UPAR Directly Interacts with LDLR-like Proteins to Induce Angiogenic Endothelial Cell Behavior

The plasma clearance of intestinally derived remnant lipoproteins by the liver is a process that likely involves three steps. Our model suggests that the initial rapid clearance by the liver begins with sequestration of the remnants within the space of Disse, where apolipoprotein E secreted by hepatocytes enhances remnant binding and uptake. Heparan sulfate proteoglycans (HSPG), which are also abundant in the space of Disse, mediate this enhanced binding. Next, the remnants undergo further processing in the space of Disse by hepatic and lipoprotein lipases, which may also serve as ligands mediating remnant uptake. The final step, endocytosis by hepatocytes, appears to be mediated, at least in part, by the low density lipoprotein (LDL) receptor and by the LDL receptor-related protein (LRP). Cell-surface HSPG play a critical role in remnant uptake, not only in the important initial sequestration or capture step in the space of Disse, but also as an essential or integral component of the HSPG-LRP pathway. In addition, HSPG appear to function alone as a receptor and display unique handling properties for specific isoforms of apolipoprotein E.

Angiogenesis, the sprouting of blood vessels form pre-existing vasculature after injury or in neoplastic diseases, is initiated by growth factor-induced endothelial cell migration. Recently, the major angiogenic growth factor VEGF165 has become the target of therapeutic interventions. However, this approach has been clinically proven to be of limited efficacy, which might be due to the fact that tumour angiogenesis is not only induced by VEGF, but also by a variety of other growth factors. Thus, the identification of a common downstream mediator of growth-factor-induced endothelial cell migration is mandatory to effectively interfere with (tumour-) angiogenesis. We found that the urokinase-type plasminogen activator (uPA)-system, which affects proteolytic as well as adhesive capacities, represents an essential regulatory mechanism in growth factor-induced endothelial cell migration and invasion. This mechanism was not limited to VEGF165, but mediated pro-angiogenic endothelial cell behaviour induced by various growth factors. Thus, VEGF165, VEGF-E, FGF-2, EGF as well as HGF induced a PI3k-dependent activation of pro-uPA when bound to uPAR, which led to an increase in cell surface fibrinolytic activity. As a consequence, uPAR became internalised and redistributed via LDLR-proteins. Interference with these events led to a reduced migratory response of endothelial cells towards VEGF in vitro as well as endothelial cell invasion in vivo. These data give first evidence that the uPA-system, which represents the only level-of-evidence-1 cancer biomarker system for prognosis and/or prediction in node negative breast cancer, might directly affect (tumour-) angiogenesis.

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.

ObjectiveInsulin resistance is associated with impaired receptor dependent hepatic uptake of triglyceride-rich lipoproteins (TRL), promoting hypertriglyceridemia and atherosclerosis. Next to low-density lipoprotein (LDL) receptor (LDLR) and syndecan-1, the LDLR-related protein 1 (LRP1) stimulated by insulin action contributes to the rapid clearance of TRL in the postprandial state. Here, we investigated the hypothesis that the adaptor protein phosphotyrosine interacting domain-containing protein 1 (PID1) regulates LRP1 function, thereby controlling hepatic endocytosis of postprandial lipoproteins. MethodsLocalization and interaction of PID1 and LRP1 in cultured hepatocytes was studied by confocal microscopy of fluorescent tagged proteins, by indirect immunohistochemistry of endogenous proteins, by GST-based pull down and by immunoprecipitation experiments. The in vivo relevance of PID1 was assessed using whole body as well as liver-specific Pid1-deficient mice on a wild type or Ldlr-deficient (Ldlr−/−) background. Intravital microscopy was used to study LRP1 translocation in the liver. Lipoprotein metabolism was investigated by lipoprotein profiling, gene and protein expression as well as organ-specific uptake of radiolabelled TRL. ResultsPID1 co-localized in perinuclear endosomes and was found associated with LRP1 under fasting conditions. We identified the distal NPxY motif of the intracellular C-terminal domain (ICD) of LRP1 as the site critical for the interaction with PID1. Insulin-mediated NPxY-phosphorylation caused the dissociation of PID1 from the ICD, causing LRP1 translocation to the plasma membrane. PID1 deletion resulted in higher LRP1 abundance at the cell surface, higher LDLR protein levels and, paradoxically, reduced total LRP1. The latter can be explained by higher receptor shedding, which we observed in cultured Pid1-deficient hepatocytes. Consistently, PID1 deficiency alone led to increased LDLR-dependent endocytosis of postprandial lipoproteins and lower plasma triglycerides. In contrast, hepatic PID1 deletion on an Ldlr−/− background reduced lipoprotein uptake into liver and caused plasma TRL accumulation. ConclusionsBy acting as an insulin-dependent retention adaptor, PID1 serves as a regulator of LRP1 function controlling the disposal of postprandial lipoproteins. PID1 inhibition provides a novel approach to lower plasma levels of pro-atherogenic TRL remnants by stimulating endocytic function of both LRP1 and LDLR in the liver.

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

CD87 (urokinase-type plasminogen activator receptor, uPAR) expression has been correlated with tumor progression most likely via its functional ole as a central receptor for extracellular proteolysis, but also via its role in initiating intracellular signal transduction. The role of CD87 in angiogenesis has recently been elucidated. Thus, we observed that in VEGF-stimulated ndothelial cells CD87 is essential for endothelial cell survival as well as for n efficient growth factor- induced endothelial cell migration. Thereby, the PI-anchored protein CD87 formed a complex with its ligand urokinase uPA) as well as the inhibitor plasminogen activator inhibitor-1 (PAI-1), which became redistributed to the leading edge via low density lipoprotein receptor- (LDLR-) like proteins. In this study, we analyzed the functional role of CD87 in integrin redistribution during growth factor-induced endothelial cell migration. CD87, thereby, directly interacted with integrin adhesion receptors (a5b1) as well as with LDLR- proteins. Either by interfering with CD87/integrin nteraction or by interfering with CD87/LDLR-protein interaction by using specific interfering peptides, we observed disruption of integrin redistribution to the leading edge in migrating cells. As a consequence diminished focal adhesion formations were observed and endothelial cell migration was diminished. In CD87-deficient HEK 293 cells as well as in endothelial cells derived from CD87 -/- mice, expression of CD87 mutants (mutL3/uPAR), which lacked the binding motif for LDLR, led to an impaired cell spreading as well as a diminished migratory response towards VEGF, while full length CD87 (wt/CD87) reconstituted this phenotype. That CD87 plays a central role in growth factor induced endothelial cell invasion and capillary-like tube formation in vivo was demonstrated by a directed in vivo angiogenesis assay (DIVAA), which was performed either in CD87 deficient or wild type animals in the presence or absence of interfering peptides. Our data give novel insights into the central role of CD87 in pro- angiogenic endothelial cell behavior as well as in its role in angiogenesis. Thus, CD87 might lead to a novel therapeutic strategies in tumor- angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4360. doi:1538-7445.AM2012-4360

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

Rho GDP-dissociation inhibitor (RhoGDI), a downregulator of Rho family GTPases, prevents nucleotide exchange and membrane association. It is responsible for the activation of Rho GTPases, which regulate a variety of cellular processes, such as migration. Although RhoGDI2 has been identified as a tumor suppressor gene involved in cellular migration and invasion, little is known about its role in vascular endothelial cell (EC) migration. CR6-interacting factor 1 (CRIF1) is a CR6/GADD45-interacting protein with important mitochondrial functions and regulation of cell growth. We examined the expression of RhoGDI2 in CRIF1-deficient human umbilical vein endothelial cells (HUVECs) and its role in cell migration. Expression of RhoGDI2 was found to be considerably higher in CRIF1-deficient HUVECs along with suppression of cell migration. Moreover, the phosphorylation levels of Akt and CREB were decreased in CRIF1-silenced cells. The Akt-CREB signaling pathway was implicated in the changes in endothelial cell migration caused by CRIF1 downregulation. In addition to RhoGDI2, we identified another factor that promotes migration and invasion of ECs. Adrenomedullin2 (ADM2) is an autocrine/paracrine factor that regulates vascular tone and other vascular functions. Endogenous ADM2 levels were elevated in CRIF1-silenced HUVECs with no effect on cell migration. However, siRNA-mediated depletion of RhoGDI2 or exogenous ADM2 administration significantly restored cell migration via the Akt-CREB signaling pathway. In conclusion, RhoGDI2 and ADM2 play important roles in the migration of CRIF1-deficient endothelial cells.

The aggressive and malignant nature of AIDS-associated Kaposi's sarcoma (KS) lesions have largely been ascribed to Tat, the HIV-1 transactivator protein. Among other activities, HIV-Tat induces the migration and invasion of KS and endothelial cells. Since cell invasion is strictly correlated to the activity of lytic enzymes, we elucidated the role of the cell-associated plasminogen activation system in Tat-dependent and in constitutive invasion and proliferation of KS and of microvascular endothelial cells (MVEC). We demonstrate that KS cells and MVEC express the u-PA receptor (u-PAR) and release plasminogen activators and plasminogen activator inhibitor type-1 (PAI-1). The urokinase-type plasminogen activator (u-PA) is chemotactic, chemoinvasive and mitogenic for KS cells and for MVEC. Conditioned medium from KS cells induced invasion and proliferation of MVEC through the u-PA/u-PAR system. Tat is motogenic and mitogenic on KS cells and MVEC, and stimulates morphogenesis of MVEC. These activities were inhibited following antagonization of u-PA and u-PAR, which also reduced constitutive proliferation and invasion of KS cells and MVEC. These data indicate that the u-PA/u-PAR/PAI-1 system is involved in KS-induced endothelial cell invasion, proliferation, and differentiation. Further, exogenous Tat protein could up-regulate the fibrinolytic system, increasing its influence on KS and endothelial cell proliferation and migration, potentially promoting KS progression. These observations suggest the potential for application of u-PA/u-PAR system inhibitors for control of AIDS-associated KS, that has a high risk of recurrence with highly active antiretroviral therapy failure, and of other KS forms.

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

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.

Endothelial cell (EC) migration plays an important role in embryonic vasculogenesis, angiogenesis, and wound healing. EC migration can be regulated by extracellular matrix (ECM) and hemodynamic forces through haptotaxis (induced by an ECM gradient) and mechanotaxis (induced by mechanical forces). Previously, the effects of haptotaxis or mechanotaxis alone on EC migration have been studied; however, the dual effect of haptotaxis and mechanotaxis on EC migration is not known. We developed a micropatterning technique to generate step changes of collagen surface density to monitor haptotactic EC migration. To investigate the crosstalk between haptotaxis and mechanotaxis on EC migration, we used an in vitro flow system to apply a well-defined fluid shear stress on ECs cultured on the micropatterned collagen. The study on the effects of haptotaxis and mechanotaxis on EC migration will provide a rational basis for promoting vascular wound healing, angiogenesis, and vascularization in engineered tissues.

High and low molecular weight kininogens (HK and LK) are able to bind to platelets to inhibit thrombin binding to and activation of platelets. The heavy chain domain on the kininogens that contains these functions has been determined. Domain 3 (D3) but not domains 1 or 2, completely inhibited 125I-HK binding to platelets (Ki = 24 +/- 7 nM, n = 4). 125I-D3 specifically bound to unstimulated platelets and human umbilical vein endothelial cells. On platelets, it was blocked by unlabeled D3 and HK but not prekallikrein, factor XII, C1s, or C1 inhibitor. Further, one monoclonal antibody (HKH13) directed to kininogens' D3 blocked 125I-HK and 125I-D3 binding to platelets. The binding of 125I-D3 to platelets was fully reversible by addition of 35 molar excess of unlabeled D3. D3 binding to platelets was saturable with an apparent Kd of 39 +/- 8 nM (n = 4) and 1227 +/- 404 binding sites/platelet. D3, like HK and LK, inhibited thrombin-induced platelet activation by preventing thrombin binding to platelets. Another monoclonal antibody (HKH12), directed to D3, which did not block HK binding to platelets, reduced HK's ability to inhibit 125I-alpha-thrombin binding. This result suggests that the region on D3 that inhibits 125I-alpha-thrombin binding to platelets is different from that which directly binds to platelets. These studies indicate that D3 of the kininogens contains both a binding region for platelets and endothelial cells and another region that inhibits thrombin-induced platelet activation.

The absorption of cholesterol by the small intestine is a major route for the net entry of cholesterol into the body and can therefore affect the plasma low density lipoprotein-cholesterol (LDL-C) concentration. These studies used ezetimibe, a potent inhibitor of cholesterol absorption, to delineate the biochemical and molecular changes in intrahepatic metabolism and biliary lipid secretion when there is a major reduction in chylomicron cholesterol delivery to the liver. In female LDL receptor (LDLR)-deficient (LDLR-/-) mice fed a basal diet containing ezetimibe (0-10 mg/day/kg body weight), cholesterol absorption was reduced up to 91%, fecal neutral sterol excretion was increased up to 4.7-fold, and plasma total cholesterol concentrations decreased by up to 18%. Blocking cholesterol absorption prevented the accumulation of very low density lipoproteins and LDL in the circulation of LDLR-/- mice fed a lipid-rich diet. In female LDLR+/+ mice fed the lipid-rich diet with ezetimibe, the relative mRNA level for the LDLR in the liver was 2-fold greater than in matching mice given the lipid-rich diet alone. We conclude that in the mouse the reduction in plasma LDL-C levels induced by blocking cholesterol absorption reflects both a diminished rate of LDL-C production and a modest increase in hepatic LDLR expression.