Anthony A. Lanahan

Heterozygous Deficiency of PHD2 Restores Tumor Oxygenation and Inhibits Metastasis via Endothelial Normalization

A key function of blood vessels, to supply oxygen, is impaired in tumors because of abnormalities in their endothelial lining. PHD proteins serve as oxygen sensors and may regulate oxygen delivery. We therefore studied the role of endothelial PHD2 in vessel shaping by implanting tumors in PHD2(+/-) mice. Haplodeficiency of PHD2 did not affect tumor vessel density or lumen size, but normalized the endothelial lining and vessel maturation. This resulted in improved tumor perfusion and oxygenation and inhibited tumor cell invasion, intravasation, and metastasis. Haplodeficiency of PHD2 redirected the specification of endothelial tip cells to a more quiescent cell type, lacking filopodia and arrayed in a phalanx formation. This transition relied on HIF-driven upregulation of (soluble) VEGFR-1 and VE-cadherin. Thus, decreased activity of an oxygen sensor in hypoxic conditions prompts endothelial cells to readjust their shape and phenotype to restore oxygen supply. Inhibition of PHD2 may offer alternative therapeutic opportunities for anticancer therapy.

VEGF Receptor 2 Endocytic Trafficking Regulates Arterial Morphogenesis

VEGF is the key growth factor regulating arterial morphogenesis. However, molecular events involved in this process have not been elucidated. Synectin null mice demonstrate impaired VEGF signaling and a marked reduction in arterial morphogenesis. Here, we show that this occurs due to delayed trafficking of VEGFR2-containing endosomes that exposes internalized VEGFR2 to selective dephosphorylation by PTP1b on Y(1175) site. Synectin involvement in VEGFR2 intracellular trafficking requires myosin-VI, and myosin-VI knockout in mice or knockdown in zebrafish phenocopy the synectin null phenotype. Silencing of PTP1b restores VEGFR2 activation and significantly recovers arterial morphogenesis in myosin-VI(-/-) knockdown zebrafish and synectin(-/-) mice. We conclude that activation of the VEGF-mediated arterial morphogenesis cascade requires phosphorylation of the VEGFR2 Y(1175) site that is dependent on trafficking of internalized VEGFR2 away from the plasma membrane via a synectin-myosin-VI complex. This key event in VEGF signaling occurs at an intracellular site and is regulated by a novel endosomal trafficking-dependent process.

The Neuropilin 1 Cytoplasmic Domain Is Required for VEGF-A-Dependent Arteriogenesis

Neuropilin 1 (NRP1) plays an important but ill-defined role in VEGF-A signaling and vascular morphogenesis. We show that mice with a knockin mutation that ablates the NRP1 cytoplasmic tail (Nrp1(cyto)) have normal angiogenesis but impaired developmental and adult arteriogenesis. The arteriogenic defect was traced to the absence of a PDZ-dependent interaction between NRP1 and VEGF receptor 2 (VEGFR2) complex and synectin, which delayed trafficking of endocytosed VEGFR2 from Rab5+ to EAA1+ endosomes. This led to increased PTPN1 (PTP1b)-mediated dephosphorylation of VEGFR2 at Y(1175), the site involved in activating ERK signaling. The Nrp1(cyto) mutation also impaired endothelial tubulogenesis in vitro, which could be rescued by expressing full-length NRP1 or constitutively active ERK. These results demonstrate that the NRP1 cytoplasmic domain promotes VEGFR2 trafficking in a PDZ-dependent manner to regulate arteriogenic ERK signaling and establish a role for NRP1 in VEGF-A signaling during vascular morphogenesis.

Neuropilin-1-VEGFR-2 Complexing Requires the PDZ-binding Domain of Neuropilin-1

Vascular endothelial growth factor (VEGF) acts as a hierarchically high switch of the angiogenic cascade by interacting with its high affinity VEGF receptors and with neuropilin co-receptors. VEGF165 binds to both Neuropilin-1 (NP-1) and VEGFR-2, and it is believed that ligand binding forms an extracellular bridge between both molecules. This leads to complex formation, thereby enhancing VEGFR-2 phosphorylation and subsequent signaling. We found that inhibition of VEGF receptor (VEGFR) phosphorylation reduced complex formation between NP-1 and VEGFR-2, suggesting a functional role of the cytoplasmic domain of VEGFR-2 for complex formation. Correspondingly, deleting the PDZ-binding domain of NP-1 decreased complex formation, indicating that extracellular VEGF165 binding is not sufficient for VEGFR-2-NP-1 interaction. Synectin is an NP-1 PDZ-binding domain-interacting molecule. Experiments in Synectin-deficient endothelial cells revealed reduced VEGFR-2-NP-1 complex formation, suggesting a role for Synectin in VEGFR-2-NP-1 signaling. Taken together, the experiments have identified a novel mechanism of NP-1 interaction with VEGFR-2, which involves the cytoplasmic domain of NP-1.

ERK1/2-Akt1 crosstalk regulates arteriogenesis in mice and zebrafish

Arterial morphogenesis is an important and poorly understood process. In particular, the signaling events controlling arterial formation have not been established. We evaluated whether alterations in the balance between ERK1/2 and PI3K signaling pathways could stimulate arterial formation in the setting of defective arterial morphogenesis in mice and zebrafish. Increased ERK1/2 activity in mouse ECs with reduced VEGF responsiveness was achieved in vitro and in vivo by downregulating PI3K activity, suppressing Akt1 but not Akt2 expression, or introducing a constitutively active ERK1/2 construct. Such restoration of ERK1/2 activation was sufficient to restore impaired arterial development and branching morphogenesis in synectin-deficient mice and synectin-knockdown zebrafish. The same approach effectively stimulated arterial growth in adult mice, restoring arteriogenesis in mice lacking synectin and in atherosclerotic mice lacking both LDL-R and ApoB48. We therefore conclude that PI3K-ERK1/2 crosstalk plays a key role in the regulation of arterial growth and that the augmentation of ERK signaling via suppression of the PI3K signaling pathway can effectively stimulate arteriogenesis.

Vascular Endothelial Growth Factor and Semaphorin Induce Neuropilin-1 Endocytosis via Separate Pathways

The neuropilin (Nrp)1 receptor is essential for both nervous and vascular system development. Nrp1 is unusually versatile, because it transmits both chemoattractive and repulsive signals in response to vascular endothelial growth factor (VEGF)-A and class 3 semaphorins, respectively. Both Nrp1 and VEGF receptor 2 undergo ligand-dependent endocytosis. We sought to establish the endocytic pathway of Nrp1 and to determine whether uptake is required for its signaling. Whereas Nrp1 underwent clathrin-dependent endocytosis in response to VEGFA165 treatment, semaphorin 3C (sema3C) induced lipid raft–dependent endocytosis. The myosin VI PDZ (postsynaptic density 95, Disk large, Zona occludens-1) adaptor protein synectin was essential for Nrp1 trafficking. Sema3C failed to inhibit migration of synectin−/− endothelial cells, mirroring the lower migratory response of these cells to VEGFA165. These results show that the endocytic pathway of Nrp1 is determined by its ligand and that the trafficking of Nrp1 is essential for its signaling.

Inhibition of tumor angiogenesis and growth by a small-molecule multi-FGF receptor blocker with allosteric properties.

Receptor tyrosine kinases (RTK) are targets for anticancer drug development. To date, only RTK inhibitors that block orthosteric binding of ligands and substrates have been developed. Here, we report the pharmacologic characterization of the chemical SSR128129E (SSR), which inhibits fibroblast growth factor receptor (FGFR) signaling by binding to the extracellular FGFR domain without affecting orthosteric FGF binding. SSR exhibits allosteric properties, including probe dependence, signaling bias, and ceiling effects. Inhibition by SSR is highly conserved throughout the animal kingdom. Oral delivery of SSR inhibits arthritis and tumors that are relatively refractory to anti-vascular endothelial growth factor receptor-2 antibodies. Thus, orally-active extracellularly acting small-molecule modulators of RTKs with allosteric properties can be developed and may offer opportunities to improve anticancer treatment.

Transcriptional Profiling in Coronary Artery Disease Indications for Novel Markers of Coronary Collateralization

Background— The development of collateral circulation plays an important role in protecting tissues from ischemic damage, and its stimulation has emerged as one of principal approaches to therapeutic angiogenesis. Clinical observations have documented substantial differences in the extent of collateralization among patients with coronary artery disease (CAD), with some individuals demonstrating marked abundance and others showing nearly complete absence of these vessels. Recent studies have suggested that circulating monocytes play a major role in collateral growth. The present study was undertaken to determine transcriptional profiles of circulating monocytes in CAD patients with different extents of collateral growth. Methods and Results— Monocyte transcriptomes from CAD patients with and without collateral vessels were obtained by use of high-throughput expression profiling. Using a newly developed redundancy-based data mining method, we have identified a set of molecular markers characteristic of a “noncollateralgenic” phenotype. Moreover, we show that these transcriptional abnormalities are independent of the severity of CAD or any other known clinical parameter thought to affect collateral development and correlated with protein expression levels in monocytes and plasma. Conclusions— Monocyte transcription profiling identifies sets of patients with extensive versus poorly developed collateral circulation. Thus, genetic factors may heavily influence coronary collateral vessel growth in CAD and affect prognosis and response to therapeutic interventions.

Syndecan 4 Regulates FGFR1 Signaling in Endothelial Cells by Directing Macropinocytosis

Macropinocytosis controls the kinetics of endothelial signaling initiated by a fibroblast growth factor receptor. Limiting the Signal Through Macropinocytosis Fibroblast growth factor 2 (FGF2) triggers migration and proliferation of endothelial cells by binding to fibroblast growth factor receptor 1 (FGFR1) and the co-receptor syndecan 4 (S4). Activation of FGFR1 initiates signaling through mitogen-activated protein kinases (MAPKs). Elfenbein et al. found that S4 decreased the internalization of FGFR1 through a process called macropinocytosis. Furthermore, S4-mediated macropinocytosis of FGFR1 decreased the amplitude and increased the deactivation kinetics of MAPK signaling. Thus, these results indicate that S4 controls the duration of MAPK activation in response to binding of FGF2 to FGFR1. Fibroblast growth factor 2 (FGF2) induces endothelial cell migration and angiogenesis through two classes of receptors: receptor tyrosine kinases, such as FGF receptor 1 (FGFR1), and heparan sulfate proteoglycans, such as syndecan 4 (S4). We examined the distinct contributions of FGFR1 and S4 in shaping the endothelial response to FGF2. S4 determined the kinetics and magnitude of FGF2-induced mitogen-activated protein kinase (MAPK) signaling by promoting the macropinocytosis of the FGFR1-S4-FGF2 signaling complex. Internalization of the S4 receptor complex was independent of clathrin and dynamin, proceeded from lipid raft–enriched membranes, and required activation of the guanosine triphosphatases RhoG and Rab5. Genetic knockout of S4, disruption of S4 function, or inhibition of Rab5 led to increased endocytosis and MAPK signaling. These data define the mechanism by which FGFR1 and S4 coordinate downstream signaling upon FGF2 stimulation: FGFR1 initiates MAPK signaling, whereas S4-dependent FGFR1 macropinocytosis modulates the kinetics of MAPK activation. Our studies identify S4 as a regulator of MAPK signaling and address the question of how distinct classes of FGFRs individually contribute to signal transduction in endothelial cells.

Endothelial Nuclear Factor-κB–Dependent Regulation of Arteriogenesis and Branching

Background— Arteriogenesis and collateral formation are complex processes requiring integration of multiple inputs to coordinate vessel branching, growth, maturation, and network size. Factors regulating these processes have not been determined. Methods and Results— We used an inhibitor of NF&kgr;B activation (I&kgr;B&agr;SR) under control of an endothelial-specific inducible promoter to selectively suppress endothelial nuclear factor-&kgr;B activation during development, in the adult vasculature, or in vitro. Inhibition of nuclear factor-&kgr;B activation resulted in formation of an excessively branched arterial network that was composed of immature vessels and provided poor distal tissue perfusion. Molecular analysis demonstrated reduced adhesion molecule expression leading to decreased monocyte influx, reduced hypoxia-inducible factor-1&agr; levels, and a marked decrease in &dgr;-like ligand 4 expression with a consequent decrease in Notch signaling. The latter was the principal cause of increased vascular branching as treatment with Jagged-1 peptide reduced the size of the arterial network to baseline levels. Conclusions— These findings identify nuclear factor-&kgr;B as a key regulator of adult and developmental arteriogenesis and collateral formation. Nuclear factor-&kgr;B achieves this by regulating hypoxia-inducible factor-1&agr;–dependent expression of vascular endothelial growth factor-A and platelet-derived growth factor-BB, which are necessary for the development and maturation of the arterial collateral network, and by regulating &dgr;-like ligand 4 expression, which in turn determines the size and complexity of the network.