Oxidized LDL signals through Rho-GTPase to induce endothelial cell stiffening and promote capillary formation.

Endothelial biomechanics is emerging as a key factor in endothelial function. Here, we address the mechanisms of endothelial stiffening induced by oxidized LDL (oxLDL) and investigate the role of oxLDL in lumen formation. We show that oxLDL-induced endothelial stiffening is mediated by CD36-dependent activation of RhoA and its downstream target, Rho kinase (ROCK), via inhibition of myosin light-chain phosphatase (MLCP) and myosin light-chain (MLC)2 phosphorylation. The LC-MS/MS analysis identifies 7-ketocholesterol (7KC) as the major oxysterol in oxLDL. Similarly to oxLDL, 7KC induces RhoA activation, MLCP inhibition, and MLC2 phosphorylation resulting in endothelial stiffening. OxLDL also facilitates formation of endothelial branching networks in 3D collagen gels in vitro and induces increased formation of functional blood vessels in a Matrigel plug assay in vivo. Both effects are RhoA and ROCK dependent. An increase in lumen formation was also observed in response to pre-exposing the cells to 7KC, an oxysterol that induces endothelial stiffening, but not to 5α,6α epoxide that does not affect endothelial stiffness. Importantly, loading cells with cholesterol prevented oxLDL-induced RhoA activation and the downstream signaling cascade, and reversed oxLDL-induced lumen formation. In summary, we show that oxLDL-induced endothelial stiffening is mediated by the CD36/RhoA/ROCK/MLCP/MLC2 pathway and is associated with increased endothelial angiogenic activity.

Induced Pluripotent Stem (iPS) Cell Culture Methods and Induction of Differentiation into Endothelial Cells.

The study of stem cell behavior and differentiation in a developmental context is complex, time-consuming, and expensive, and for this reason, cell culture remains a method of choice for developmental and regenerative biology and mechanistic studies. Similar to ES cells, iPS cells have the ability to differentiate into endothelial cells (ECs), and the route for differentiation appears to mimic the developmental process that occurs during the formation of an embryo. Traditional EC induction methods from embryonic stem (ES) cells rely mostly on the formation of embryoid body (EB), which employs feeder or feeder-free conditions in the presence or absence of supporting cells. Similar to ES cells, iPS cells can be cultured in feeder layer or feeder-free conditions. Here, we describe the iPS cell culture methods and induction differentiation of these cells into ECs. We use anti-mouse Flk1 and anti-mouse VE-cadherin to isolate and characterize mouse ECs, because these antibodies are commercially available and their use has been described in the literature, including by our group. The ECs produced by this method have been used by our laboratory, and we have demonstrated their in vivo potential. We also discuss how iPS cells differ in their ability to differentiate into endothelial cells in culture.

Low-dose 6-bromoindirubin-3'-oxime induces partial dedifferentiation of endothelial cells to promote increased neovascularization.

Endothelial cell (EC) dedifferentiation in relation to neovascularization is a poorly understood process. In this report, we addressed the role of Wnt signaling in the mechanisms of neovascularization in adult tissues. Here, we show that a low-dose of 6-bromoindirubin-3'-oxime (BIO), a competitive inhibitor of glycogen synthase kinase-3ß, induced the stabilization of ß-catenin and its subsequent direct interaction with the transcription factor NANOG in the nucleus of ECs. This event induced loss of VE-cadherin from the adherens junctions, increased EC proliferation accompanied by asymmetric cell division (ACD), and formed cellular aggregates in hanging drop assays indicating the acquisition of a dedifferentiated state. In a chromatin immunoprecipitation assay, nuclear NANOG protein bound to the NANOG- and VEGFR2-promoters in ECs, and the addition of BIO activated the NANOG-promoter-luciferase reporter system in a cell-based assay. Consequently, NANOG-knockdown decreased BIO-induced NOTCH-1 expression, thereby decreasing cell proliferation, ACD, and neovascularization. In a Matrigel plug assay, BIO induced increased neovascularization, secondary to the presence of vascular endothelial growth factor (VEGF). Moreover, in a mouse model of hind limb ischemia, BIO augmented neovascularization that was coupled with increased expression of NOTCH-1 in ECs and increased smooth muscle α-actin(+) cell recruitment around the neovessels. Thus, these results demonstrate the ability of a low-dose of BIO to augment neovascularization secondary to VEGF, a process that was accompanied by a partial dedifferentiation of ECs via ß-catenin and the NANOG signaling pathway.

Flk1+ and VE-cadherin+ endothelial cells derived from iPSCs recapitulates vascular development during differentiation and display similar angiogenic potential as ESC-derived cells.

RATIONALE: Induced pluripotent stem (iPS) cells have emerged as a source of potentially unlimited supply of autologous endothelial cells (ECs) for vascularization. However, the regenerative function of these cells relative to adult ECs and ECs derived from embryonic stem (ES) cells is unknown. The objective was to define the differentiation characteristics and vascularization potential of Fetal liver kinase (Flk)1(+) and Vascular Endothelial (VE)-cadherin(+) ECs derived identically from mouse (m)ES and miPS cells. METHODS AND RESULTS: Naive mES and miPS cells cultured in type IV collagen (IV Col) in defined media for 5 days induced the formation of adherent cell populations, which demonstrated similar expression of Flk1 and VE-cadherin and the emergence of EC progenies. FACS purification resulted in 100% Flk1(+) VE-cadherin(+) cells from both mES and miPS cells. Emergence of Flk1(+)VE-cadherin(+) cells entailed expression of the vascular developmental transcription factor Er71, which bound identically to Flk1, VE-cadherin, and CD31 promoters in both populations. Immunostaining with anti-VE-cadherin and anti-CD31 antibodies and microscopy demonstrated the endothelial nature of these cells. Each cell population (unlike mature ECs) organized into well-developed vascular structures in vitro and incorporated into CD31(+) neovessels in matrigel plugs implanted in nude mice in vivo. CONCLUSION: Thus, iPS cell-derived Flk1(+)VE-cadherin(+) cells expressing the Er71 are as angiogenic as mES cell-derived cells and incorporate into CD31(+) neovessels. Their vessel forming capacity highlights the potential of autologous iPS cells-derived EC progeny for therapeutic angiogenesis.

Focal adhesion kinase regulation of neovascularization.

In this review, we discuss the role of focal adhesion kinase (FAK), an intracellular tyrosine kinase, in endothelial cells in relation to neovascularization. Genetic and in vitro studies have identified critical factors, receptor systems, and their intracellular signaling components that regulate the neovasculogenic phenotypes of endothelial cells. Among these factors, FAK appears to regulate several aspects of endothelial cellular behavior, including migration, survival, cytoskeletal organization, as well as cell proliferation. Upon adhesion of endothelial cells to extracellular matrix (ECM) ligands, integrins cluster on the plane of plasma-membrane, while cytoplasmic domains of integrins interact with cytoskeletal proteins and signaling molecules including FAK. However, FAK not only serves as a critical component of integrin signaling, but is also a downstream element of the VEGF/VEGF-receptor and other ligand-receptor systems that regulate neovascularization. A complete understanding of FAK-mediated neovascularization, therefore, should address the molecular and cellular mechanisms that regulate the biology of FAK. Continued research on FAK may, therefore, yield novel therapies to improve treatment modalities for the pathological neovascularization associated with diseases.

Lipid phosphate phosphatase-3 regulates tumor growth via ß-catenin and CYCLIN-D1 signaling.

BACKGROUND: The acquisition of proliferative and invasive phenotypes is considered a hallmark of neoplastic transformation; however, the underlying mechanisms are less well known. Lipid phosphate phosphatase-3 (LPP3) not only catalyzes the dephosphorylation of the bioactive lipid sphingosine-1-phosphate (S1P) to generate sphingosine but also may regulate embryonic development and angiogenesis via the Wnt pathway. The goal of this study was to determine the role of LPP3 in tumor cells. RESULTS: We observed increased expression of LPP3 in glioblastoma primary tumors and in U87 and U118 glioblastoma cell lines. We demonstrate that LPP3-knockdown inhibited both U87 and U118 glioblastoma cell proliferation in culture and tumor growth in xenograft assays. Biochemical experiments provided evidence that LPP3-knockdown reduced ß-catenin, CYCLIN-D1, and CD133 expression, with a concomitant increase in phosphorylated ß-catenin. In a converse experiment, the forced expression of LPP3 in human colon tumor (SW480) cells potentiated tumor growth via increased ß-catenin stability and CYCLIN-D1 synthesis. In contrast, elevated expression of LPP3 had no tumorigenic effects on primary cells. CONCLUSIONS: These results demonstrate for the first time an unexpected role of LPP3 in regulating glioblastoma progression by amplifying ß-catenin and CYCLIN-D1 activities.

NANOG induction of fetal liver kinase-1 (FLK1) transcription regulates endothelial cell proliferation and angiogenesis.

NANOG is a master transcription factor associated with the maintenance of stem cell pluripotency. Here, we demonstrate that transcription factor NANOG is expressed in cultured endothelial cells (ECs) and in a subset of tumor cell lines. Importantly, we provide evidence that WNT3A stimulation of ECs induces the transcription of NANOG which mediates the expression of vascular endothelial growth factor receptor-2, also known as fetal liver kinase-1 (FLK1). We defined ATTA as a minimal binding site for NANOG. Accordingly, a luciferase reporter assay showed that NANOG binds to and activates 4 ATTA binding sites identified in the FLK1 promoter after WNT3A stimulation. Consistent with this data, we found that, under basal conditions and in response to WNT3A stimulation, NANOG binding to these ATTA sequences markedly induced the expression of FLK1. Thus, our data indicate an essential role in angiogenesis for NANOG binding to these 4 ATTA sites. Surprisingly, NANOG depletion not only decreased FLK1 expression but also reduced cell proliferation and angiogenesis. These findings show the necessary and sufficient role of NANOG in inducing the transcription of FLK1 to regulate the angiogenic phenotypes of ECs.

Kruppel-like factor-4 transcriptionally regulates VE-cadherin expression and endothelial barrier function.

RATIONALE: Vascular endothelial (VE)-cadherin localized at adherens junctions (AJs) regulates endothelial barrier function. Because WNT (wingless) signaling-induced activation of the transcription factor Krüppel-like factor (KLF)4 may have an important role in mediating the expression of VE-cadherin and AJ integrity, we studied the function of KLF4 in regulating VE-cadherin expression and the control of endothelial barrier function. OBJECTIVE: The goal of this study was to determine the transcriptional role of KLF4 in regulating VE-cadherin expression and endothelial barrier function. METHODS AND RESULTS: Expression analysis, microscopy, chromatin immunoprecipitation, electrophoretic mobility shift assays, and VE-cadherin-luciferase reporter experiments demonstrated that KLF4 interacted with specific domains of VE-cadherin promoter and regulated the expression of VE-cadherin at AJs. KLF4 knockdown disrupted the endothelial barrier, indicating that KLF4 is required for normal barrier function. In vivo studies in mice showed augmented lipopolysaccharide-induced lung injury and pulmonary edema following Klf4 depletion. CONCLUSION: Our data show the key role of KLF4 in the regulation of VE-cadherin expression at the level of the AJs and in the acquisition of VE-cadherin-mediated endothelial barrier function. Thus, KLF4 maintains the integrity of AJs and prevents vascular leakage in response to inflammatory stimuli.