Endothelial Cell Flow-Mediated Quiescence Is Temporally Regulated and Utilizes the Cell Cycle Inhibitor p27.

BACKGROUND: Endothelial cells regulate their cell cycle as blood vessels remodel and transition to quiescence downstream of blood flow-induced mechanotransduction. Laminar blood flow leads to quiescence, but how flow-mediated quiescence is established and maintained is poorly understood. METHODS: Primary human endothelial cells were exposed to laminar flow regimens and gene expression manipulations, and quiescence depth was analyzed via time-to-cell cycle reentry after flow cessation. Mouse and zebrafish endothelial expression patterns were examined via scRNA-seq (single-cell RNA sequencing) analysis, and mutant or morphant fish lacking p27 were analyzed for endothelial cell cycle regulation and in vivo cellular behaviors. RESULTS: Arterial flow-exposed endothelial cells had a distinct transcriptome, and they first entered a deep quiescence, then transitioned to shallow quiescence under homeostatic maintenance conditions. In contrast, venous flow-exposed endothelial cells entered deep quiescence early that did not change with homeostasis. The cell cycle inhibitor p27 (CDKN1B) was required to establish endothelial flow-mediated quiescence, and expression levels positively correlated with quiescence depth. p27 loss in vivo led to endothelial cell cycle upregulation and ectopic sprouting, consistent with loss of quiescence. HES1 and ID3, transcriptional repressors of p27 upregulated by arterial flow, were required for quiescence depth changes and the reduced p27 levels associated with shallow quiescence. CONCLUSIONS: Endothelial cell flow-mediated quiescence has unique properties and temporal regulation of quiescence depth that depends on the flow stimulus. These findings are consistent with a model whereby flow-mediated endothelial cell quiescence depth is temporally regulated downstream of p27 transcriptional regulation by HES1 and ID3. The findings are important in understanding endothelial cell quiescence misregulation that leads to vascular dysfunction and disease.

Trafficking dynamics of VEGFR1, VEGFR2, and NRP1 in human endothelial cells.

The vascular endothelial growth factor (VEGF) family of cytokines are key drivers of blood vessel growth and remodeling. These ligands act via multiple VEGF receptors (VEGFR) and co-receptors such as Neuropilin (NRP) expressed on endothelial cells. These membrane-associated receptors are not solely expressed on the cell surface, they move between the surface and intracellular locations, where they can function differently. The location of the receptor alters its ability to 'see' (access and bind to) its ligands, which regulates receptor activation; location also alters receptor exposure to subcellularly localized phosphatases, which regulates its deactivation. Thus, receptors in different subcellular locations initiate different signaling, both in terms of quantity and quality. Similarly, the local levels of co-expression of other receptors alters competition for ligands. Subcellular localization is controlled by intracellular trafficking processes, which thus control VEGFR activity; therefore, to understand VEGFR activity, we must understand receptor trafficking. Here, for the first time, we simultaneously quantify the trafficking of VEGFR1, VEGFR2, and NRP1 on the same cells-specifically human umbilical vein endothelial cells (HUVECs). We build a computational model describing the expression, interaction, and trafficking of these receptors, and use it to simulate cell culture experiments. We use new quantitative experimental data to parameterize the model, which then provides mechanistic insight into the trafficking and localization of this receptor network. We show that VEGFR2 and NRP1 trafficking is not the same on HUVECs as on non-human ECs; and we show that VEGFR1 trafficking is not the same as VEGFR2 trafficking, but rather is faster in both internalization and recycling. As a consequence, the VEGF receptors are not evenly distributed between the cell surface and intracellular locations, with a very low percentage of VEGFR1 being on the cell surface, and high levels of NRP1 on the cell surface. Our findings have implications both for the sensing of extracellular ligands and for the composition of signaling complexes at the cell surface versus inside the cell.

A defined clathrin-mediated trafficking pathway regulates sFLT1/VEGFR1 secretion from endothelial cells.

FLT1/VEGFR1 negatively regulates VEGF-A signaling and is required for proper vessel morphogenesis during vascular development and vessel homeostasis. Although a soluble isoform, sFLT1, is often mis-regulated in disease and aging, how sFLT1 is trafficked and secreted from endothelial cells is not well understood. Here we define requirements for constitutive sFLT1 trafficking and secretion in endothelial cells from the Golgi to the plasma membrane, and we show that sFLT1 secretion requires clathrin at or near the Golgi. Perturbations that affect sFLT1 trafficking blunted endothelial cell secretion and promoted intracellular mis-localization in cells and zebrafish embryos. siRNA-mediated depletion of specific trafficking components revealed requirements for RAB27A, VAMP3, and STX3 for post-Golgi vesicle trafficking and sFLT1 secretion, while STX6, ARF1, and AP1 were required at the Golgi. Live-imaging of temporally controlled sFLT1 release from the endoplasmic reticulum showed clathrin-dependent sFLT1 trafficking at the Golgi into secretory vesicles that then trafficked to the plasma membrane. Depletion of STX6 altered vessel sprouting in 3D, suggesting that endothelial cell sFLT1 secretion influences proper vessel sprouting. Thus, specific trafficking components provide a secretory path from the Golgi to the plasma membrane for sFLT1 in endothelial cells that utilizes a specialized clathrin-dependent intermediate, suggesting novel therapeutic targets.

Excess centrosomes disrupt vascular lumenization and endothelial cell adherens junctions.

Proper blood vessel formation requires coordinated changes in endothelial cell polarity and rearrangement of cell-cell junctions to form a functional lumen. One important regulator of cell polarity is the centrosome, which acts as a microtubule organizing center. Excess centrosomes perturb aspects of endothelial cell polarity linked to migration, but whether centrosome number influences apical-basal polarity and cell-cell junctions is unknown. Here, we show that excess centrosomes alter the apical-basal polarity of endothelial cells in angiogenic sprouts and disrupt endothelial cell-cell adherens junctions. Endothelial cells with excess centrosomes had narrower lumens in a 3D sprouting angiogenesis model, and zebrafish intersegmental vessels had reduced perfusion following centrosome overduplication. These results indicate that endothelial cell centrosome number regulates proper lumenization downstream of effects on apical-basal polarity and cell-cell junctions. Endothelial cells with excess centrosomes are prevalent in tumor vessels, suggesting how centrosomes may contribute to tumor vessel dysfunction.

Ultrasound Measurement of Vascular Density to Evaluate Response to Anti-Angiogenic Therapy in Renal Cell Carcinoma.

BACKGROUND: Functional and molecular changes often precede gross anatomical changes, so early assessment of a tumor's functional and molecular response to therapy can help reduce a patient's exposure to the side effects of ineffective chemotherapeutics or other treatment strategies. OBJECTIVE: Our intent was to test the hypothesis that an ultrasound microvascular imaging approach might provide indications of response to therapy prior to assessment of tumor size. METHODS: Mice bearing clear-cell renal cell carcinoma xenograft tumors were treated with antiangiogenic and Notch inhibition therapies. An ultrasound measurement of microvascular density was used to serially track the tumor response to therapy. RESULTS: Data indicated that ultrasound-derived microvascular density can indicate response to therapy a week prior to changes in tumor volume and is strongly correlated with physiological characteristics of the tumors as measured by histology ([Formula: see text]). Furthermore, data demonstrated that ultrasound measurements of vascular density can determine response to therapy and classify between-treatment groups with high sensitivity and specificity. CONCLUSION/SIGNIFICANCE: Results suggests that future applications utilizing ultrasound imaging to monitor tumor response to therapy may be able to provide earlier insight into tumor behavior from metrics of microvascular density rather than anatomical tumor size measurements.

Von Hippel-Lindau mutations disrupt vascular patterning and maturation via Notch.

Von Hippel-Lindau (VHL) gene mutations induce neural tissue hemangioblastomas, as well as highly vascularized clear cell renal cell carcinomas (ccRCCs). Pathological vessel remodeling arises from misregulation of HIFs and VEGF, among other genes. Variation in disease penetrance has long been recognized in relation to genotype. We show Vhl mutations also disrupt Notch signaling, causing mutation-specific vascular abnormalities, e.g., type 1 (null) vs. type 2B (murine G518A representing human R167Q). In conditional mutation retina vasculature, Vhl-null mutation (i.e., UBCCreER/+Vhlfl/fl) had little effect on initial vessel branching, but it severely reduced arterial and venous branching at later stages. Interestingly, this mutation accelerated arterial maturation, as observed in retina vessel morphology and aberrant α-smooth muscle actin localization, particularly in vascular pericytes. RNA sequencing analysis identified gene expression changes within several key pathways, including Notch and smooth muscle cell contractility. Notch inhibition failed to reverse later-stage branching defects but rescued the accelerated arterialization. Retinal vessels harboring the type 2B Vhl mutation (i.e., UBCCreER/+Vhlfl/2B) displayed stage-specific changes in vessel branching and an advanced progression toward an arterial phenotype. Disrupting Notch signaling in type 2B mutants increased both artery and vein branching and restored arterial maturation toward nonmutant levels. By revealing differential effects of the null and type 2B Vhl mutations on vessel branching and maturation, these data may provide insight into the variability of VHL-associated vascular changes - particularly the heterogeneity and aggressiveness in ccRCC vessel growth - and also suggest Notch pathway targets for treating VHL syndrome.

Ultrasound Molecular Imaging of VEGFR-2 in Clear-Cell Renal Cell Carcinoma Tracks Disease Response to Antiangiogenic and Notch-Inhibition Therapy.

Metastatic clear-cell renal cell carcinoma (ccRCC) affects thousands of patients worldwide each year. Antiangiogenic therapy has been shown to have beneficial effects initially, but resistance is eventually developed. Therefore, it is important to accurately track the response of cancer to different therapeutics in order to appropriately adjust the therapy to maximize efficacy. Change in tumor volume is the current gold standard for determining efficacy of treatment. However, functional variations can occur much earlier than measurable volume changes. Contrast-enhanced ultrasound (CEUS) is an important tool for assessing tumor progression and response to therapy, since it can monitor functional changes in the physiology. In this study, we demonstrate how ultrasound molecular imaging (USMI) can accurately track the evolution of the disease and molecular response to treatment. Methods A cohort of NSG (NOD/scid/gamma) mice was injected with ccRCC cells and treated with either the VEGF inhibitor SU (Sunitinib malate, Selleckchem, TX, USA) or the Notch pathway inhibitor GSI (Gamma secretase inhibitor, PF-03084014, Pfizer, New York, NY, USA), or started on SU and later switched to GSI (Switch group). The therapies used in the study focus on disrupting angiogenesis and proper vessel development. SU inhibits signaling of vascular endothelial growth factor (VEGF), which is responsible for the sprouting of new vasculature, and GSI inhibits the Notch pathway, which is a key factor in the correct maturation of newly formed vasculature. Microbubble contrast agents targeted to VEGFR-2 (VEGF Receptor) were delivered as a bolus, and the bound agents were imaged in 3D after the free-flowing contrast was cleared from the body. Additionally, the tumors were harvested at the end of the study and stained for CD31. Results The results show that MI can detect changes in VEGFR-2 expression in the group treated with SU within a week of the start of treatment, while differences in volume only become apparent after the mice have been treated for three weeks. Furthermore, USMI can detect response to therapy in 92% of cases after 1 week of treatment, while the detection rate is only 40% for volume measurements. The amount of targeting for the GSI and Control groups was high throughout the duration of the study, while that of the SU and Switch groups remained low. However, the amount of targeting in the Switch group increased to levels similar to those of the Control group after the treatment was switched to GSI. CD31 staining indicates significantly lower levels of patent vasculature for the SU group compared to the Control and GSI groups. Therefore, the results parallel the expected physiological changes in the tumor, since GSI promotes angiogenesis through the VEGF pathway, while SU inhibits it. Conclusion This study demonstrates that MI can track disease progression and assess functional changes in tumors before changes in volume are apparent, and thus, CEUS can be a valuable tool for assessing response to therapy in disease. Future work is required to determine whether levels of VEGFR-2 targeting correlate with eventual survival outcomes.

Excess centrosomes induce p53-dependent senescence without DNA damage in endothelial cells.

Tumor blood vessels support tumor growth and progression. Centrosomes are microtubule organization centers in cells, and often up to 30% of tumor endothelial cells (ECs) acquire excess (>2) centrosomes. Although excess centrosomes can lead to aneuploidy and chromosome instability in tumor cells, how untransformed ECs respond to excess centrosomes is poorly understood. We found that the frequency of primary human ECs with excess centrosomes was quickly reduced in a p53-dependent manner. Excess centrosomes in ECs were associated with p53 phosphorylation at Ser33, increased p21 levels, and decreased cell proliferation and expression of senescence markers, but independent of DNA damage and apoptosis. Aspects of the senescence-associated phenotype were also observed in mouse ECs that were isolated from tumors with excess centrosomes. Primary ECs with excess centrosomes in vascular sprouts also had elevated Ser33 p53 phosphorylation and expressed senescence markers. Our work demonstrates that nontransformed ECs respond differently to excess centrosomes than do most tumor cells-they undergo senescence in vascular sprouts and vessels, which suggests that pathologic outcomes of centrosome overduplication depend on the transformation status of cells.-Yu, Z., Ruter, D. L., Kushner, E. J., Bautch, V. L. Excess centrosomes induce p53-dependent senescence without DNA damage in endothelial cells.

Dynamic alterations in decoy VEGF receptor-1 stability regulate angiogenesis.

Blood vessel expansion is driven by sprouting angiogenesis of endothelial cells, and is essential for development, wound healing and disease. Membrane-localized vascular endothelial growth factor receptor-1 (mVEGFR1) is an endothelial cell-intrinsic decoy receptor that negatively modulates blood vessel morphogenesis. Here we show that dynamic regulation of mVEGFR1 stability and turnover in blood vessels impacts angiogenesis. mVEGFR1 is highly stable and constitutively internalizes from the plasma membrane. Post-translational palmitoylation of mVEGFR1 is a binary stabilization switch, and ligand engagement leads to depalmitoylation and lysosomal degradation. Trafficking of palmitoylation enzymes via Rab27a regulates mVEGFR1 stability, as reduced levels of Rab27a impaired palmitoylation of mVEGFR1, decreased its stability, and elevated blood vessel sprouting and in vivo angiogenesis. These findings identify a regulatory axis affecting blood vessel morphogenesis that highlights exquisite post-translational regulation of mVEGFR1 in its role as a molecular rheostat.

Blood vessel anastomosis is spatially regulated by Flt1 during angiogenesis.

Blood vessel formation is essential for vertebrate development and is primarily achieved by angiogenesis - endothelial cell sprouting from pre-existing vessels. Vessel networks expand when sprouts form new connections, a process whose regulation is poorly understood. Here, we show that vessel anastomosis is spatially regulated by Flt1 (VEGFR1), a VEGFA receptor that acts as a decoy receptor. In vivo, expanding vessel networks favor interactions with Flt1 mutant mouse endothelial cells. Live imaging in human endothelial cells in vitro revealed that stable connections are preceded by transient contacts from extending sprouts, suggesting sampling of potential target sites, and lowered Flt1 levels reduced transient contacts and increased VEGFA signaling. Endothelial cells at target sites with reduced Flt1 and/or elevated protrusive activity were more likely to form stable connections with incoming sprouts. Target cells with reduced membrane-localized Flt1 (mFlt1), but not soluble Flt1, recapitulated the bias towards stable connections, suggesting that relative mFlt1 expression spatially influences the selection of stable connections. Thus, sprout anastomosis parameters are regulated by VEGFA signaling, and stable connections are spatially regulated by endothelial cell-intrinsic modulation of mFlt1, suggesting new ways to manipulate vessel network formation.

Tumor-Derived Factors and Reduced p53 Promote Endothelial Cell Centrosome Over-Duplication.

Approximately 30% of tumor endothelial cells have over-duplicated (>2) centrosomes, which may contribute to abnormal vessel function and drug resistance. Elevated levels of vascular endothelial growth factor A induce excess centrosomes in endothelial cells, but how other features of the tumor environment affect centrosome over-duplication is not known. To test this, we treated endothelial cells with tumor-derived factors, hypoxia, or reduced p53, and assessed centrosome numbers. We found that hypoxia and elevated levels of bone morphogenetic protein 2, 6 and 7 induced excess centrosomes in endothelial cells through BMPR1A and likely via SMAD signaling. In contrast, inflammatory mediators IL-8 and lipopolysaccharide did not induce excess centrosomes. Finally, down-regulation in endothelial cells of p53, a critical regulator of DNA damage and proliferation, caused centrosome over-duplication. Our findings suggest that some tumor-derived factors and genetic changes in endothelial cells contribute to excess centrosomes in tumor endothelial cells.

Flt-1 (VEGFR-1) coordinates discrete stages of blood vessel formation.

AIMS: In developing blood vessel networks, the overall level of vessel branching often correlates with angiogenic sprout initiations, but in some pathological situations, increased sprout initiations paradoxically lead to reduced vessel branching and impaired vascular function. We examine the hypothesis that defects in the discrete stages of angiogenesis can uniquely contribute to vessel branching outcomes. METHODS AND RESULTS: Time-lapse movies of mammalian blood vessel development were used to define and quantify the dynamics of angiogenic sprouting. We characterized the formation of new functional conduits by classifying discrete sequential stages-sprout initiation, extension, connection, and stability-that are differentially affected by manipulation of vascular endothelial growth factor-A (VEGF-A) signalling via genetic loss of the receptor flt-1 (vegfr1). In mouse embryonic stem cell-derived vessels genetically lacking flt-1, overall branching is significantly decreased while sprout initiations are significantly increased. Flt-1(-/-) mutant sprouts are less likely to retract, and they form increased numbers of connections with other vessels. However, loss of flt-1 also leads to vessel collapse, which reduces the number of new stable conduits. Computational simulations predict that loss of flt-1 results in ectopic Flk-1 signalling in connecting sprouts post-fusion, causing protrusion of cell processes into avascular gaps and collapse of branches. Thus, defects in stabilization of new vessel connections offset increased sprout initiations and connectivity in flt-1(-/-) vascular networks, with an overall outcome of reduced numbers of new conduits. CONCLUSIONS: These results show that VEGF-A signalling has stage-specific effects on vascular morphogenesis, and that understanding these effects on dynamic stages of angiogenesis and how they integrate to expand a vessel network may suggest new therapeutic strategies.

Excess centrosomes perturb dynamic endothelial cell repolarization during blood vessel formation.

Blood vessel formation requires dynamic movements of endothelial cells (ECs) within sprouts. The cytoskeleton regulates migratory polarity, and centrosomes organize the microtubule cytoskeleton. However, it is not well understood how excess centrosomes, commonly found in tumor stromal cells, affect microtubule dynamics and interphase cell polarity. Here we find that ECs dynamically repolarize during sprouting angiogenesis, and excess centrosomes block repolarization and reduce migration and sprouting. ECs with excess centrosomes initially had more centrosome-derived microtubules but, paradoxically, fewer steady-state microtubules. ECs with excess centrosomes had elevated Rac1 activity, and repolarization was rescued by blockade of Rac1 or actomyosin blockers, consistent with Rac1 activity promoting cortical retrograde actin flow and actomyosin contractility, which precludes cortical microtubule engagement necessary for dynamic repolarization. Thus normal centrosome numbers are required for dynamic repolarization and migration of sprouting ECs that contribute to blood vessel formation.

Feeding cancer's sweet tooth: specialized tumour vasculature shuttles glucose in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal neoplasm characterized by a 'fortress' of thick collagen fibres, abundant myofibroblasts, and paradoxically reduced vascularization compared to normal pancreas. Despite these features, PDAC shows no reduction in the uptake of glucose that fuels tumour cell survival. In new work published in The Journal of Pathology, Saiyin and colleagues have identified a novel adaptation of PDAC tumour endothelium; namely, 'hairy-like' basal microvilli that increase the total vascular surface area and correlate with regions of highest glucose uptake. Since basal microvilli are not present on normal pancreatic blood vessels, their presence may add diagnostic value and blocking their function is a potential new treatment strategy for PDAC. This novel finding of basal microvilli on PDAC endothelium is a striking example of how phenotypic plasticity in tumour blood vessels contributes to tumour growth and progression, independent of conventional modes of angiogenesis.

Antiangiogenic VEGF-A in peripheral artery disease.

Vascular endothelial growth factor A (VEGF-A) is a potent proangiogenic cytokine elevated in patients with peripheral artery disease (PAD). A new study links impaired vascular regrowth in PAD to increased expression of an antiangiogenic splice variant of VEGF-A.

Excess centrosomes disrupt endothelial cell migration via centrosome scattering.

Supernumerary centrosomes contribute to spindle defects and aneuploidy at mitosis, but the effects of excess centrosomes during interphase are poorly understood. In this paper, we show that interphase endothelial cells with even one extra centrosome exhibit a cascade of defects, resulting in disrupted cell migration and abnormal blood vessel sprouting. Endothelial cells with supernumerary centrosomes had increased centrosome scattering and reduced microtubule (MT) nucleation capacity that correlated with decreased Golgi integrity and randomized vesicle trafficking, and ablation of excess centrosomes partially rescued these parameters. Mechanistically, tumor endothelial cells with supernumerary centrosomes had less centrosome-localized γ-tubulin, and Plk1 blockade prevented MT growth, whereas overexpression rescued centrosome γ-tubulin levels and centrosome dynamics. These data support a model whereby centrosome-MT interactions during interphase are important for centrosome clustering and cell polarity and further suggest that disruption of interphase cell behavior by supernumerary centrosomes contributes to pathology independent of mitotic effects.

Flt-1 (vascular endothelial growth factor receptor-1) is essential for the vascular endothelial growth factor-Notch feedback loop during angiogenesis.

OBJECTIVE: Vascular endothelial growth factor (VEGF) signaling induces Notch signaling during angiogenesis. Flt-1/VEGF receptor-1 negatively modulates VEGF signaling. Therefore, we tested the hypothesis that disrupted Flt-1 regulation of VEGF signaling causes Notch pathway defects that contribute to dysmorphogenesis of Flt-1 mutant vessels. APPROACH AND RESULTS: Wild-type and flt-1(-/-) mouse embryonic stem cell-derived vessels were exposed to pharmacological and protein-based Notch inhibitors with and without added VEGF. Vessel morphology, endothelial cell proliferation, and Notch target gene expression levels were assessed. Similar pathway manipulations were performed in developing vessels of zebrafish embryos. Notch inhibition reduced flt-1(-/-) embryonic stem cell-derived vessel branching dysmorphogenesis and endothelial hyperproliferation, and rescue of flt-1(-/-) vessels was accompanied by a reduction in elevated Notch targets. Surprisingly, wild-type vessel morphogenesis and proliferation were unaffected by Notch suppression, Notch targets in wild-type endothelium were unchanged, and Notch suppression perturbed zebrafish intersegmental vessels but not caudal vein plexuses. In contrast, exogenous VEGF caused wild-type embryonic stem cell-derived vessel and zebrafish intersegmental vessel dysmorphogenesis that was rescued by Notch blockade. CONCLUSIONS: Elevated Notch signaling downstream of perturbed VEGF signaling contributes to aberrant flt-1(-/-) blood vessel formation. Notch signaling may be dispensable for blood vessel formation when VEGF signaling is below a critical threshold.

CASZ1 promotes vascular assembly and morphogenesis through the direct regulation of an EGFL7/RhoA-mediated pathway.

The formation of the vascular system is essential for embryonic development and homeostasis. However, transcriptional control of this process is not fully understood. Here we report an evolutionarily conserved role for the transcription factor CASZ1 (CASTOR) in blood vessel assembly and morphogenesis. In the absence of CASZ1, Xenopus embryos fail to develop a branched and lumenized vascular system, and CASZ1-depleted human endothelial cells display dramatic alterations in adhesion, morphology, and sprouting. Mechanistically, we show that CASZ1 directly regulates Epidermal Growth Factor-Like Domain 7 (Egfl7). We further demonstrate that defects of CASZ1- or EGFL7-depleted cells are in part due to diminished RhoA expression and impaired focal adhesion localization. Moreover, these abnormal endothelial cell behaviors in CASZ1-depleted cells can be rescued by restoration of Egfl7. Collectively, these studies show that CASZ1 is required to directly regulate an EGFL7/RhoA-mediated pathway to promote vertebrate vascular development.

Building blood vessels in development and disease.

PURPOSE OF REVIEW: This review will examine developmental angiogenesis and tumor-related changes to endothelial cells. RECENT FINDINGS: Processes that govern developmental angiogenesis become dysfunctional in the tumor environment, leading to abnormal tumor endothelial cells and blood vessels. Recent findings suggest that tumor endothelial cells are permanently modified compared with normal counterparts. SUMMARY: Coordination of numerous intracellular and extracellular programs promotes the formation of new blood vessels that are necessary for both development and certain diseases. Developmental angiogenesis uses canonical signaling modalities to effectively assemble endothelial cells into predictable vessel structures, and disruption of critical signaling factors has dramatic effects on blood vessel development. Solid tumors co-opt developmental cues to promote formation of tumor vessels that sustain their growth, but these angiogenic signals are not well regulated and produce endothelial cell dysfunction. Aberrant growth factor signaling contributes to phenotypic changes and acquired irreversible intracellular signaling, cytoskeletal and genetic modifications in endothelial cells of tumor vessels. Permanently altered tumor endothelial cells may represent a significant population.