Haptotaxis of Endothelial Cell Migration Under Flow

Vascular endothelial growth factor (VEGF) is involved in the promotion of endothelial cell proliferation, migration, and capillary formation. These activities are mainly mediated by the VEGFR2 receptor tyrosine kinase that upon stimulation, promotes the activation of numerous proteins including phospholipase Cgamma (PLCgamma), phosphatidylinositol 3-kinase (PI3K), Akt, Src, and ERK1/2. However, the VEGFR2-proximal signaling events leading to the activation of these targets remain ill defined. We have identified the Gab1 adapter as a novel tyrosine-phosphorylated protein in VEGF-stimulated cells. In bovine aortic endothelial cells, Gab1 associates with VEGFR2, Grb2, PI3K, SHP2, Shc, and PLCgamma, and its overexpression enhances VEGF-dependent cell migration. Importantly, silencing of Gab1 using small interfering RNAs leads to the impaired activation of PLCgamma, ERK1/2, Src, and Akt; blocks VEGF-induced endothelial cell migration; and perturbs actin reorganization and capillary formation. In addition, co-expression of VEGFR2 with Gab1 mutants unable to bind SHP2 or PI3K in human embryonic kidney 293 cells and bovine aortic endothelial cells mimics the defects observed in Gab1-depleted cells. Our work thus identifies Gab1 as a novel critical regulatory component of endothelial cell migration and capillary formation and reveals its key role in the activation of VEGF-evoked signaling pathways required for angiogenesis.

Introduction: Surgical wounds are characterized by increased tissue lactate concentrations. As we know now, accumulated lactate in wounds is not a by-product of hypoxia or anaerobic glycolysis in wound healing. In fact, lactate is mostly produced aerobically. Lactate accumulation commonly occurs in the presence of oxygen, especially under inflammatory conditions, and is capable of initiating signals for angiogenesis and connective tissue deposition. The significance of the high lactate levels that characterize healing wounds is not fully understood yet. Therefore, the aim of this study was to investigate the effect of lactate on migration and collagen synthesis in endothelial cells. Material and Methods: Human umbilical vein endothelial cells (HUVEC) were incubated with different concentrations of lactate. Gene expression analysis was performed using oligonucleotid-microarrays. Differentially expressed and selected genes of interest were additionally evaluated using qRT-PCR. HPLC was carried out to detect the hydroxyproline-concentration in cell supernatants. The generation of superoxide (O2-) was evaluated in vivo using a nitroblue tetrazolium test (NBT). Cell migration was assessed using a modified Boyden chamber. VEGF expression in cell supernatant was detected via ELISA. Results are given as mean ±SD or as fold-change to the corresponding control. Results: Lactate induced gene expression of collagen-I al (16 and 24 h: 2-fold) and collagen-IV al mRNA (4 h: 2.5-fold). Similarly, we detected high concentrations of hydroxyproline in the supernatants of lactate stimulated cells (72 h: control 0.20+0.04 vs. lactate 10 mM 0.32+0.02* vs. Laktat 15 mM 0.32+0.03* μg/ml hydroxyproline; *p<0.05). Endothelial migration was increased by the supernatant of lactate stimulated HUVEC in a time-dependent manner (4 h: 37+17%* and 8 h: 52+16%* vs. control; *p<0.05). Concerning lactate stimulated HUVEC, microarray analysis revealed enhanced expression of NADPH oxidaes (NOX-4) (1 h: 7-fold; 8 h: 4-fold), a diminished expression of thioredoxin (Trx) (1 h: 4-fold; 4 h: 3-fold) and superoxide dismutase 2 (SOD2) (4 h: 2-fold; 24 h: 8-fold). The NBT in vivo revealed increased formation of formazaan crystals in lactate stimulated HUVEC. A significant overexpression of VEGF in the supernatant was detected supplying HUVEC with an exogenous superoxide-donor (SIN-1, 1mM) (64+21%; p<0.05). This effect was reversed using a NADPH-inhibitor. Conclusion: Lactate enhances collagen production and migration in endothelial cells. We suggest that this detected effect is- at least partially- mediated by increased generation of superoxide radicals via NADPH activity. The correlation between lactate and modulation of the activity of NADPH provides new insights into the wound healing physiology. Hence, this data might serve for new treatment strategies of chronic non-healing wounds.

Paclitaxel Impairs Endothelial Cell Adhesion But Not Cytokine-Induced Cellular Adhesion Molecule Expression

Adverse microenvironmental stimuli can trigger the endoplasmic reticulum (ER) stress pathway, which initiates the unfolded protein response (UPR), to restore protein-folding homeostasis. Several studies show induction of ER stress during obesity. Chronic UPR has been linked to different mechanisms of disease in obese and diabetic individuals, including insulin resistance (IR) and impaired angiogenesis. Endothelial cell (EC) migration is an initial step for angiogenesis, which is associated with remodeling of existing blood vessels. EC migration occurs according to the leader–follower model, involving coordinated processes of chemotaxis, haptotaxis, and mechanotaxis. Thus, a fine-tuning of EC migration is necessary to provide the right timing to form the required vessels during angiogenesis. ER stress modulates EC migration at different levels, usually impairing migration and angiogenesis, although different effects may be observed depending on the tissue and/or microenvironment. In the context of pregnancy, maternal obesity (MO) induces IR in the offspring. Interestingly, several proteins associated with obesity-induced IR are also involved in EC migration, providing a potential link with the ER stress-dependent alterations observed in obese individuals. Different signaling cascades that converge on cytoskeleton regulation directly impact EC migration, including the Akt and/or RhoA pathways. In addition, ER is the main intracellular reservoir for Ca2+, which plays a pivotal role during EC migration. Therefore, ER stress-related alterations in Ca2+ signaling or Ca2+ levels might also produce distorted EC migration. However, the above findings have been studied in the context of adult obesity, and no information has been reported regarding the effect of MO on fetal EC migration. Here we summarize the state of knowledge about the possible mechanisms by which ER stress and IR might impact EC migration and angiogenesis in fetal endothelium exposed to MO during pregnancy.

Purpose: Microbeam radiation therapy (MRT) is a novel experimental radiotherapy regimen, which delivers high doses of synchrotron-generated X-rays in the form of quasi-parallel arrays of microbeam separated by microplanar spaces. The repair or healing of irradiated regions (Peak) via migration of endothelial cells (EC) from unirradiated regions (Valley) plays an important role in the response of tumors and normal tissues to MRT. It is known that intratumor microenvironment is acidic. We investigated the influence of environmental acidity on the response of EC to ionizing radiation.Materials and methods: Effects of irradiation on the viability, clonogenicity and migration rate of endothelial cells were studied using human umbilical vascular endothelial cells and mouse endothelial cells in pH 7.3 and 6.4 environments.Results: An exposure to acidic environment (pH 6.4) for 2–4 days exerted little effect on the viability of EC. On the other hand, acidic environment significantly retarded the migration of control and irradiated EC. The migration of EC into 2000 μm-wide wound was slower than that into 1000 μm-side wounds.Conclusion: The microenvironmental acidity and the size of beam opening in MRT may greatly affect the repair of irradiated peak regions via migration of EC from unirradiated valley regions.

Effects of media conditioned by a non-metastasizing human salivary gland adenocarcinoma cell clone and metastasizing clones from salivary gland and various other tissues on the proliferation, migration and protease production of bovine aortic endothelial cells in vitro

Sub-Marginal Endothelial Cells Dynamically Regulate Endothelial Sheet Migration.

The effect of fibrin on endothelial cell migration in vitro

Maturation of Embryonic Stem Cells Into Endothelial Cells in an In Vitro Model of Vasculogenesis

Sprouting angiogenesis requires endothelial cell (EC) migration. In glioblastoma (GBM), cancer stem cells (CSC) in the perivascular niche secrete factors that promote EC migration. Here, we identify a direct interaction between the CSCs and ECs that is mediated by RGD-peptide-dependent binding of L1CAM on CSCs with integrin αvβ3 on ECs. Using in vitro assays, we found that this interaction increased migration-associated signaling events in ECs, including activation of integrin αvβ3, ERK, JNK and Akt, and VCAM-1 and E-selectin mRNA expression. Comparison of the effects of co-culturing CSCs with ECs versus the effects of conditioned media from CSCs co-cultured with ECs indicated that the effects of the direct interaction and soluble factors differed quantitatively and qualitatively. The direct interactions stimulated the migration of ECs and primed them to the migratory effects of soluble factors produced by the CSCs. The direct interaction also enhanced expression of pro-survival genes in CSCs and ECs. In GBM biopsies, Sox2-positive tumor cells were localized in close proximity to ECs and injection of β3-negative ECs or L1CAM-negative CSCs into brain slices in organotypic culture resulted in reduced interactions between ECs and CSCs. RGD-peptide treatment of mice with established intracerebral GBM xenografts increased the mean distance of Sox2-positive tumor cells from ECs, decreased integrin αvβ3 activation and decreased vessel surface area. These data show that direct interactions between CSCs and ECs have potent effects on EC migration and angiogenesis in GBM and have implications for the design of angiogenesis-targeted therapies. Citation Format: Monica E. Burgett, Justin D. Lathia, Patrick Roth, Amy S. Nowacki, Ping Huang, Amit Vasanji, Meizhang Li, Tatiana Byzova, Tom Mikkelsen, Shideng Bao, Jeremy Rich, Michael Weller, Candece L. Gladson. L1CAM and integrin αvβ3 mediate direct cell contact between cancer stem cells and endothelial cells: Promotion of endothelial cell migration and survival. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4178. doi:10.1158/1538-7445.AM2015-4178

The expression of urokinase-type plasminogen activator (uPA) receptor (uPAR) correlates with the malignant phenotype of various cancers. The soluble form of uPAR (s-uPAR) is present in the circulation of cancer patients, but the role of s-uPAR in endothelial cell migration is poorly understood. Therefore, we examined the role of tumor-associated s-uPAR on endothelial cell motility and angiogenesis. Here, we present evidence that tumor-associated s-uPAR augments the migration of human umbilical vein endothelial cells (HUVECs). When grown on tumor-conditioned medium, the membrane fraction of HUVECs had increased localization of s-uPAR onto its cell membrane. Colocalization studies for GM1 ganglioside receptor and uPAR further demonstrated s-uPAR recruitment onto lipid rafts of HUVECs. Immunoblot analysis for uPAR in lipid raft fractions confirmed s-uPAR recruiting onto HUVECs' membrane. Further, s-uPAR induced Rac1-mediated cell migration while either function-blocking uPAR antibodies or dominant-negative mutant Rac1 expression in HUVECs-mitigated s-uPAR-enhanced cell migration. In addition, orthotopic implantation of uPAR-overexpressing cells resulted in a significant increase in circulating s-uPAR in blood serum and invasive nature of tumor and tumor vasculature in mice. Collectively, this data provide insight into tumor-associated s-uPAR-directed migration of endothelial cells and its subsequent influence on tumor angiogenesis.

To explore the specific role of TUG1 in regulating the occurrence and progression of diabetic atherosclerosis and its underlying mechanism. TUG1 expressions in coronary artery disease (CAD) tissues, normal arterial tissues, endothelial cells induced by high-dose glucose and tumor necrosis factor-α (TNF-α) were detected by quantitative Real-time polymerase chain reaction (qRT-PCR). The effects of TUG1 on proliferation, migration and cell cycle of human umbilical vein endothelial cells (HUVECs) were detected by cell counting kit-8 (CCK-8), transwell assay and flow cytometry, respectively. Subsequently, protein expressions of proliferation-related genes, cell cycle-related genes and Wnt pathway-related genes were detected by Western blot after altering TUG1 expression in HUVECs. Further rescue experiments were carried out to explore whether TUG1 could regulate diabetic atherosclerosis via Wnt pathway. Overexpressed TUG1 was found in CAD tissues and endothelial cells induced by high-dose glucose and TNF-α compared with those of controls. TUG1 overexpression remarkably promoted proliferation, migration and cell cycle of HUVECs. Protein expressions of β-catenin and c-Myc were upregulated by overexpression of TUG1. Rescue experiments indicated that XAV-939, the inhibitor of Wnt pathway, could partially reverse the increased proliferative and migratory changes in HUVECs induced by TUG1 overexpression. We found that overexpressed TUG1 stimulates proliferation and migration of endothelial cells via Wnt pathway, thereby promoting the occurrence and progression of diabetic atherosclerosis.

Human guanylate binding protein-1 (GBP-1) is a large GTPase that is induced by inflammatory cytokines and acts antiangiogenically through the inhibition of endothelial cell proliferation and migration. In this study, we detected that GBP-1-expressing cells show a significantly reduced spreading and migration on fibronectin matrices. Investigating possible mechanisms of these effects, we found that integrin alpha(4) (ITGA4) was consistently up-regulated at both the RNA and protein level in GBP-1-expressing cell cultures. Inhibition of cell spreading and migration by GBP-1 was dependent on the binding of ITGA4 to fibronectin. The inflammatory cytokines IL-1beta and TNF-alpha induced ITGA4 expression in HUVECs and inhibited spreading and migration. Knockdown of GBP-1 by shRNA abrogated inflammatory cytokine induced ITGA4 expression and restored spreading and migration capabilities of the cells. These results show that inhibition of endothelial cell spreading and migration by inflammatory cytokines is mediated by GBP-1 through induction of ITGA4 expression. Endothelial cell migration is a key process during angiogenesis. Therefore, ITGA4 may be a novel molecular target to modulate angiogenesis in human disease.

Objective: The processes of proliferation and migration in endothelial cells are fundamental to the development of atherosclerosis. Recent studies underscore the critical involvement of long noncoding RNAs (lncRNAs) in atherosclerosis by influencing the proliferation and migration of human umbilical vein endothelial cells (HUVECs). Nonetheless, the precise molecular mechanism of lncRNA matrix metalloproteinase 2 antisense RNA 1 (MMP2-AS1) remains inadequately characterized. Methods: To investigate this, we established a HUVEC model subjected to transforming growth factor-β1, oxidized low-density lipoprotein, and hypoxia, which resulted in the upregulation of lncRNA MMP2-AS1 expression. Further experimentation demonstrated that knockdown of MMP2-AS1 effectively mitigates the YB-1-mediated enhancement of HUVEC proliferation and migration. Results: Therefore, the primary aim of this research was to explore the role of the MMP2-AS1/YB-1 signaling pathway in atherosclerosis. The findings indicate that MMP2-AS1 regulates HUVEC proliferation and migration through YB-1 targeting. Conclusions: These results suggest that the MMP2-AS1/YB-1 axis holds potential as a therapeutic target for atherosclerosis treatment.

Nox4- and Nox2-dependent oxidant production is required for VEGF-induced SERCA cysteine-674 S-glutathiolation and endothelial cell migration