Nintedanib induces senolytic effect via STAT3 inhibition.

Selective removal of senescent cells, or senolytic therapy, has been proposed to be a potent strategy for overcoming age-related diseases and even for reversing aging. We found that nintedanib, a tyrosine kinase inhibitor, selectively induced the death of primary human dermal fibroblasts undergoing RS. Similar to ABT263, a well-known senolytic agent, nintedanib triggered intrinsic apoptosis in senescent cells. Additionally, at the concentration producing the senolytic effect, nintedanib arrested the cell cycle of nonsenescent cells in the G1 phase without inducing cytotoxicity. Interestingly, the mechanism by which nintedanib activated caspase-9 in the intrinsic apoptotic pathway differed from that of ABT263 apoptosis induction; specifically, nintedanib did not decrease the levels of Bcl-2 family proteins in senescent cells. Moreover, nintedanib suppressed the activation of the JAK2/STAT3 pathway, which caused the drug-induced death of senescent cells. STAT3 knockdown in senescent cells induced caspase activation. Moreover, nintedanib reduced the number of senescence-associated ß-galactosidase-positive senescent cells in parallel with a reduction in STAT3 phosphorylation and ameliorated collagen deposition in a mouse model of bleomycin-induced lung fibrosis. Consistently, nintedanib exhibited a senolytic effect through bleomycin-induced senescence of human pulmonary fibroblasts. Overall, we found that nintedanib can be used as a new senolytic agent and that inhibiting STAT3 may be an approach for inducing the selective death of senescent cells. Our findings pave the way for expanding the senolytic toolkit for use in various aging statuses and age-related diseases.

Targeting and clearance of senescent foamy macrophages and senescent endothelial cells by antibody-functionalized mesoporous silica nanoparticles for alleviating aorta atherosclerosis.

Senescent cells drive atherosclerosis at all stages and contribute to cardiovascular disease. However, the markers in these senescent aortic plaques have not been well studied, creating a huge obstacle in the exploration of a precise and efficient system for atherosclerosis treatment. Recently, CD9 has been found to induce cellular senescence and aggravated atherosclerotic plaque formation in apolipoprotein E knockout (ApoE-/-) mice. In the present study, this result has been leveraged to develop CD9 antibody-modified, hyaluronic acid-coated mesoporous silica nanoparticles with a hyaluronidase-responsive drug release profile. In invitro models of senescent foamy macrophages and senescent endothelial cells stimulated with oxidized high-density-lipoprotein, the CD9 antibody-modified mesoporous silica nanoparticles exhibit high cellular uptake; reduce the reactive oxygen species level, high-density lipoprotein oxidation, and production of TNF-α and IL-6; and attenuate the senescence process, contributing to improved cell viability. In vivo experiment demonstrated that these nanoparticles can successfully target the senescent lesion areas, deliver the anti-senescence drug rosuvastatin to the senescent atherosclerotic plaques (mainly endothelial cells and macrophages), and alleviate the progression of atherosclerosis in ApoE-/- mice. By providing deep insight regarding the markers in senescent atherosclerotic plaque and developing a nano-system targeting this lesion area, the study proposes a novel and an accurate therapeutic approach for mitigating atherosclerosis through senescent cell clearance.

Identification of SYK inhibitor, R406 as a novel senolytic agent.

The selective removal of senescent cells by senolytics is suggested as a potential approach to reverse aging and extend lifespan. Using high-throughput screening with replicative senescence of human diploid fibroblasts (HDFs), we identified a novel senolytic drug R406 that showed selective toxicity in senescent cells. Using flow cytometry and caspase expression analysis, we confirmed that R406 caused apoptotic cell death along with morphological changes in senescent cells. Interestingly, R406 altered the cell survival-related molecular processes including the inhibition of phosphorylation of the focal adhesion kinase (FAK) and p38 mitogen-activated protein kinase (MAPK) in senescent cells. This pattern was not observed in other known senolytic agent ABT263. Correspondingly, apoptotic cell death in senescent cells was induced by simultaneously blocking the FAK and p38 pathways. Taken together, we suggest that R406 acts as a senolytic drug by inducing apoptosis and reducing cell attachment capacity.

CD9 induces cellular senescence and aggravates atherosclerotic plaque formation.

CD9, a 24 kDa tetraspanin membrane protein, is known to regulate cell adhesion and migration, cancer progression and metastasis, immune and allergic responses, and viral infection. CD9 is upregulated in senescent endothelial cells, neointima hyperplasia, and atherosclerotic plaques. However, its role in cellular senescence and atherosclerosis remains undefined. We investigated the potential mechanism for CD9-mediated cellular senescence and its role in atherosclerotic plaque formation. CD9 knockdown in senescent human umbilical vein endothelial cells significantly rescued senescence phenotypes, while CD9 upregulation in young cells accelerated senescence. CD9 regulated cellular senescence through a phosphatidylinositide 3 kinase-AKT-mTOR-p53 signal pathway. CD9 expression increased in arterial tissues from humans and rats with age, and in atherosclerotic plaques in humans and mice. Anti-mouse CD9 antibody noticeably prevented the formation of atherosclerotic lesions in ApoE-/- mice and Ldlr-/- mice. Furthermore, CD9 ablation in ApoE-/- mice decreased atherosclerotic lesions in aorta and aortic sinus. These results suggest that CD9 plays critical roles in endothelial cell senescence and consequently the pathogenesis of atherosclerosis, implying that CD9 is a novel target for prevention and treatment of vascular aging and atherosclerosis.

Senotherapeutics: emerging strategy for healthy aging and age-related disease.

Cellular senescence (CS) is one of hallmarks of aging and accumulation of senescent cells (SCs) with age contributes to tissue or organismal aging, as well as the pathophysiologies of diverse age-related diseases (ARDs). Genetic ablation of SCs in tissues lengthened health span and reduced the risk of age-related pathologies in a mouse model, suggesting a direct link between SCs, longevity, and ARDs. Therefore, senotherapeutics, medicines targeting SCs, might be an emerging strategy for the extension of health span, and prevention or treatment of ARDs. Senotherapeutics are classified as senolytics which kills SCs selectively; senomorphics which modulate functions and morphology of SCs to those of young cells, or delays the progression of young cells to SCs in tissues; and immune-system mediators of the clearance of SCs. Some senolytics and senomorphics have been proven to markedly prevent or treat ARDs in animal models. This review will present the current status of the development of senotherapeutics, in relation to aging itself and ARDs. Finally, future directions and opportunities for senotherapeutics use will discussed. This knowledge will provide information that can be used to develop novel senotherapeutics for health span and ARDs. [BMB Reports 2019; 52(1): 47-55].

Coptis japonica Makino extract suppresses angiogenesis through regulation of cell cycle-related proteins.

Angiogenesis, neovascularization from pre-existing vessels, is a key step in tumor growth and metastasis, and anti-angiogenic agents that can interfere with these essential steps of cancer development are a promising strategy for human cancer treatment. In this study, we characterized the anti-angiogenic effects of Coptis japonica Makino extract (CJME) and its mechanism of action. CJME significantly inhibited the proliferation, migration, and invasion of vascular endothelial growth factor (VEGF)-stimulated HUVECs. Furthermore, CJME suppressed VEGF-induced tube formation in vitro and VEGF-induced microvessel sprouting ex vivo. According to our study, CJME blocked VEGF-induced cell cycle transition in G1. CJME decreased expression of cell cycle-regulated proteins, including Cyclin D, Cyclin E, Cdk2, and Cdk4 in response to VEGF. Taken together, the results of our study indicate that CJME suppresses VEGF-induced angiogenic events such as proliferation, migration, and tube formation via cell cycle arrest in G1.

Antiangiogenic Activity of Acer tegmentosum Maxim Water Extract in Vitro and in Vivo.

Angiogenesis, the formation of new blood vessels, is critical for tumor growth and metastasis. Notably, tumors themselves can lead to angiogenesis by inducing vascular endothelial growth factor (VEGF), which is one of the most potent angiogenic factors. Inhibition of angiogenesis is currently perceived as one of the most promising strategies for the blockage of tumor growth. In this study, we investigated the effects of Acer tegmentosum maxim water extract (ATME) on angiogenesis and its underlying signal mechanism. We studied the antiangiogenic activity of ATME by using human umbilical vein endothelial cells (HUVECs). ATME strongly inhibited VEGF-induced endothelial cell proliferation, migration, invasion, and tube formation, as well as vessel sprouting in a rat aortic ring sprouting assay. Moreover, we found that the p44/42 mitogen activated protein (MAP) kinase signaling pathway is involved in the inhibition of angiogenesis by ATME. Moreover, when we performed the in vivo matrigel plug assay, VEGF-induced angiogenesis was potently reduced when compared to that for the control group. Taken together, these results suggest that ATME exhibits potent antiangiogenic activity in vivo and in vitro and that these effects are regulated by the extracellular regulated kinase (ERK) pathway.

Water extract of Cinnamomum cassia suppresses angiogenesis through inhibition of VEGF receptor 2 phosphorylation.

Angiogenesis, the process of new blood vessel formation, has been a major target for cancer therapy. Antiangiogenic herbal medicines are useful in the treatment of cancer. In this study, we found that a water extract of Cinnamomum cassia (CCWE) was a potent inhibitor of angiogenesis. In cultured human umbilical vein endothelial cells, CCWE suppressed vascular endothelial growth factor (VEGF)-induced proliferation, migration, invasion, tube formation, and intracellular signaling events such as phosphorylation of ERK, p38 and VEGFR2, and activation of matrix metalloproteinase. Furthermore, CCWE inhibited VEGF-induced vessel sprouting of rat aorta ex vivo. These findings might be of particular interest for drug development because VEGF signaling is a potential target for treatment of angiogenesis-associated diseases.

Meso-dihydroguaiaretic acid inhibits rat aortic vascular smooth muscle cell proliferation by suppressing phosphorylation of platelet-derived growth factor receptor beta.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) plays an essential functional role in the pathogenesis of vascular disorders, such as atherosclerosis, restenosis, and neointimal hyperplasia. In this study, we examined the effects of meso-dihydroguaiaretic acid (MDGA) on platelet-derived growth factor (PDGF)-BB-induced proliferation and the molecular basis of its underlying mechanism of action in rat aortic VSMCs. Incubation of resting VSMCs with MDGA for 24 h significantly diminished PDGF-BB-induced DNA synthesis in a dose-dependent manner. We also examined the effects of MDGA on PDGF-BB signal transduction. Pre-treatment of VSMCs with MDGA inhibited PDGF-BB-induced phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), p38, and C-Jun N-terminal kinase (JNK). MDGA also effectively inhibited phosphorylation of Akt, phospholipase C gamma 1 (PLCγ1), and PDGF receptor beta (PDGFRß). These results indicate that MDGA may inhibit proliferation of VSMCs by suppressing autophosphorylation of PDGFRß, and may be useful in the treatment of VSMC-associated vascular disease such as atherosclerosis, restenosis, and neointimal hyperplasia after angioplasty.

Effect of nodakenin on atopic dermatitis-like skin lesions.

Nodakenin, derived from the roots of Angelica gigas Nakai, is an important natural resource and medicinal material with anti-allergic and anti- inflammatory activities. We have previously shown that nodakenin inhibits IgE/Ag-induced degranulation in mast cells. In this study, we investigated the inhibitory effect of nodakenin on 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis (AD)- like skin lesions in ICR mice. Scratching behavior, skin severity score, blood IgE level, and skin thickness were improved in DNCB-induced AD-like ICR mice. Our results showed that nodakenin suppressed the increase of AD-like skin lesions in ICR mice. These results suggest that nodakenin may be a potential therapeutic resource for AD as well as an adjunctive agent to control itching associated with AD.

Role of endogenous ENaC and TRP channels in the myogenic response of rat posterior cerebral arteries.

AIMS: Mechanogated ion channels are predicted to mediate pressure-induced myogenic vasoconstriction in small resistance arteries. Recent findings have indicated that transient receptor potential (TRP) channels and epithelial sodium channels (ENaC) are involved in mechanotransduction. The purpose of this study was to investigate the role of TRP channels and ENaC in the myogenic response. Our previous study suggested that ENaC could be a component of the mechanosensitive ion channels in rat posterior cerebral arteries (PCA). However, the specific ion channel proteins mediating myogenic constriction are unknown. Here we found, for the first time, that ENaC interacted with TRPM4 but not with TRPC6 using immunoprecipitation and confocal microscopy. METHODS AND RESULTS: Treatment with a specific ßENaC inhibitor, amiloride, a specific TRPM4 inhibitor, 9-phenanthrol, and a TRPC6 inhibitor, SKF96365, resulted in inhibition of the pressure-induced myogenic response. Moreover, the myogenic response was inhibited in rat PCA transfected with small interfering RNA of ßENaC, TRPM4, and TRPC6. Co-treatment with amiloride and 9-phenanthrol showed a similar inhibitory effect on myogenic contraction compared to single treatment with amiloride or 9-phenanthrol. The myogenic response was not affected by 9-phenanthrol or amiloride treatment in PCA transfected with ßENaC or TRPM4 siRNA, respectively. However, pressure-induced myogenic response was fully inhibited by co-treatment with amiloride, 9-phenanthrol, and SKF96365, and by treatment with SKF96365 in PCA transfected with ßENaC siRNA. CONCLUSION: Our results suggest that ENaC, TRPM4, and TRPC6 play important roles in the pressure-induced myogenic response, and that ENaC and TRPM4 interact in rat PCA.

The role of sphingosine kinase 1/sphingosine-1-phosphate pathway in the myogenic tone of posterior cerebral arteries.

AIMS: The goal of the current study was to determine whether the sphingosine kinase 1 (SK1)/sphingosine-1-phosphate (S1P) pathway is involved in myogenic vasoconstriction under normal physiological conditions. In the present study, we assessed whether endogenous S1P generated by pressure participates in myogenic vasoconstriction and which signaling pathways are involved in SK1/S1P-induced myogenic response under normal physiological conditions. METHODS AND RESULTS: We measured pressure-induced myogenic response, Ca(2+) concentration, and 20 kDa myosin light chain phosphorylation (MLC(20)) in rabbit posterior cerebral arteries (PCAs). SK1 was expressed and activated by elevated transmural pressure in rabbit PCAs. Translocation of SK1 by pressure elevation was blocked in the absence of external Ca(2+) and in the presence of mechanosensitive ion channel and voltage-sensitive Ca(2+) channel blockers. Pressure-induced myogenic tone was inhibited in rabbit PCAs treated with sphingosine kinase inhibitor (SKI), but was augmented by treatment with NaF, which is an inhibitor of sphingosine-1-phosphate phosphohydrolase. Exogenous S1P further augmented pressure-induced myogenic responses. Pressure induced an increase in Ca(2+) concentration leading to the development of myogenic tone, which was inhibited by SKI. Exogenous S1P further increased the pressure-induced increased Ca(2+) concentration and myogenic tone, but SKI had no effect. Pressure- and exogenous S1P-induced myogenic tone was inhibited by pre-treatment with the Rho kinase inhibitor and NADPH oxidase inhibitors. Pressure- and exogenous S1P-induced myogenic tone were inhibited by pre-treatment with S1P receptor blockers, W146 (S1P1), JTE013 (S1P2), and CAY10444 (S1P3). MLC(20) phosphorylation was increased when the transmural pressure was raised from 40 to 80 mmHg and exogenous S1P further increased MLC(20) phosphorylation. The pressure-induced increase of MLC(20) phosphorylation was inhibited by pre-treatment of arteries with SKI. CONCLUSIONS: Our results suggest that the SK1/S1P pathway may play an important role in pressure-induced myogenic responses in rabbit PCAs under normal physiological conditions.

Increase of L-type Ca2+ current by protease-activated receptor 2 activation contributes to augmentation of spontaneous uterine contractility in pregnant rats.

We evaluated the effects of protease-activated receptor (PAR)-2 on spontaneous myometrial contraction (SMC) in isolated term pregnant myometrial strips of rat, and elucidated the cellular mechanisms of this effect using a conventional voltage-clamp method. In isometric tension measurements, trypsin and SL-NH(2), PAR-2 agonists, significantly augmented SMC in frequency and amplitude; however, boiled trypsin (BT) and LR-NH(2) had no effect on SMC. These stimulatory effects of PAR-2 agonists on SMC were nearly completely occluded by pre-application of Bay K 8644, an L-type voltage-gated Ca(2+) channel activator, thus showing the involvement of L-type voltage-gated Ca(2+) channels in PAR-2-induced augmentation of SMC. In addition, PAR-2 agonists significantly enhanced L-type voltage-gated Ca(2+) currents (I(Ca-L)), as measured by a conventional voltage-clamp method, and this increase was primarily mediated by activation of phospholipase C (PLC) and protein kinase C (PKC) via G-protein activation. Taken together, we have demonstrated that PAR-2 may actively regulate SMC during pregnancy by modulating Ca(2+) influx through L-type voltage-gated Ca(2+) channels, and that this increase of I(Ca-L) may be primarily mediated by PLC and PKC activation. These results suggest a cellular mechanism for the pathophysiological effects of PAR-2 activation on myometrial contractility during pregnancy and provide basic and theoretical information about developing new agents for the treatment of premature labor and other obstetric complications.

Epithelial Na+ channel proteins are mechanotransducers of myogenic constriction in rat posterior cerebral arteries.

It has been suggested that mechanosensitive ion channels initiate myogenic responses in vessels; however, the molecular identity of the mechanosensitive ion channel complex is unknown. Although previous reports have suggested that epithelial Na(+) channel (ENaC) proteins are mechanotransducers in arteries, experimental evidence demonstrating that ENaC proteins are mechanotransducers are not fully elucidated. The goal of the present study was to determine whether the ENaC is a mechanotransducer for the myogenic response by providing supporting evidence in the rat posterior cerebral artery (PCA). We measured the effect of ENaC inhibition on the pressure-induced myogenic response, Ca(2+) concentration and 20 kDa myosin light chain (MLC(20)) phosphorylation. We detected expression of ßENaC and γENaC subunits in rat PCA by Western blots and immunofluorescence. Inhibition of ENaCs with amiloride, ethyl isopropyl amiloride or benzamil blocked the myogenic response. Moreover, the myogenic response was inhibited in rat PCA transfected with ßENaC and γENaC small interfering RNA. The myogenic response was inhibited by elimination of external Na(+), which was replaced with N-methyl-d-glucamine. Amiloride and nifedipine inhibited the pressure-induced increase in Ca(2+) concentration. Finally, MLC(20) increased when the intraluminal pressure was raised, and the pressure-induced increase in MLC(20) phosphorylation was inhibited by pretreatment with amiloride, and in arteries transfected with ßENaC or γENaC small interfering RNA. Our results suggest that ENaCs may play an important role as mechanosensitive ion channels initiating pressure-induced myogenic responses in rat PCA.

Neuromedin B induces angiogenesis via activation of ERK and Akt in endothelial cells.

Neuromedin B (NMB) is one of the bombesin-like peptides in mammals. Recently, bombesin-like peptides have been characterized as growth factors in highly vascularized tumors. In this study, we report that NMB potently stimulates in vivo neovascularization in a mouse Matrigel plug and the sprouting of endothelial cells ex vivo in rat aortic rings. In addition, NMB increases the migration and tube formation in human umbilical vein endothelial cells (HUVECs). Moreover, treatment of HUVECs with NMB activates the extracellular signal-regulated kinase 1/2 (ERK(1/2)), Akt, and endothelial nitric oxide synthase (eNOS) and increases the level of NO production in a dose- and time-dependent manner. Furthermore, ERK activation and angiogenic sprouting in response to NMB are significantly blocked by the MEK inhibitor. Inhibition of phosphatidylinositol 3-kinase (PI3K) suppresses the NMB-stimulated tubular formation of HUVECs, along with reduction in the phosphorylation of Akt and eNOS. Taken together, these results indicate that NMB is a novel angiogenic peptide, and its angiogenic activity is mediated by activating the MEK/ERK- and PI3K/Akt/eNOS-dependent pathways. This study suggests that NMB may play important roles in mediating a variety of pathophysiological angiogenesis.

VEGF-specific short hairpin RNA-expressing oncolytic adenovirus elicits potent inhibition of angiogenesis and tumor growth.

RNA interference is being developed to treat cancer. Although highly target specific, its use has been limited by its short duration of expression. To overcome this shortcoming, we constructed an oncolytic adenovirus (Ad)-based short hairpin RNA (shRNA) expression system (Ad-DeltaB7-shVEGF) against vascular endothelial growth factor (VEGF), a key mediator in angiogenesis. To demonstrate the VEGF-specific nature of this Ad-based shRNA, replication-incompetent Ad expressing VEGF-specific shRNA (Ad-DeltaE1-shVEGF) was also generated. Ad-DeltaE1-shVEGF was highly effective in reducing VEGF expression, and elicited an antiangiogenic effect in vitro and in vivo. Similarly, Ad-DeltaB7-shVEGF exhibited potent antiangiogenic effects in the matrigel plug assay. Moreover, Ad-DeltaB7-shVEGF demonstrated a greater antitumor effect and enhanced survival compared to the cognate control oncolytic Ad, Ad-DeltaB7. Ad-DeltaB7-shVEGF induced significant reduction in tumor vasculature, verifying the antiangiogenic mechanism. Furthermore, both the duration and magnitude of gene silencing by Ad-DeltaB7-shVEGF was greater than Ad-DeltaE1-shVEGF. These results suggest that the combined effects of oncolytic viral therapy and cancer cell-specific expression of VEGF-targeted shRNA elicits greater antitumor effect than an oncolytic Ad alone.

[6]-Gingerol, a pungent ingredient of ginger, inhibits angiogenesis in vitro and in vivo.

[6]-Gingerol, a pungent ingredient of ginger (Zingiber officinale Roscoe, Zingiberaceae), has anti-bacterial, anti-inflammatory, and anti-tumor-promoting activities. Here, we describe its novel anti-angiogenic activity in vitro and in vivo. In vitro, [6]-gingerol inhibited both the VEGF- and bFGF-induced proliferation of human endothelial cells and caused cell cycle arrest in the G1 phase. It also blocked capillary-like tube formation by endothelial cells in response to VEGF, and strongly inhibited sprouting of endothelial cells in the rat aorta and formation of new blood vessel in the mouse cornea in response to VEGF. Moreover, i.p. administration, without reaching tumor cytotoxic blood levels, to mice receiving i.v. injection of B16F10 melanoma cells, reduced the number of lung metastasis, with preservation of apparently healthy behavior. Taken together, these results demonstrate that [6]-gingerol inhibits angiogenesis and may be useful in the treatment of tumors and other angiogenesis-dependent diseases.

Capsaicin inhibits in vitro and in vivo angiogenesis.

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), a natural product of Capsicum species, is known to induce excitation of nociceptive terminals involved in pain perception. Recent studies have also shown that capsaicin not only has chemopreventive properties against certain carcinogens and mutagens but also exerts anticancer activity. Here, we demonstrated the antiangiogenic activity of capsaicin using in vitro and in vivo assay systems. In vitro, capsaicin inhibited vascular endothelial growth factor (VEGF) -induced proliferation, DNA synthesis, chemotactic motility, and capillary-like tube formation of primary cultured human endothelial cells. Capsaicin inhibited both VEGF-induced vessel sprouting in rat aortic ring assay and VEGF-induced vessel formation in the mouse Matrigel plug assay. Moreover, capsaicin was able to suppress tumor-induced angiogenesis in chick chorioallantoic membrane assay. Capsaicin caused G(1) arrest in endothelial cells. This effect correlated with the down-regulation of the expression of cyclin D1 that led to inhibition of cyclin-dependent kinase 4-mediated phosphorylation of retinoblastoma protein. Signaling experiments show that capsaicin inhibits VEGF-induced p38 mitogen-activated protein kinase, p125(FAK), and AKT activation, but its molecular target is distinct from the VEGF receptor KDR/Flk-1. Taken together, these results demonstrate that capsaicin is a novel inhibitor of angiogenesis and suggest that it may be valuable to develop pharmaceutical drugs for treatment of angiogenesis-dependent human diseases such as tumors.

Complestatin prevents apoptotic cell death: inhibition of a mitochondrial caspase pathway through AKT/PKB activation.

Complestatin, a bicyclo hexapeptide from Streptomyces, was isolated as a possible regulator of neuronal cell death. In this study, we report an anti-apoptotic activity of complestatin and its underlying molecular mechanism. Complestatin blocked TRAIL (TNF-related apoptosis-inducing ligand)-induced apoptosis and activation of caspase-3 and -8 at micromolar concentration levels without inhibiting the catalytic activities of these caspases. Complestatin potently induced a rapid and sustained AKT/PKB activation and Bad phosphorylation, resulting in inhibition of mitochondrial cytochrome c release. These anti-apoptotic activities of complestatin were significantly abrogated in cells expressing dominant negative AKT/PKB. Taken together, our results suggest that complestatin prevents apoptotic cell death via AKT/PKB-dependent inhibition of the mitochondrial apoptosis signal pathway. The novel property of complestatin may be valuable for developing new pharmaceutical means that will control unwanted cell death.

Vascular endothelial growth factor up-regulates expression of receptor activator of NF-kappa B (RANK) in endothelial cells. Concomitant increase of angiogenic responses to RANK ligand.

Vascular endothelial growth factor (VEGF) is known as a key regulator of angiogenesis during endochondral bone formation. Recently, we demonstrated that TNF-related activation-induced cytokine (TRANCE or RANKL), which is essential for bone remodeling, also had an angiogenic activity. Here we report that VEGF up-regulates expression of receptor activator of NF-kappa B (RANK) and increases angiogenic responses of endothelial cells to TRANCE. Treatment of human umbilical vein endothelial cells (HUVECs) with VEGF increased both RANK mRNA and surface protein expression. Although placenta growth factor specific to VEGF receptor-1 had no significant effect on RANK expression, inhibition of downstream signaling molecules of the VEGF receptor-2 (Flk-1/KDR) such as Src, phospholipase C, protein kinase C, and phosphatidylinositol 3'-kinase suppressed VEGF-stimulated RANK expression in HUVECs. Moreover, the MEK inhibitor PD98059 or expression of dominant negative MEK1 inhibited induction of RANK by VEGF but not the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). VEGF potentiated TRANCE-induced ERK activation and tube formation via RANK up-regulation in HUVECs. Together, these results show that VEGF enhances RANK expression in endothelial cells through Flk-1/KDR-protein kinase C-ERK signaling pathway, suggesting that VEGF plays an important role in modulating the angiogenic action of TRANCE under physiological or pathological conditions.