Bradykinin enhances cell migration in human chondrosarcoma cells through BK receptor signaling pathways

Bradykinin (BK) is an inflammatory mediator, and shows elevated levels in regions of severe injury and inflammatory diseases. BK has recently been shown to be involved in carcinogenesis and cancer progression. In this study, we found that BK increased the migration and the expression of α2β1 integrin in human chondrosarcoma cells. We also found that human chondrosarcoma tissues had significantly higher expression of the B1 and B2 receptors comparing to normal cartilage. BK‐mediated migration and integrin up‐regulation was attenuated by B1 and B2 BK receptor siRNA or antagonist. Activations of phospholipase C (PLC), protein kinase Cδ (PKCδ), and NF‐κB pathways after BK treatment was demonstrated, and BK‐induced integrin expression and migration activity was inhibited by the specific inhibitor of PLC, PKCδ, and NF‐κB cascades. Taken together, our results indicated that BK enhances the migration of chondrosarcoma cells by increasing α2β1 integrin expression through the BK receptors/PLC/PKCδ/NF‐κB signal transduction pathway. J. Cell. Biochem. 109: 82–92, 2010. © 2009 Wiley‐Liss, Inc.     

Thrombin induces heme oxygenase-1 expression in human synovial fibroblasts through protease-activated receptor signaling pathways

Introduction

Thrombin is a key factor in the stimulation of fibrin deposition, angiogenesis, and proinflammatory processes. Abnormalities in these processes are primary features of osteoarthritis (OA). Heme oxygenase (HO)-1 is a stress-inducible rate-limiting enzyme in heme degradation that confers cytoprotection against oxidative injury. Here, we investigated the intracellular signaling pathways involved in thrombin-induced HO-1 expression in human synovial fibroblasts (SFs).

Methods

Thrombin-mediated HO-1 expression was assessed with quantitative real-time (q)PCR. The mechanisms of action of thrombin in different signaling pathways were studied by using Western blotting. Knockdown of protease-activated receptor (PAR) proteins was achieved by transfection with siRNA. Chromatin immunoprecipitation assays were used to study in vivo binding of Nrf2 to the HO-1 promoter. Transient transfection was used to examine HO-1 activity.

Results

Osteoarthritis synovial fibroblasts (OASFs) showed significant expression of thrombin, and expression was higher than in normal SFs. OASFs stimulation with thrombin induced concentration- and time-dependent increases in HO-1 expression. Pharmacologic inhibitors or activators and genetic inhibition by siRNA of protease-activated receptors (PARs) revealed that the PAR1 and PAR3 receptors, but not the PAR4 receptor, are involved in thrombin-mediated upregulation of HO-1. Thrombin-mediated HO-1 expression was attenuated by thrombin inhibitor (PPACK), PKCδ inhibitor (rottlerin), or c-Src inhibitor (PP2). Stimulation of cells with thrombin increased PKCδ, c-Src, and Nrf2 activation.

Conclusion

Our results suggest that the interaction between thrombin and PAR1/PAR3 increases HO-1 expression in human synovial fibroblasts through the PKCδ, c-Src, and Nrf2 signaling pathways.     

Transformation of NIH 3T3 cells by enhanced PAR expression

Prostate androgen regulated (PAR) is a 1038bp novel gene located on chromosome 1 in epidermal differentiation complex. The gene is ubiquitously expressed in normal tissues and is overexpressed in most of their malignant counterparts. PAR cellular function is unknown. Here we report the effect of increased PAR expression induced by transfection of PAR cDNA on NIH3T3 cell phenotype. PAR-NIH3T3 transfectants expressing 3- to 4-fold higher PAR levels compared to controls grew faster in tissue cultures, formed colonies in soft agar, and exhibited a shortening of G1 and S phases of cell cycle and formed tumors in SCID mice. Transfection of NIH3T3 cells with increased ectopic PAR expression with a 22 mer oligonucleotide in antisense orientation with PAR mRNA abrogated their ability to form colonies in soft agar. The data presented here along with our previously reported results on DU145 cells transfected with antisense PAR cDNA suggest that PAR gene behaves like a proto-oncogene.     

Activation dependent expression of MMPs in peripheral blood mononuclear cells involves protein kinase A

Monocyte/Macrophages are integral cellular components of inflammation. Matrix metalloproteinases (MMPs) produced by these cells play a crucial role in every aspect of inflammation. Results of the investigations on activation dependent upregulation of MMPs in human peripheral blood mononuclear cells in culture using different lectins as an in vitro model system to mimic inflammatory monocytes are presented. Under normal physiological conditions the monocytes produced only very low amount of MMPs in an indomethacin insensitive PG/cAMP independent manner. Zymographic analysis and ELISA showed that treatment of monocyte with lectins like concanavalin A (ConA), wheat germ agglutinin (WGA) and Artocarpus lakoocha agglutinin (ALA) caused upregulation of MMPs and the maximum effect was produced by ALA. ALA significantly upregulated MMP-9 in a concentration and time dependent manner. Immunoblot analysis and RT-PCR confirmed ALA mediated upregulation of MMP-9 production. Inhibition of ALA effect by indomethacin and reversal of the indomethacin effect by Bt₂cAMP indicated involvement of cAMP dependent signaling pathway. Further support for the prostaglandin mediated effect was obtained by the upregulation of cyclooxygenase by ALA. H-89, an inhibitor of protein kinase A (PKA), inhibited the expression of MMP-9 indicating that ALA mediated upregulation of MMP-9 is mediated through PKA pathway. Increase in MMP production and increase in cyclooxygenase activity and inhibition of the effect of ALA on MMP production by indomethacin suggested that the ALA activated monocytes in culture can be used as an in vitro model system to study the intracellular signaling process involved in the mediation of inflammatory response.     

Thrombin responses in human endothelial cells. Contributions from receptors other than PAR1 include the transactivation of PAR2 by thrombin-cleaved PAR1

The recent identification of two new thrombin receptors, PAR3 and PAR4, led us to re-examine the basis for endothelial cell responses to thrombin. Human umbilical vein endothelial cells (HUVEC) are known to express PAR1 and the trypsin/tryptase receptor, PAR2. Northern blots detected both of those receptors and, to a lesser extent, PAR3, but PAR4 message was undetectable and there was no response to PAR4 agonist peptides. To determine whether PAR3 or any other receptor contributes to thrombin signaling in HUVEC, PAR1 cleavage was blocked with two selective antibodies and PAR1 activation was inhibited with the antagonist, BMS200261. The antibodies completely inhibited HUVEC responses to thrombin, but BMS200261 was only partly effective, even though separate studies established that the antagonist completely inhibits PAR1 signaling at the concentrations used. Since peptides mimicking the PAR1 tethered ligand domain can also activate PAR2, we asked whether the remaining thrombin response in the presence of the antagonist could be due in part to the intermolecular transactivation of PAR2 by cleaved PAR1. Evidence that transactivation can occur was obtained in COS-7 cells co-expressing PAR2 and a variant of PAR1 that can be cleaved, but not signal. There was a substantial response to thrombin only in cells expressing both receptors. Conversely, in HUVEC, complete blockade of the thrombin response by the PAR1 antagonist occurred only when signaling through PAR2 was also blocked. From these observations we conclude that 1) PAR1 is the predominant thrombin receptor expressed in HUVEC and cleavage of PAR1 is required for endothelial cell responses to thrombin; 2) although PAR3 may be expressed, there is still no evidence that it mediates thrombin responses; 3) PAR4 is not expressed on HUVEC; and 4) transactivation of PAR2 by cleaved PAR1 can contribute to endothelial cell responses to thrombin, particularly when signaling through PAR1 is blocked. Such transactivation may limit the effectiveness of PAR1 antagonists, which compete with the tethered ligand domain rather than preventing PAR1 cleavage.     

Costimulation of chemokine receptor signaling by matrix metalloproteinase-9 mediates enhanced migration of IFN-alpha dendritic cells

Type I IFNs induce differentiation of dendritic cells (DCs) with potent Ag-presenting capacity, termed IFN-alpha DCs, that have been implicated in the pathogenesis of systemic lupus erythematosus. In this study, we found that IFN-alpha DCs exhibit enhanced migration across the extracellular matrix (ECM) in response to chemokines CCL3 and CCL5 that recruit DCs to inflammatory sites, but not the lymphoid-homing chemokine CCL21. IFN-alpha DCs expressed elevated matrix metalloproteinase-9 (MMP-9), which mediated increased migration across ECM. Unexpectedly, MMP-9 and its cell surface receptors CD11b and CD44 were required for enhanced CCL5-induced chemotaxis even in the absence of a matrix barrier. MMP-9, CD11b, and CD44 selectively modulated CCL5-dependent activation of JNK that was required for enhanced chemotactic responses. These results establish the migratory phenotype of IFN-alpha DCs and identify an important role for costimulation of chemotactic responses by synergistic activation of JNK. Thus, cell motility is regulated by integrating signaling inputs from chemokine receptors and molecules such as MMP-9, CD11b, and CD44 that also mediate cell interactions with inflammatory factors and ECM.     

L-asparaginase-induced apoptosis in ALL cells involves IP3 receptor signaling

L-asparaginase treatment is used in the clinic to treat acute lymphoblastic leukemia (ALL) patients. Lee et al. (2019, Blood 133:2222-2232) demonstrated that L-asparaginase induces apoptosis by activating inositol 1,4,5-trisphosphate (IP₃)-induced Ca²⁺ signaling in a Huntingin-associated protein 1 (HAP1)-dependent manner. Moreover, HAP1 levels inversely correlate with the sensitivity of the ALL cells to L-asparaginase. HAP1 can therefore be used as biomarker for evaluating L-asparaginase resistance.     

Improved hematopoietic differentiation of human pluripotent stem cells via estrogen receptor signaling pathway

Background

Aside from its importance in reproduction, estrogen (E2) is known to regulate the proliferation and differentiation of hematopoietic stem cells in rodents. However, the regulatory role of E2 in human hematopoietic system has not been investigated. The purpose of this study is to investigate the effect of E2 on hematopoietic differentiation using human pluripotent stem cells (hPSCs).

Results

E2 improved hematopoietic differentiation of hPSCs via estrogen receptor alpha (ER-α)-dependent pathway. During hematopoietic differentiation of hPSCs, ER-α is persistently maintained and hematopoietic phenotypes (CD34 and CD45) were exclusively detected in ER-α positive cells. Interestingly, continuous E2 signaling is required to promote hematopoietic output from hPSCs. Supplementation of E2 or an ER-α selective agonist significantly increased the number of hemangioblasts and hematopoietic progenitors, and subsequent erythropoiesis, whereas ER-β selective agonist did not. Furthermore, ICI 182,780 (ER antagonist) completely abrogated the E2-induced hematopoietic augmentation. Not only from hPSCs but also from human umbilical cord bloods, does E2 signaling potentiate hematopoietic development, suggesting universal function of E2 on hematopoiesis.

Conclusions

Our study identifies E2 as positive regulator of human hematopoiesis and suggests that endocrine factors such as E2 influence the behavior of hematopoietic stem cells in various physiological conditions.

     

Thrombin inhibits migration of human hepatic myofibroblasts

Several lines of data recently pointed out a role of the serine proteinase thrombin in liver fibrogenesis, but its mechanism of action is unknown. The aim of this study was to evaluate the effect of thrombin on the migration of human liver myofibroblasts. We show here that thrombin inhibits both basal migration and platelet-derived growth factor (PDGF)-BB-induced migration of myofibroblasts. By using a thrombin antagonist, a protease-activated receptor (PAR)-1 mimetic peptide, and a PAR-1 antibody, we show that this effect is dependent on the catalytic activity of thrombin and on PAR-1 activation. Thrombin's effect on basal migration was dependent on cyclooxygenase 2 (COX-2) activation because it was blocked by the COX-2 inhibitors NS-398 and nimesulide, and pharmacological studies showed that it was relayed through prostaglandin E(2) and its EP(2) receptor. On the other hand, thrombin-induced inhibition of PDGF-BB-induced migration was not dependent on COX-2. We show that thrombin inhibits PDGF-induced Akt-1 phosphorylation. This effect was consecutive to inhibition of PDGF-beta receptor activation through active dephosphorylation. Thus thrombin, through two distinct mechanisms, inhibits both basal- and PDGF-BB-induced migration of human hepatic liver myofibroblasts. The fine tuning of myofibroblast migration may be one of the mechanisms used by thrombin to regulate liver fibrogenesis.     

The differentiation of skeletal muscle cells involves a protein-tyrosine phosphatase-alpha-mediated C-Src signaling pathway

Protein-tyrosine phosphatase-alpha (PTPalpha) plays an important role in various cellular signaling events, including proliferation and differentiation. In this study, we established L6 cell lines either underexpressing or overexpressing PTPalpha by stable transfection of cells with antisense PTPalpha or with full-length wild-type human or mouse or double catalytic site Cys --> Ala mutant (DM8) PTPalpha cDNA. Expression of PTPalpha in these cell lines was determined by immunoblotting and immunofluorescence. Cells harboring antisense PTPalpha exhibited a significantly reduced growth rate and thymidine incorporation when compared with the wild-type L6 cells. In contrast, cells overexpressing PTPalpha showed more rapid (2-fold) proliferation. Myoblasts with diminished PTPalpha failed to undergo fusion and did not form myotubes in reduced serum whereas overexpression of PTPalpha promoted myogenesis 2 days earlier than wild-type L6 cells. Overexpression of phosphatase-inactive mutant PTPalpha recapitulated the phenotype of the antisense cells. The different myogenic activities of these cell lines were correlated with the expression of myogenin and creatine kinase activity. Consistent with previous reports, PTPalpha positively regulated the activity of the protein-tyrosine kinase Src. Treatment of L6 cells with PP2 or SU6656, specific inhibitors of Src family kinases, and transient transfection of dominant-inhibitory Src inhibited the formation of myotubes and expression of myogenin. Moreover, enhanced expression of PTPalpha and activation of Src was detected during myogenesis. Together, these data indicate that PTPalpha is involved in the regulation of L6 myoblast growth and skeletal muscle cell differentiation via an Src-mediated signaling pathway.     

Profiling gene expression induced by protease-activated receptor 2 (PAR2) activation in human kidney cells

Protease-Activated Receptor-2 (PAR2) has been implicated through genetic knockout mice with cytokine regulation and arthritis development. Many studies have associated PAR2 with inflammatory conditions (arthritis, airways inflammation, IBD) and key events in tumor progression (angiogenesis, metastasis), but they have relied heavily on the use of single agonists to identify physiological roles for PAR2. However such probes are now known not to be highly selective for PAR2, and thus precisely what PAR2 does and what mechanisms of downstream regulation are truly affected remain obscure. Effects of PAR2 activation on gene expression in Human Embryonic Kidney cells (HEK293), a commonly studied cell line in PAR2 research, were investigated here by comparing 19,000 human genes for intersecting up- or down-regulation by both trypsin (an endogenous protease that activates PAR2) and a PAR2 activating hexapeptide (2f-LIGRLO-NH(2)). Among 2,500 human genes regulated similarly by both agonists, there were clear associations between PAR2 activation and cellular metabolism (1,000 genes), the cell cycle, the MAPK pathway, HDAC and sirtuin enzymes, inflammatory cytokines, and anti-complement function. PAR-2 activation up-regulated four genes more than 5 fold (DUSP6, WWOX, AREG, SERPINB2) and down-regulated another six genes more than 3 fold (TXNIP, RARG, ITGB4, CTSD, MSC and TM4SF15). Both PAR2 and PAR1 activation resulted in up-regulated expression of several genes (CD44, FOSL1, TNFRSF12A, RAB3A, COPEB, CORO1C, THBS1, SDC4) known to be important in cancer. This is the first widespread profiling of specific activation of PAR2 and provides a valuable platform for better understanding key mechanistic roles of PAR2 in human physiology. Results clearly support the development of both antagonists and agonists of human PAR2 as potential disease modifying therapeutic agents.     

Thrombin receptor induction by injury-related factors in human skeletal muscle cells

Thrombin is involved in tissue repair through its proteolytic activation of a specific thrombin receptor (PAR-1). Previous studies have shown that serine proteases and their inhibitors are involved in neuromuscular junction plasticity. We hypothesized that thrombin could also be involved during skeletal muscle inflammation. Thus we investigated the expression of PAR-1 in human myoblasts and myotubes in vitro and its regulation by injury-related factors. The functionality of this receptor was tested by measuring thrombin's ability to elicit Ca2+ signals. Western blot analysis and immunocytochemistry demonstrated the presence of PAR-1 in myoblasts but not in myotubes unless they were treated by tumor necrosis factor-alpha (10 ng/ml), interleukin-1beta (5 ng/ml), or transforming growth factor-beta(1) (10 ng/ml). The addition of 10 nM alpha-thrombin evoked a strong Ca2+ signal in myoblasts while a limited response in myotubes was observed. However, in the additional presence of injury-related factors, the amplitude of the Ca2+ response was significantly enhanced, representing 88, 65, 48% of their respective basal level, compared to 27% of that obtained in controls. Moreover, immunochemical studies on human skeletal muscle biopsies of patients suffering from inflammatory myopathies showed an overexpression of PAR-1. These results suggest that PAR-1 synthesis may be induced in response to muscle injury, thereby implicating thrombin signaling in certain muscle inflammatory diseases.     

Lymphocyte migration through brain endothelial cell monolayers involves signaling through endothelial ICAM-1 via a rho-dependent pathway

Lymphocyte extravasation into the brain is mediated largely by the Ig superfamily molecule ICAM-1. Several lines of evidence indicate that at the tight vascular barriers of the central nervous system (CNS), endothelial cell (EC) ICAM-1 not only acts as a docking molecule for circulating lymphocytes, but is also involved in transducing signals to the EC. In this paper, we examine the signaling pathways in brain EC following Ab ligation of endothelial ICAM-1, which mimics adhesion of lymphocytes to CNS endothelia. ICAM-1 cross-linking results in a reorganization of the endothelial actin cytoskeleton to form stress fibers and activation of the small guanosine triphosphate (GTP)-binding protein Rho. ICAM-1-stimulated tyrosine phosphorylation of the actin-associated molecule cortactin and ICAM-1-mediated, Ag/IL-2-stimulated T lymphocyte migration through EC monolayers were inhibited following pretreatment of EC with cytochalasin D. Pretreatment of EC with C3 transferase, a specific inhibitor of Rho proteins, significantly inhibited the transmonolayer migration of T lymphocytes, endothelial Rho-GTP loading, and endothelial actin reorganization, without affecting either lymphocyte adhesion to EC or cortactin phosphorylation. These data show that brain vascular EC are actively involved in facilitating T lymphocyte migration through the tight blood-brain barrier of the CNS and that this process involves ICAM-1-stimulated rearrangement of the endothelial actin cytoskeleton and functional EC Rho proteins.     

IL-6 increases MMP-13 expression and motility in human chondrosarcoma cells

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. IL-6 is a multifunctional cytokine that is associated with the disease status and outcomes of cancers. However, the effect of IL-6 on the migration activity of human chondrosarcoma cells is mostly unknown. Here, we found that IL-6 increased the migration and expression of MMP-13 in human chondrosarcoma cells. We also found that human chondrosarcoma tissues had significant expression of IL-6, which was higher than that in normal cartilage. IL-6-mediated migration and MMP-13 up-regulation were attenuated by anti-IL-6 receptor antibody, Ras, Raf-1, and a MEK inhibitor. Activation of the Ras, Raf-1, MEK, ERK, and NF-κB signaling pathways after IL-6 treatment was demonstrated, and IL-6-induced MMP-13 expression and migration activity were inhibited by the specific inhibitor and mutant Ras, Raf-1, MEK, ERK, and NF-κB cascades. In addition, migration-prone sublines demonstrated that cells with increasing migration ability had greater expression of IL-6 and MMP-13. Taken together, these results indicate that IL-6 and IL-6 receptor interaction enhances migration of chondrosarcoma through an increase in MMP-13 production.     

IL-8 increases integrin expression and cell motility in human chondrosarcoma cells

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Interleukin-8 (IL-8), a chemokine with a defining CXC amino acid motif, is known to possess tumorigenic and proangiogenic properties. Over-expression of IL-8 has been detected in many human tumors. However, the effects of IL-8 in migration and integrin expression in chondrosarcoma cells are largely unknown. In this study, we found that IL-8 increased the migration and the expression of αvβ3 integrin in human chondrosarcoma cells. Activations of phosphatidylinositol 3-kinase (PI3K), Akt, and AP-1 pathways after IL-8 treatment were demonstrated, and IL-8-induced expression of integrin and migration activity was inhibited by the specific inhibitor and mutant of PI3K, Akt, and AP-1 cascades. Taken together, our results indicated that IL-8 enhances the migration of chondrosarcoma cells by increasing αvβ3 integrin expression through the PI3K/Akt/AP-1 signal transduction pathway.     

High density lipoprotein-induced signaling of the MAPK pathway involves scavenger receptor type BI-mediated activation of Ras

High density lipoprotein (HDL) stimulates multiple signaling pathways. HDL-induced activation of the mitogen-activated protein kinase (MAPK) pathway can be mediated by protein kinase C (PKC) and/or pertussis toxin-sensitive G-proteins. Although HDL-induced activation of MAPK involves Raf-1, Mek, and Erk1/2, the upstream contribution of p21(ras) (Ras) on the activation of Raf-1 and MAPK remains elusive. Here we examine the effect of HDL on Ras activity and demonstrate that HDL induces PKC-independent activation of Ras that is completely blocked by pertussis toxin, thus implicating heterotrimeric G-proteins. In addition, the HDL-induced activation of Ras is inhibited by a neutralizing antibody against scavenger receptor type BI. We conclude that the binding of HDL to scavenger receptor type BI activates Ras in a PKC-independent manner with subsequent induction of the MAPK signaling cascade.     

Luteinizing hormone signaling in preovulatory follicles involves early activation of the epidermal growth factor receptor pathway

LH activates a cascade of signaling events that are propagated throughout the ovarian preovulatory follicle to promote ovulation of a mature egg. Critical to LH-induced ovulation is the induction of epidermal growth factor (EGF)-like growth factors and transactivation of EGF receptor (EGFR) signaling. Because the timing of this transactivation has not been well characterized, we investigated the dynamics of LH regulation of the EGF network in cultured follicles. Preovulatory follicles were cultured with or without recombinant LH and/or specific inhibitors. EGFR and MAPK phosphorylation were examined by immunoprecipitation and Western blot analyses. By semiquantitative RT-PCR, increases in amphiregulin and epiregulin mRNAs were detected 30 min after recombinant LH stimulation of follicles and were maximal after 2 h. LH-induced EGFR phosphorylation also increased after 30 min and reached a maximum at 2 h. EGFR activation precedes oocyte maturation and is cAMP dependent, because forskolin similarly activated EGFR. LH-induced EGFR phosphorylation was sensitive to AG1478, an EGFR kinase inhibitor, and to inhibitors of matrix metalloproteases GM6001 and TNFalpha protease inhibitor-1 (TAPI-1), suggesting the involvement of EGF-like growth factor shedding. LH- but not amphiregulin-induced oocyte maturation and EGFR phosphorylation were sensitive to protein synthesis inhibition. When granulosa cells were cultured with a combination of neutralizing antibodies against amphiregulin, epiregulin, and betacellulin, EGFR phosphorylation and MAPK activation were inhibited. In cultured follicles, LH-induced MAPK activation was partially inhibited by AG1478 and GM6001, indicating that this pathway is regulated in part by the EGF network but also involves additional pathways. Thus, complex mechanisms are involved in the rapid amplification and propagation of the LH signal within preovulatory follicles and include the early activation of the EGF network.     

High molecular weight kininogen activates B2 receptor signaling pathway in human vascular endothelial cells

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 [Ca(2+)](i) level in HPAEC. Gd(3+) and verapamil potentiated the HK-induced increase in [Ca(2+)](i). HK-induced Ca(2+) increase stimulated endothelial nitric oxide (NO) and prostacyclin (PGI(2)) production. The inhibitors of B(2) 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.