP-9, blocked the vacuole and tube formation by EPCs. Thus, MMP-2 is selectively required for EPC vasculogenesis. In a concentration-dependent manner, HKa significantly inhibited tube formation by EPCs and the conversion of pro-MMP-2 to MMP-2. Moreover, HKa completely blocked the association between pro-MMP-2 and alphavbeta3 integrin, and its inhibition of MMP-2 activation was dependent on the presence of alphavbeta3 integrin. In a purified system, HKa did not directly inhibit MMP-2 activity. HKa inhibits tube forming capacity of EPCs by suppression of MMP-2 activation, which may constitute a novel link between activation of the KKS and EPC dysfunction.

Human plasma kallikrein-kinin system: physiological and biochemical parameters.

The plasma kallikrein-kinin system (KKS) plays a critical role in human physiology. The KKS encompasses coagulation factor XII (FXII), the complex of prekallikrein (PK) and high molecular weight kininogen (HK). The conversion of plasma prekallikrein to kallikrein by the activated FXII and in response to numerous different stimuli leads to the generation of bradykinin (BK) and activated HK (HKa, an antiangiogenic peptide). BK is a proinflammatory peptide, a pain mediator and potent vasodilator, leading to robust accumulation of fluid in the interstitium. Systemic production of BK, HKa with the interplay between BK bound-BK receptors and the soluble form of HKa are key to angiogenesis and hemodynamics. KKS has been implicated in the pathogenesis of inflammation, hypertension, endotoxemia, and coagulopathy. In all these cases increased BK levels is the hallmark. In some cases, the persistent production of BK due to the deficiency of the blood protein C1-inhibitor, which controls FXII, is detrimental to the survival of the patients with hereditary angioedema (HAE). In others, the inability of angiotensin converting enzyme (ACE) to degrade BK leads to elevated BK levels and edema in patients on ACE inhibitors. Thus, the mechanisms that interfere with BK liberation or degradation would lead to blood pressure dysfunction. In contrast, anti-kallikrein treatment could have adverse effects in hemodynamic changes induced by vasoconstrictor agents. Genetic models of kallikrein deficiency are needed to evaluate the quantitative role of kallikrein and to validate whether strategies designed to activate or inhibit kallikrein may be important for regulating whole-body BK sensitivity.