Endothelial monolayers have shown the ability to signal each other through gap junctions. Gap junction-mediated cell-cell interactions have been implicated in the modulation of endothelial cell functions during vascular inflammation. Inflammatory mediators alter the mechanical properties of endothelial cells, although the exact role of gap junctions in this process remains unclear. Here, we sought to study the role of gap junctions in the regulation of endothelial stiffness, an important physical feature that is associated with many vascular pathologies. The endothelial cellular stiffness of living endothelial cells was determined by using atomic force microscopy. We found that tumor necrosis factor-α transiently increased endothelial cellular stiffness, which is regulated by cytoskeletal rearrangement and cell-cell interactions. We explored the role of gap junctions in endothelial cellular stiffening by utilizing gap junction blockers, carbenoxolone, inhibitory anti-connexin 32 antibody or anti-connexin 43 antibody. Blockade of gap junctions induced the cellular stiffening associated with focal adhesion formation and cytoskeletal rearrangement, and prolonged tumor necrosis factor-α-induced endothelial cellular stiffening. These results suggest that gap junction-mediated cell-cell interactions play an important role in the regulation of endothelial cellular stiffness.
Protein C inhibitor (PCI), a member of the serine protease inhibitor family, is produced in various human tissues, including the liver, kidney and testis. In addition to inhibiting the anticoagulant protein C pathway, PCI also inhibits urinary plasminogen activator (uPA), which is a well-known mediator of tumor cell invasion. In the present study, to clarify the biologic significance of PCI in the kidney, we compared the expression of PCI between human renal cell carcinoma (RCC) tissue and nontumor kidney tissue. The PCI antigen level in RCC tissue was found to be significantly lower than in nontumor kidney tissue, and expression of PCI mRNA was detected in normal renal proximal tubular epithelial cells (RPTEC), but not in RCC or in an RCC cell line (Caki-1 cells). Key words: protein C inhibitor; urinary plasminogen activator; renal cell carcinoma; tumor invasionProtein C inhibitor (PCI) was originally identified as an inhibitor of the anticoagulant protease, activated protein C, 1 and was subsequently found to inhibit other proteases involved in blood coagulation and fibrinolysis, such as thrombin, 2 thrombin-thrombomodulin complex, 3 factor Xa, 2 factor XIa, 4 plasma kallikrein 4 and urinary plasminogen activator (uPA). 5 Human plasma PCI is believed to be produced mainly by the liver, 6 but is also synthesized in the kidneys and the reproductive organs, including the testis, seminal vesicles and ovary. 7,8 Thus, besides its function in the regulation of blood coagulation, PCI may also play a role in the regulation of reproduction. Recently, Uhrin et al. 9 reported that PCI knockout male mice are apparently healthy but infertile, and that this infertility is caused by abnormal spermatogenesis induced by destruction of the Sertoli cell barrier. In the kidneys, PCI is mainly synthesized by proximal tubular epithelial cells, 10 and most urinary PCI is present in complex with uPA, 11 which it inhibits; hence, PCI is also referred to as plasminogen activator inhibitor-3 (PAI-3). However, the physiologic role of uPA inhibition by PCI in the urinary tract is unknown.It is known that metastasis and invasion of various tumor cells are mediated by uPA and its receptor. 12 uPA is inhibited by PAI-1 and PAI-2, 13 the former of which has been reported to be essential for regulation of tumor cell invasion and metastasis by promoting angiogenesis. 14,15 Previously, it was shown that the expression of uPA is lower, that the expression of uPA receptor is moderately higher and that the expression of PAI-1 is significantly higher in human renal cell carcinoma (RCC) than in nontumor kidney tissue. 16,17 Furthermore, Swiercz et al. 18 suggested that the expression of uPA, its receptor and PAI-1 correlate with the aggressive phenotype of RCC. These studies suggest the importance of the plasminogen activator system, including uPA, its receptor and PAI-1, in the process of RCC tumor cell invasion; however, there is no direct evidence yet that uPA regulates the invasiveness of RCC cells. It is also known that uPA and/or plasmin may ...
Protein C inhibitor (PCI) regulates the anticoagulant protein C pathway and also inhibits urinary plasminogen activator (uPA), a mediator of tumor cell invasion. In the present study, we evaluated the effect of human PCI and its inactive derivatives on tumor growth and metastasis of human breast cancer (MDA-231) cells, and on angiogenesis in vivo. The invasiveness of MDA-231 cells was inhibited by recombinant intact PCI, but not by reactive sitemodified PCI (R354APCI) or by the N-terminal fragment of protease-cleaved PCI (NTPCI). The in vitro invasiveness of MDA-231 cells expressing intact PCI (MDA-PCI) was significantly decreased as compared to MDA-231 cells expressing R354APCI (MDA-R354APCI) or NTPCI (MDA-NTPCI). Further, in vivo growth and metastatic potential of MDA-PCI, MDA-R354APCI and MDA-NTPCI cells in severe combined immunodeficient (SCID) mice were significantly decreased as compared to MDA-Mock cells. Angiogenesis was also significantly decreased in Matrigel implant containing MDA-PCI, MDA-R354APCI or MDA-NTPCI cells as compared to that containing MDA-Mock cells. In vivo angiogenesis in rat cornea and in vitro tube formation were also inhibited by recombinant intact PCI, R354APCI and NTPCI. Furthermore, the anti-angiogenic activity of PCI was strong as cleaved antithrombin (AT), and slightly stronger than that of plasminogen activator inhibitor (PAI)-1 and pigment epitheliumderived factor (PEDF). Overall, this study showed that, in addition to a reactive site-dependent mechanism, PCI may also regulate tumor growth and metastasis independently of its protease inhibitory activity by inhibiting angiogenesis. ' 2007 Wiley-Liss, Inc.Key words: protein C inhibitor; serine protease inhibitor; invasion; metastasis; angiogenesis Protein C inhibitor (PCI) was originally identified as a plasma inhibitor of the anticoagulant protease, activated protein C. 1,2 PCI is a member of the serine protease inhibitor (SERPIN) family (SERPINA5); it is also able to inhibit other proteases of the blood coagulation and fibrinolysis system including thrombin, 3 thrombin-thrombomodulin complex, 4 factor Xa, 3 factor XIa, 5 plasma kallikrein 5 and urinary plasminogen activator (uPA). 6 Human plasma PCI is mainly synthesized in the liver, 7 but it is also produced in kidneys and reproductive organs, including testes, seminal vesicles and ovaries. 8,9 Unlike humans, mice and rats produce PCI only in reproductive organs including testes and ovaries 10,11 ; thus, rodents are inappropriate for studying the physiological role of PCI in plasma and other organs. We recently developed a human PCI gene transgenic (TG) mouse in which the tissue distribution of PCI is similar to humans. Using this animal model, we demonstrated that PCI is the physiological inhibitor of APC in plasma, and that it promotes blood coagulation and inflammation in animals with endotoxemia. 12 PCI appears to have also a function in fertilization; Uhrin et al. 13 reported that PCI knock-out male mice have infertility due to abnormal spermatogenesis caused by destruc...
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