[Show abstract][Hide abstract] ABSTRACT: Despite the clear major contribution of hyperlipidemia to the prevalence of cardiovascular disease in the developed world, the direct effects of lipoproteins on endothelial cells have remained obscure and are under debate. Here we report a previously uncharacterized mechanism of vessel growth modulation by lipoprotein availability. Using a genetic screen for vascular defects in zebrafish, we initially identified a mutation, stalactite (stl), in the gene encoding microsomal triglyceride transfer protein (mtp), which is involved in the biosynthesis of apolipoprotein B (ApoB)-containing lipoproteins. By manipulating lipoprotein concentrations in zebrafish, we found that ApoB negatively regulates angiogenesis and that it is the ApoB protein particle, rather than lipid moieties within ApoB-containing lipoproteins, that is primarily responsible for this effect. Mechanistically, we identified downregulation of vascular endothelial growth factor receptor 1 (VEGFR1), which acts as a decoy receptor for VEGF, as a key mediator of the endothelial response to lipoproteins, and we observed VEGFR1 downregulation in hyperlipidemic mice. These findings may open new avenues for the treatment of lipoprotein-related vascular disorders.
Nature medicine 05/2012; 18(6):967-73. · 27.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CCN2/Connective Tissue Growth Factor (CTGF) is a matricellular protein that regulates cell adhesion, migration, and survival. CCN2 is best known for its ability to promote fibrosis by mediating the ability of transforming growth factor β (TGFβ) to induce excess extracellular matrix production. In addition to its role in pathological processes, CCN2 is required for chondrogenesis. CCN2 is also highly expressed during development in endothelial cells, suggesting a role in angiogenesis. The potential role of CCN2 in angiogenesis is unclear, however, as both pro- and anti-angiogenic effects have been reported. Here, through analysis of Ccn2-deficient mice, we show that CCN2 is required for stable association and retention of pericytes by endothelial cells. PDGF signaling and the establishment of the endothelial basement membrane are required for pericytes recruitment and retention. CCN2 induced PDGF-B expression in endothelial cells, and potentiated PDGF-B-mediated Akt signaling in mural (vascular smooth muscle/pericyte) cells. In addition, CCN2 induced the production of endothelial basement membrane components in vitro, and was required for their expression in vivo. Overall, these results highlight CCN2 as an essential mediator of vascular remodeling by regulating endothelial-pericyte interactions. Although most studies of CCN2 function have focused on effects of CCN2 overexpression on the interstitial extracellular matrix, the results presented here show that CCN2 is required for the normal production of vascular basement membranes.
PLoS ONE 01/2012; 7(2):e30562. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Vascular endothelial growth factor (VEGF) is known to activate proliferation, migration, and survival pathways in endothelial cells through phosphorylation of VEGF receptor-2 (VEGFR-2). VEGF has been incorporated into biomaterials through encapsulation, electrostatic sequestration, and covalent attachment, but the effect of these immobilization strategies on VEGF signaling has not been thoroughly investigated. Further, although growth factor internalization along with the receptor generally occurs in a physiological setting, whether this internalization is needed for receptor phosphorylation is not entirely clear. Here we show that VEGF covalently bound through a modified heparin molecule elicits an extended response of pVEGFR-2 in human umbilical vein endothelial cells (HUVECs) and that the covalent linkage reduces internalization of the growth factor during receptor endocytosis. Optical tweezer measurements show that the rupture force required to disrupt the heparin-VEGF-VEGFR-2 interaction increases from 3-8 pN to 6-12 pN when a covalent bond is introduced between VEGF and heparin. Importantly, by covalently binding VEGF to a heparin substrate, the stability (half-life) of VEGF is extended over three-fold. Here, mathematical models support the biological conclusions, further suggesting that VEGF internalization is significantly reduced when covalently bound, and indicating that VEGF is available for repeated phosphorylation events.
[Show abstract][Hide abstract] ABSTRACT: VEGF can be secreted in multiple isoforms with variable affinity for extracellular proteins and different abilities to induce vascular morphogenesis, but the molecular mechanisms behind these effects remain unclear. Here, we show molecular distinctions between signaling initiated from soluble versus matrix-bound VEGF, which mediates a sustained level of VEGFR2 internalization and clustering. Exposure of endothelial cells to matrix-bound VEGF elicits prolonged activation of VEGFR2 with differential phosphorylation of Y1214, and extended activation kinetics of p38. These events require association of VEGFR2 with beta1 integrins. Matrix-bound VEGF also promotes reciprocal responses on beta1 integrin by inducing its association with focal adhesions; a response that is absent upon exposure to soluble VEGF. Inactivation of beta1 integrin blocks the prolonged phosphorylation of Y1214 and consequent activation of p38. Combined, these results indicate that when in the context of extracellular matrix, activation of VEGFR2 is distinct from that of soluble VEGF in terms of recruitment of receptor partners, phosphorylation kinetics, and activation of downstream effectors.
The Journal of Cell Biology 02/2010; 188(4):595-609. · 10.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Growth factors are a class of signaling proteins that direct cell fate through interaction with cell-surface receptors. Although a myriad of possible cell fates stems from a growth factor binding to its receptor, the signaling cascades that result in one fate over another are still being elucidated. One possible mechanism by which nature modulates growth factor signaling is through the method of presentation of the growth factor--soluble or immobilized (matrix bound). Here we present the methodology to study signaling of soluble versus immobilized VEGF through VEGFR-2. We have designed a strategy to covalently immobilize VEGF using its heparin-binding domain to orient the molecule (bind) and a secondary functional group to mediate covalent binding (lock). This bind-and-lock approach aims to allow VEGF to assume a bioactive orientation before covalent immobilization. Surface plasmon resonance (SPR) demonstrated heparin and VEGF binding with surface densities of 60 ng/cm2 and 100 pg/cm2, respectively. ELISA experiments confirmed VEGF surface density and showed that electrostatically bound VEGF releases in cell medium and heparin solutions while covalently bound VEGF remains immobilized. Electrostatically bound VEGF and covalently bound VEGF phosphorylate VEGFR-2 in both VEGFR-2 transfected cells and VEGFR-2 endogenously producing cells. HUVECs plated on VEGF functionalized surfaces showed different morphologies between surface-bound VEGF and soluble VEGF. The surfaces synthesized in these studies allow for the study of VEGF/VEGFR-2 signaling induced by covalently bound, electrostatically bound, and soluble VEGF and may provide further insight into the design of materials for the generation of a mature and stable vasculature.
[Show abstract][Hide abstract] ABSTRACT: Vascular endothelial growth factor (VEGF) is essential for developmental and pathological angiogenesis. Here we show that in the absence of any pathological insult, autocrine VEGF is required for the homeostasis of blood vessels in the adult. Genetic deletion of vegf specifically in the endothelial lineage leads to progressive endothelial degeneration and sudden death in 55% of mutant mice by 25 weeks of age. The phenotype is manifested without detectable changes in the total levels of VEGF mRNA or protein, indicating that paracrine VEGF could not compensate for the absence of endothelial VEGF. Furthermore, wild-type, but not VEGF null, endothelial cells showed phosphorylation of VEGFR2 in the absence of exogenous VEGF. Activation of the receptor in wild-type cells was suppressed by small molecule antagonists but not by extracellular blockade of VEGF. These results reveal a cell-autonomous VEGF signaling pathway that holds significance for vascular homeostasis but is dispensable for the angiogenic cascade.
[Show abstract][Hide abstract] ABSTRACT: Growth factors are increasingly employed to promote tissue regeneration with various biomaterial scaffolds. In vitro release kinetics of protein growth factors from tissue engineering scaffolds are often investigated in aqueous environment, which is significantly different from in vivo environment. This study investigates the release of model proteins with net-positive (histone) and net-negative charge (bovine serum albumin, BSA) from various scaffolding surfaces and from encapsulated microspheres in the presence of ions, proteins, and cells. The release kinetics of proteins in media with varying concentrations of ions (NaCl) suggests stronger electrostatic interaction between the positively charged histone with the negatively charged substrates. While both proteins released slowly from hydrophobic PCL surfaces, plasma etching resulted in rapid release of BSA, but not histone. Interestingly, although negatively charged BSA released readily from negatively charged collagen (col), BSA released slowly from col-coated PCL scaffolds. Such electrostatic interaction effects were abolished in the presence of serum proteins and cells as evidenced by the rapid release of proteins from col-coated scaffolds. To achieve sustained release in the complex environment of serum proteins and cells, the model proteins were encapsulated into poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, which were embedded within col-coated PCL scaffolds. Protein release from microspheres was modulated by changing the lactide-to-glycolide ratio of PLGA polymer. BSA adsorbed to col released faster than histone encapsulated in microspheres in the presence of serum and cells. Collectively, the data suggest that growth factor release is highly influenced by scaffold surface and the presence of ions, proteins, and cells in the media. Strategies to deliver multiple growth factors and studies which investigate their release should consider these important variables.
[Show abstract][Hide abstract] ABSTRACT: Previous studies have shown that oxidized products of PAPC (Ox-PAPC) regulate cell transcription of interleukin-8, LDL receptor, and tissue factor. This upregulation takes place in part through the activation of sterol regulatory element-binding protein (SREBP) and Erk 1/2. The present studies identify vascular endothelial growth factor receptor 2 (VEGFR2) as a major regulator in the activation of SREBP and Erk 1/2 in endothelial cells activated by Ox-PAPC.
Ox-PAPC induced the phosphorylation of VEGFR2 at Tyr1175 in human aortic endothelial cells. Inhibitors and siRNA for VEGFR2 decreased the transcription of interleukin-8, LDL receptor, and tissue factor in response to Ox-PAPC and the activation of SREBP and Erk 1/2, which mediate this transcription. We provide evidence that the activation of VEGFR2 is rapid, sustained, and c-Src-dependent.
These data point to a major role of VEGFR2 in endothelial regulation by oxidized phospholipids which accumulate in atherosclerotic lesions and apoptotic cells.