Involvement of integrin-linked kinase in capillary/tube-like network formation of human vascular endothelial cells

Watanabe, Motomu; Fujioka-Kaneko, Yayoi; Kobayashi, Hisayuki; Kiniwa, Mamoru; Kuwano, Michihiko; Basaki, Yuji

doi:10.1251/bpo104

Cited by 18 publications

(10 citation statements)

References 20 publications

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“…An in vitro study using bovine aortic EC showed that siRNA-mediated silencing of ILK increased cell adhesivity to a variety of specific matrix substratum components (collagen I, vitronectin, fibronectin, and fibrinogen) but impaired cell spreading, migration, and capillary morphogenesis; these effects were attributed to destabilization of cell matrix through alterations in surface distribution of ␣v␤3 and ␣5␤1 integrins and were not accompanied by changes in Akt or Ser-9 GSK3␤ phosphorylation (27). In HUVEC, ILK was shown to associate with VEGF receptor (57), whereas transduction with dominant negative, kinase deficient ILK, or pharmacological inhibition of ILK resulted in inhibition of a number of VEGFinduced responses, including adherence to collagen 1, phosphorylation of Ser-473 Akt, migration, survival, proliferation, and capillary morphogenesis (35,57,60). In both HUVEC and endothelial progenitor cells, ILK plays a key role in cell survival signaling responses ( Ser-473 Akt or Ser-9 GSK3␤ phosphorylation) to anchorage deprivation, nutrient deprivation or hypoxia; furthermore, with the use of a model of mouse hind limb ischemia, ILK was demonstrated to promote endothelial progenitor cell recruitment and neovascularization of ischemic tissue (61,62).…”

Section: Discussionmentioning

confidence: 99%

Integrin‐linked kinase is an essential mediator for T‐cadherin‐dependent signaling via Akt and GSK3β in endothelial cells

Joshi¹,

Ivanov²,

Philippova³

et al. 2007

FASEB j.

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Glycosylphosphatidylinositol-anchored T-cadherin (T-cad) influences several parameters of angiogenesis including endothelial cell (EC) differentiation, migration, proliferation, and survival. This presupposes signal transduction networking via mediatory regulators and molecular adaptors since T-cad lacks transmembrane and cytosolic domains. Here, using pharmacological inhibition of PI3K, adenoviral-mediated T-cad-overexpression, siRNA-mediated T-cad-depletion, and agonistic antibody-mediated ligation, we demonstrate signaling by T-cad through PI3K-Akt-GSK3beta pathways in EC. T-cad-overexpressing EC exhibited increased levels and nuclear accumulation of active beta-catenin, which was transcriptionally active as shown by increased Lef/Tcf reporter activity and cyclin D1 levels. Cotransduction of EC with constitutively active GSK3beta (S9A-GSK3beta) abrogated the stimulatory effects of T-cad on active beta-catenin accumulation, proliferation, and survival. Integrin-linked kinase (ILK), a membrane proximal upstream regulator of Akt and GSK3beta, was considered a candidate signaling mediator for T-cad. T-cad was present in anti-ILK immunoprecipitates, and confocal microscopy revealed colocalization of T-cad and ILK within lamellipodia of migrating cells. ILK-siRNA abolished T-cad-dependent effects on (Ser-473)Akt/(Ser-9)GSK3beta phosphorylation, active beta-catenin accumulation, and survival. We conclude ILK is an essential mediator for T-cad signaling via Akt and GSK3beta in EC. This is the first demonstration that ILK can regulate inward signaling by GPI-anchored proteins. Furthermore, ILK-GSK3beta-dependent modulation of active beta-catenin levels by GPI-anchored T-cad represents a novel mechanism for controlling cellular beta-catenin activity.

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Section: Discussionmentioning

confidence: 99%

Integrin‐linked kinase is an essential mediator for T‐cadherin‐dependent signaling via Akt and GSK3β in endothelial cells

Joshi¹,

Ivanov²,

Philippova³

et al. 2007

FASEB j.

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“…Angiogenesis is driven by the tumor's release of vascular endothelial growth factor (VEGF), which binds to the receptors on endothelial cells of nearby blood vessels causing the endothelial cells to proliferate and bud from the existing blood vessel, and migrate toward the source of proangiogenic signal. ILK regulates hypoxia-inducible factor 1 alpha subunit (HIF-1a)-mediated VEGF expression in glioblastoma and prostate cancer cells, and is essential for VEGFstimulated endothelial cell migration, tube formation and tumor angiogenesis (Tan et al, 2004;Koul et al, 2005;Watanabe et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

Integrin-linked kinase regulates melanoma angiogenesis by activating NF-κB/interleukin-6 signaling pathway

et al. 2011

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Integrin-linked kinase (ILK) is a highly conserved serinethreonine protein kinase involved in cell-extracellular matrix interactions, cytoskeletal organization and cell signaling. Overexpression of ILK in epithelial cells leads to anchorage-independent growth with increased cell cycle progression. Previously, we have shown that ILK upregulation strongly correlates with melanoma progression, invasion and inversely correlates with 5-year survival of melanoma patients. However, the molecular mechanism by which ILK enhances melanoma progression is currently unknown. In the present study, we found that proangiogenic molecule interleukin-6 (IL-6) is the downstream target of ILK in melanoma cells. ILK overexpression increased IL-6, whereas silencing of ILK suppressed IL-6 expression at both messenger RNA and protein levels. ILK also altered the activity and subcellular localization of nuclear factor-kappaB (NF-jB) subunit p65. We further found that ILK enhanced the IL-6 gene transcription by promoting the binding of NF-kB p65 to IL-6 promoter. Moreover, ILK overexpression in melanoma cells enhanced the tube-forming ability of endothelial cells in vitro and microvessel formation in vivo. ILK-induced tube and blood vessel formation of endothelial cells was significantly reduced upon IL-6 inhibition in ILK-overexpressing melanoma cells. To delineate the mechanism by which ILK-induced IL-6 production can enhance angiogenesis, further analysis of the downstream targets of IL-6 signaling showed an increased activity of the signal transducer and activator of transcription 3 (STAT3) in ILK-overexpressing cells. As STAT3 binds to vascular endothelial growth factor (VEGF) promoter, we found that VEGF levels were elevated in ILK-overexpressing cells and declined upon transfection of IL-6 small interfering RNA, suggesting that ILK may regulate VEGF expression through IL-6 pathway by activating STAT3.

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“…Results of VEGF treatment showed that RCE cells formed a capillary-like network after continual stimulation by 10 ng/mL of VEGF 165 for 2 passages. Because the formation of a capillary-like network is the specialized function of endothelial cells [27,30,31] , the RCE cell line established here is indeed an endothelial cell line. This conclusion is coincident with that of human vascular endothelial cells differentiated from human adipose tissue derived mesenchymal stem cells reported by Guan et al [43] .…”

Section: Discussionmentioning

confidence: 99%

“…Chen et al (2002) [30] found that human umbilical vein endothelial cells being cultured on matrigel formed an elaborate capillary-like network at the light microscopic level after treatment with VEGF 165 . In 2005, Watanabe et al [31] postulated that formation of a capillary-like network was a specialized function of endothelial cells, and VEGF promoted the network formation of endothelial cells cultured in different types of gel matrix. Therefore, in vitro formation of a capillary-like network has become a morphological marker for endothelial cell identification.…”

mentioning

confidence: 99%

Establishment of a novel corneal endothelial cell line from domestic rabbit, Oryctolagus curiculus

Fan

Zhao

et al. 2007

SCI CHINA SER C

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To develop a rabbit corneal endothelial (RCE) cell line, in vitro culture of RCE cells was initiated from Oryctolagus curiculus corneas and a novel RCE cell line was established in this study. To initiate the primary culture of RCE cells, corneas from rabbit eyes were sliced and attached into glutin-coated wells with endothelial cell surface down. After being cultured at a time-gradient interval from 48 to 6 h, the corneal slices were detached and reattached into new wells, respectively. Cells in the wells containing only a pure population of RCE cells were collected and cultured in 20% FBS-DMEM/F12 medium containing chondroitin sulfate, ocular extract, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), carboxymethyl-chitosan, N-acetylglucosamine hydrochloride, glucosamine hydrochloride, culture medium of rabbit corneal stromal cells and oxidation-degradation products of chondroitin sulfate at 37 degrees C, 5% CO(2). The cultured RCE cells, in quadrangle and polygonal shapes, proliferated to confluence 3 weeks later. During the subsequent subculture, the shape of RCE cells changed gradually from polygonal to more fibroblastic. A novel RCE cell line, growing at a steady rate, with a population doubling time of 53.8 h, has been established and subcultured to passage 67. Chromosome analysis showed that the RCE cells exhibited chromosomal aneuploidy with the modal chromosome number of 44. The results of immuno-cytochemical staining with neuron specific enolase (NSE) confirmed that the RCE cells were in neuroectodermal origin. Combined with the results of vascular endothelial growth factor (VEGF) treatment and endothelial cell morphology recovery, it can be concluded that the cell line established here is an RCE cell line. This RCE cell line may serve as a useful tool in theoretical researches of mammalian corneal endothelial cells, and may also have potential application in artificial corneal endothelium development.

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Involvement of integrin-linked kinase in capillary/tube-like network formation of human vascular endothelial cells

Cited by 18 publications

References 20 publications

Integrin‐linked kinase is an essential mediator for T‐cadherin‐dependent signaling via Akt and GSK3β in endothelial cells

Integrin‐linked kinase is an essential mediator for T‐cadherin‐dependent signaling via Akt and GSK3β in endothelial cells

Integrin-linked kinase regulates melanoma angiogenesis by activating NF-κB/interleukin-6 signaling pathway

Establishment of a novel corneal endothelial cell line from domestic rabbit, Oryctolagus curiculus

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