Nobuyuki Akita

Regulation of carcinoma cell invasion by protein C inhibitor whose expression is decreased in renal cell carcinoma

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). No differences were detected between the nucleotide sequence of the major cis‐elements in the promoter region of the PCI gene from nontumor kidney and RCC tissues, RPTEC and Caki‐1 cells, an RPTEC‐derived RCC cell line. The in vitro invasiveness of Caki‐1 cells transfected with a PCI expression vector was significantly decreased compared to mock‐transfected Caki‐1 cells, and it was blocked in the presence of anti‐PCI antibody. Since PCI itself did not affect the proliferation rate of Caki‐1 cells or cell expression of uPA in vitro, the effect of uPA, PCI, heat‐inactivated PCI and plasminogen activator inhibitor (PAI)‐1 on the invasive potential of cultured RCC cells was evaluated. The in vitro invasiveness of Caki‐1 cells, which express uPA, was significantly enhanced by the addition of uPA, and it was inhibited by anti‐uPA antibody, PCI and PAI‐1, but not by heat‐inactivated PCI. In addition, uPA activity was significantly decreased and uPA‐PCI complex level was significantly increased in the culture medium of PCI expression vector‐transfected Caki‐1 cells as compared to mock‐transfected Caki‐1 cells. These findings strongly suggest that PCI regulates the invasive potential of RCC cells by inhibiting uPA secreted by these cells. The results of our study suggest that PCI might be a potential therapeutic agent for inhibiting renal tumor invasion.

Protein C inhibitor inhibits breast cancer cell growth, metastasis and angiogenesis independently of its protease inhibitory activity

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 site‐modified 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 epithelium‐derived 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.

Connexin32 protects against vascular inflammation by modulating inflammatory cytokine expression by endothelial cells

Gap junctions (GJs) play an important role in vascular function, stability, and homeostasis in endothelial cells (ECs), and GJs are comprised of members of the connexin (Cx) family. GJs of vascular ECs are assembled from Cx37, Cx40, and Cx43, and we showed that ECs also express Cx32. In this study, we investigated a potential role for Cx32 during vascular inflammation. Expression of Cx32 mRNA and protein by human umbilical venous ECs (HUVECs) decreased following treatment with tumor necrosis factor (TNF)-α, but lipopolysaccharide (LPS) and interleukin (IL)-1β did not affect Cx32 expression. Intracellular transfer of an inhibitory anti-Cx32 monoclonal antibody significantly enhanced TNF-α-induced monocyte chemotactic protein (MCP)-1 and IL-6 expression, but overexpression of Cx32 abrogated TNF-α-induced MCP-1 and IL-6 expression. LPS treatment of Cx32 knock-out mice significantly increased the serum concentrations of TNF-α, interferon-γ, IL-6 and MCP-1, compared to wild-type littermate mice. These data suggest that Cx32 protects ECs from inflammation by regulating cytokine expression and plays an important role in the maintenance of vascular function.

Endothelial connexin32 enhances angiogenesis by positively regulating tube formation and cell migration.

The gap junction proteins connexin32 (Cx32), Cx37, Cx40, and Cx43 are expressed in endothelial cells, and regulate vascular functions involving inflammation, vasculogenesis and vascular remodeling. Aberrant Cxs expression promotes the development of atherosclerosis which is modulated by angiogenesis; however the role played by endothelial Cxs in angiogenesis remains unclear. In this study, we determined the effects of endothelial Cxs, particularly Cx32, on angiogenesis. EA.hy926 cells that had been transfected to overexpress Cx32 significantly increased capillary length and the number on branches compared to Cx-transfectant cells over-expressing Cx37, Cx40, and Cx43 or mock-treated cells. Treatment via intracellular transfer of anti-Cx32 antibody suppressed tube formation of human umbilical vein endothelial cells (HUVECs) compared to controls. In vitro wound healing assays revealed that Cx32-transfectant cells significantly increased the repaired area while anti-Cx32 antibody-treated HUVECs reduced it. Ex vivo aorta ring assays and in vivo matrigel plaque assays showed that Cx32-deficient mice impaired both vascular sprouting from the aorta and cell migration into the implanted matrigel. Therefore endothelial Cx32 facilitates tube formation, wound healing, vascular sprouting, and cell migration. Our results suggest that endothelial Cx32 positively regulates angiogenesis by enhancing endothelial cell tube formation and cell migration.

Amla (Emblica officinalis Gaertn.) extract inhibits lipopolysaccharide-induced procoagulant and pro-inflammatory factors in cultured vascular endothelial cells.

Amla (Emblica officinalis Gaertn.) has been used for many centuries in traditional Indian Ayurvedic formulations for the prevention and treatment of many inflammatory diseases. The present study evaluated the anti-inflammatory and anticoagulant properties of amla fruit extract. The amla fruit extract potentially and significantly reduced lipopolysaccharide (LPS)-induced tissue factor expression and von Willebrand factor release in human umbilical vein endothelial cells (HUVEC) in vitro at clinically relevant concentrations (1-100 μg/ml). In a leucocyte adhesion model of inflammation, it also significantly decreased LPS-induced adhesion of human monocytic cells (THP-1) to the HUVEC, as well as reduced the expression of endothelial-leucocyte adhesion molecule-1 (E-selectin) in the target cells. In addition, the in vivo anti-inflammatory effects were evaluated in a LPS-induced endotoxaemia rat model. Oral administration of the amla fruit extract (50 mg/kg body weight) significantly decreased the concentrations of pro-inflammatory cytokines, TNF-α and IL-6 in serum. These results suggest that amla fruit extract may be an effective anticoagulant and anti-inflammatory agent.

Gap junction-mediated regulation of endothelial cellular stiffness

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.

Endothelial connexin 32 regulates tissue factor expression induced by inflammatory stimulation and direct cell–cell interaction with activated cells

OBJECTIVE Endothelial cell (EC) interacts with adjacent EC through gap junction, and abnormal expression or function of Cxs is associated with cardiovascular diseases. In patients with endothelial dysfunction, the up-regulation of tissue factor (TF) expression promotes the pathogenic activation of blood coagulation, however the relationship between gap junctions and TF expression in ECs remains uncharacterized. ECs express the gap junction (GJ) proteins connexin32 (Cx32), Cx37, Cx40 and Cx43. We investigated the role of endothelial gap junctions, particularly Cx32, in modulating TF expression during vascular inflammation. METHODS AND RESULTS Human umbilical vein endothelial cells (HUVECs) were stimulated with tumor necrosis factor-α (TNF-α) and TF activity was assessed in the presence of GJ blockers and an inhibitory anti-Cx32 monoclonal antibody. Treatment with GJ blockers and anti-Cx32 monoclonal antibody enhanced the TNF-α-induced TF activity and mRNA expression in HUVECs. TNF-α-activated effector HUVECs or mouse MS-1 cells were co-cultured with non-stimulated acceptor HUVECs and TF expression in acceptor HUVECs was detected. Effector EC induced TF expression in adjacent acceptor HUVECs through direct cell-cell interaction. Cell-cell interaction induced TF expression was reduced by anti-intercellular adhesion molecule-1 (ICAM1) monoclonal antibody. Soluble ICAM1-Fc fusion protein promotes TF expression. GJ blockers and anti-Cx32 monoclonal antibody enhanced TF expression induced by cell-cell interaction and ICAM1-Fc treatment. CONCLUSION Blockade of endothelial Cx32 increased TF expression induced by TNF-α stimulation and cell-cell interaction which was at least partly dependent upon ICAM1. These results suggest that direct Cx32-mediated interaction modulates TF expression in ECs during vascular inflammation.

Host protein C inhibitor inhibits tumor growth, but promotes tumor metastasis, which is closely correlated with hypercoagulability.

INTRODUCTION Protein C inhibitor (PCI), a member of the serine protease inhibitor family, is expressed in various human tissues, including liver and kidneys. In the plasma, PCI physiologically inhibits an anticoagulant serine protease, activated protein C (APC). PCI expressed by cancer cells suppresses tumor invasion by inhibiting urokinase-type plasminogen activator, and inhibits tumor growth and metastasis, which are independent of its protease-inhibitory activity. In the present study, we clarified the effects of host PCI on growth and metastasis of B16 melanoma (B16) cells by comparing between wild-type mice and mice transgenic for human PCI gene (hPCI-TG), which have a tissue distribution of PCI similar to that observed in humans. MATERIALS AND METHODS Growth of intracutaneously-injected B16 cells was evaluated by measuring the tumor volume, and metastatic behavior of intravenously-injected B16 cells by counting the number of metastatic lung nodules. RESULTS Growth of intracutaneously injected B16 cells was significantly faster in wild-type mice than in hPCI-TG mice; however, hPCI-TG mice developed more metastatic nodules of B16 cells in the lungs. Immunohistochemical analysis using anti-mouse fibrinogen antibody revealed more fibrin deposition in the lung in hPCI-TG mice than in wild-type mice. Furthermore, the more invasive behavior observed in hPCI-TG mice was reduced by rabbit anti-human PCI IgG, APC, or soluble TM administration for 3 consecutive days including the day that B16 cells were injected. CONCLUSIONS Our results suggest that like PCI expressed in tumor cells, host PCI also inhibits tumor growth, but host PCI promotes tumor metastasis via its procoagulant properties.

Activated protein C suppresses osteoclast differentiation via endothelial protein C receptor, protease-activated receptor-1, sphingosine 1-phosphate receptor, and apolipoprotein E receptor 2

INTRODUCTION Bone remodeling relies on a delicate balance between formation and resorption of bone tissues, processes in which bone-forming osteoblasts and bone-resorbing osteoclasts play central roles. Recently, we reported that anticoagulant activated protein C (APC) promotes osteoblast proliferation, but the role of the blood coagulation system in bone remodeling remains unclear. In this study, to further elucidate the relationship between bone remodeling and blood coagulation, we investigated the effect of APC on osteoclast differentiation. MATERIALS AND METHODS Normal human osteoclast precursor cells were cultured in their growth medium including soluble RANKL, M-CSF, and FBS, and on days 4 and 7, the culture medium was replaced with the same medium containing various concentrations of APC, protein C (PC), sphingosine 1-phosphate (S1P) receptor agonist, FTY720, or APC+various substances without FBS. On day 8, TRAP-positive multinucleated cells (≥3 nuclei) were counted manually using a light microscope. The effects of APC on NF-κB and NFATc1 activation were evaluated using specific ELISA. RESULTS APC suppressed RANKL-induced osteoclast differentiation, and this APC-induced suppression of osteoclast differentiation was inhibited by zymogen protein C and aprotinin, a serine protease inhibitor. Immunohistochemistry and RT-PCR analyses suggested that endothelial protein C receptor (EPCR) and protease-activated receptor-1 (PAR-1) were expressed in osteoclast precursor cells and osteoclasts. Both anti-PAR-1 antibody and anti-EPCR antibody (RCR-252), which blocks APC binding to EPCR, inhibited the APC-induced suppression of osteoclast differentiation. FTY720 had no effect on osteoclast differentiation. However, FTY 720 and S1P receptor antagonist, VP 23019, inhibited the APC-induced suppression of osteoclast differentiation. On the other hand, recombinant soluble human ApoER2 and anti-human ApoER2 inhibited the APC-induced suppression of osteoclast differentiation. Further, APC had no effect on NF-κB and NFATc1 activation. CONCLUSIONS APC suppresses human osteoclast differentiation mainly by inhibiting the formation of multinucleated cells via EPCR, PAR-1, S1P receptor, and ApoER2 in a manner that depends on APC protease activity.