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Department of Pharmacology, University of Mississippi, University, Mississippi 38677-1848Light Microscopy Core, University of Mississippi, University, Mississippi 38677-1848Research Institute of Pharmaceutical Sciences, University of Mississippi, University, Mississippi 38677-1848
* This work was supported, in whole or in part, by NCRR/National Institutes of Health Grant P20RR021929 (to Z. S.-M.), National Science Foundation Grant MRI 0619774, and American Heart Association Grant 0330193N.
The nonenzymatic cofactor high molecular weight kininogen (HK) is a precursor of bradykinin (BK). The production of BK from HK by plasma kallikrein has been implicated in the pathogenesis of inflammation and vascular injury. However, the functional role of HK in the absence of prekallikrein (PK), the proenzyme of plasma kallikrein, on vascular endothelial cells is not fully defined. In addition, no clinical abnormality is seen in PK-deficient patients. Therefore, an investigation into the effect of HK, in the absence of PK, on human pulmonary artery endothelial cell (HPAEC) function was performed. HK caused a marked and dose-dependent increase in the intracellular calcium [Ca2+]i level in HPAEC. Gd3+ and verapamil potentiated the HK-induced increase in [Ca2+]i. HK-induced Ca2+ increase stimulated endothelial nitric oxide (NO) and prostacyclin (PGI2) production. The inhibitors of B2 receptor-dependent signaling pathway impaired HK-mediated signal transduction in HPAEC. HK had no effect on endothelial permeability at physiological concentration. This study demonstrated that HK regulates endothelial cell function. HK could play an important role in maintaining normal endothelial function and blood flow and serve as a cardioprotective peptide.
consists of three proenzymes; factor XII (FXII, Hageman factor), prekallikrein (PK, Fletcher factor), and factor XI (FXI, plasma thromboplastin antecedent) as well as one cofactor; high molecular weight kininogen (HK, Fitzgerald factor). KKS is involved in the regulation of hemodynamics, inflammation, complement activation, angiogenesis, thrombosis, and fibrinolysis. Basically, all these proposed roles represent a range of overlapping effects that contribute to various extents toward vasodilation and healing. Therefore, the plasma KKS can be considered to have a spectrum of physiological effects, ranging at one extreme from a hemostatic state of vasodilation and promotion of smooth blood flow, all the way to a prothrombotic state. It is conceivable to suggest that other mechanisms proposed about respiratory, retinal, and renal systems can fit into this spectrum of physiological effects (
). There is accumulating evidence suggesting that when the plasma KKS is activated, the results are a sequential release of proteolytic enzymes and vasoactive peptides, generation of both angiogenic and anti-angiogenic molecules, stabilization of thrombus, and an increase in protease inhibitor activity in blood (
). The activation of HK-PK complex on endothelial cells triggers vasodilation through smooth muscle relaxation, inhibits platelet aggregation, and induces proinflammatory responses. Of note, the direct assembly of HK, PK, and FXII on vascular smooth muscle cells (VSMC) also results in the activation of PK to kallikrein (
). The induction of these physiological reactions is caused by the release of the vasoactive peptide bradykinin (BK) from HK by kallikrein. Bradykinin B2 receptor activation by BK mediates the activation of endothelial nitric-oxide synthase (eNOS) and phospholipase A2 (PLA2) leading to production of nitric oxide (NO) and prostacylin (PGI2). Evidence suggests that BK phosphorylates p44/42 mitogen-activated protein kinase in VMSC, which is blocked by BK antagonist HOE-140 (
Besides having a direct effect on blood vessels, the HK-PK complex has also been shown to mediate the effects of other pro-inflammatory molecules. Recent study suggests that the inhibitors of both BK and factor XII activity protect from mast cell-induced effects not only in patients but also in genetically engineered mouse models. The authors proposed that this class of inhibitors could be useful to treat allergic diseases (
). Tryptase is a serine protease produced by mast cells during inflammation. Evidence suggests that tryptase-induced increase in vascular permeability is dependent on PK activation to kallikrein and subsequent release of BK from HK (
). Imamura et al. showed that while HK-deficient plasma completely lacks tryptase-induced vascular permeability enhancement activity, tryptase produces a 30% increase in vascular permeability in PK-deficient plasma. Therefore, we asked the question, what is the cause of incomplete abolishment of vascular permeability enhancement from PK-deficient plasma? Possible explanations for this observation could be heterozygosity of the donor cell genome, direct release of BK from HK by tryptase or the presence of a tryptase-independent mechanism. Finally, HK may have the ability to induce vascular permeability. Because the functional role of HK, in the absence of PK, on vascular endothelial cells is not fully defined and because no clinical abnormality is seen in PK-deficient patients, we determined whether HK regulates endothelial cell function (
). Thus, we characterized the effect of HK on cultured endothelial cells in the absence of PK.
We report that HK regulates endothelial cell function in the absence of PK. The major findings of this study are: 1) HK induced a concentration- and time-dependent increase in [Ca2+]i in endothelial cells; 2) endothelial cell activation by HK was mediated via its binding to B2 receptors and promoted NO and PGI2 production without influencing endothelial monolayer permeability at physiological concentration; 3) BK-free HK was ineffective in stimulating the production of PGI2 suggesting that the BK sequence within intact HK is important for the interaction of HK with B2 receptor and is responsible for HK-induced endothelial cell activation; 4) HK-induced increase in [Ca2+]i likely mediates responses such as endothelial cell volume regulation; and 5) Verapamil and Gd3+ enhanced HK-induced increase in [Ca2+]i in HPAEC.
The present study aimed at determining the effect of HK on endothelial cell function in the absence of PK. Upon the discovery that HK influences the kinetics of intracellular Ca2+ in HPAEC, we studied the mechanisms involved in HK-induced increase in [Ca2+]i. Using the inhibitors of both intracellular Ca2+ mobilization and extracellular Ca2+ influx, we identified that HK modulates endothelial cell [Ca2+]i levels through the release of intracellular Ca2+ from IP3-sensitive vesicles as well as the influx of extracellular Ca2+.
Studies have shown that extracellular Ca2+ can enter endothelial cells via five distinct pathways: (i) receptor operated Ca2+ channels (ii) store operated Ca2+ channels (iii) stretch-activated non-selective cation channels (iv) voltage-gated L-type Ca2+ channels and (v) Ca2+ leak channels (
). Based on previous studies and our current findings it could be suggested that the interaction of HK with endothelial cell membrane is very selective. The underlying mechanisms of HK interaction with cell membrane proteins have not been completely unraveled. However, this current study suggests that while other mechanisms could be involved, HK-induced influx of extracellular Ca2+ is primarily mediated through receptor (B2) and store operated Ca2+ channels. The proposed mechanism of modulation of endothelial cell function by HK and the identified signaling pathway intermediates are summarized in Fig. 5.
Ca2+ regulates cell volume by modulating the release of ions such as potassium and chloride (Fig. 5). Intermediate calcium-activated potassium channels (IKCa) and small calcium-activated potassium channels (SKCa) are expressed in endothelial cells and contribute to NO generation (
). Understanding the effect of HK on the intracellular signals that control cell volume was important because both extracellular and intracellular Ca2+ have been shown to appreciably affect the cell volume. Calcium-activated potassium channels are activated by an elevation of [Ca2+]i and membrane depolarization. HK-induced increase in [Ca2+]i was not affected by quinine (IKCa blocker) or apamin (SKCa blocker), suggesting that endothelial cell activation by HK does not involve the activation of calcium-activated potassium channels. However, hypertonic stimulation of HPAEC significantly reduced the HK-mediated Ca2+ response. These findings led us to suggest that HK may play a role in regulating endothelial cell volume via modulation of calcium-activated chloride channel activity.
The binding of HK to endothelial cells via cytokeratin 1 (CK1) (
). These studies indicated that HK binds to the soluble extracellular form of uPAR with a much lower affinity than does HKa. The direct binding of HK to these cell membrane proteins indicated that gC1qR is dominant for binding using surface plasmon resonance (
). Furthermore, these cell membrane proteins are not directly coupled with G proteins and there is no evidence suggesting that the interaction of HK with uPAR, CK1 or gC1qR leads to changes in [Ca2+]i in endothelial cells. Further, it has been shown that optimum binding of HK to endothelial cells under physiological condition requires three regions on HK domain 3, domain 5 and BK (domain 4) (
). The existence of a novel endothelial cell binding site that recognizes a part of BK in the context of its parent molecule HK and the close apposition of HK and BK receptors to regulate the bioavailability of BK at endothelial cell surface has been previously suggested by Hasan (
). Because BK, acting via Gq coupled B2 receptors, is known to induce a robust increase in [Ca2+]i and since HK is the precursor of BK, we investigated whether the effects of HK on [Ca2+]i are mediated through its interaction with B2 receptors. We demonstrated that HK mediated its effects through the activation of B2 receptors and that B2 receptor antagonist (HOE140), PLC inhibitor (U73122) and IP3-R inhibitor (2-APB) inhibited HK-induced endothelial cell activation (Fig. 3, A and B). Competition studies using biotin-HK, HKH20 (as a control), HOE140 and BK confirmed that HK binds to B2 receptors, although with a much lesser affinity than to uPAR, CK1, and gC1qR. Although HKH20 inhibited biotin-HK binding to cell surface in a dose-dependent manner, HKH20 appears to act as an atypical synthetic peptide that does not just interact with cytokeratin 1 (
) but tends to influence the binding of HK to B2 receptors at high concentration (Fig. 3C). Of note, HKH20 did not completely abolish HK binding to cells, confirming previous finding that HK binds to cells also via domain 3 (
). Alternatively, HKH20 may directly interact with B2. Further investigations are needed to address this potential interaction. However, this property of HKH20 could be useful in treating BK-induced inflammatory (
). Nonetheless, HK interaction with B2 is strong enough to trigger the B2 receptor signaling cascade. This is a novel finding with an important physiological implication. HK, although to a lesser extent than BK, might have a direct cardioprotective effect. It has been reported that deficiency of plasma HK in the genetically susceptible Lewis rat results in decreased chronic enterocolitis and systemic inflammation (
). These studies support our hypothesis and points to the importance of HK-induced transient signals in endothelial cells, although the in vivo significance of this finding is difficult to determine at this point.
HK-deficient rats are protected from peptidoglycan-polysaccharide (PG-PS)-induced inflammatory arthritis as well as endototxin-induced hypotension (
). We, therefore, hypothesized that HK promotes inflammation. To test this hypothesis, we determined the effect of HK on NO and PGI2 generation as well as induction of endothelial permeability. We showed that HK stimulated B2 receptor-mediated increase in the production of NO and PGI2, the two mediators involved in inflammation. However, contrary to our hypothesis, HK at physiological concentration had no effect on endothelial monolayer permeability as determined using an in vitro assay.
In conclusion, the present study provides evidence that HK has the capability to stimulate production of endogenous vasodilators NO and PGI2 by endothelial cells. The significance of this finding might be at least 3-fold: 1) HK regulates endothelial function, 2) HK-induced increase in the production of NO, PGI2 might have a cardioprotective role in patients with PK deficiency, and 3) inhibition of both kallikrein and HK should produce more complete blockade of this pathway in patients with hereditary angioedema (HAE).
We thank Dr. Sean Wilson and Scotty Taylor for technical assistance.