Pathological angiogenesis is induced by sustained Akt signaling and inhibited by rapamycin.

Endothelial cells in growing tumors express activated Akt, which when modeled by transgenic endothelial expression of myrAkt1 was sufficient to recapitulate the abnormal structural and functional features of tumor blood vessels in nontumor tissues. Sustained endothelial Akt activation caused increased blood vessel size and generalized edema from chronic vascular permeability, while acute permeability in response to VEGF-A was unaffected. These changes were reversible, demonstrating an ongoing requirement for Akt signaling for the maintenance of these phenotypes. Furthermore, rapamycin inhibited endothelial Akt signaling, vascular changes from myrAkt1, tumor growth, and tumor vascular permeability. Akt signaling in the tumor vascular stroma was sensitive to rapamycin, suggesting that rapamycin may affect tumor growth in part by acting as a vascular Akt inhibitor.

Vascular tumors have increased p70 S6-kinase activation and are inhibited by topical rapamycin.

Vascular tumors are endothelial cell neoplasms whose cellular and molecular mechanisms, leading to tumor formation, are poorly understood, and current therapies have limited efficacy with significant side effects. We have investigated mechanistic (mammalian) target of rapamycin (mTOR) signaling in benign and malignant vascular tumors, and the effects of mTOR kinase inhibitor as a potential therapy for these lesions. Human vascular tumors (infantile hemangioma and angiosarcoma) were analyzed by immunohistochemical stains and western blot for the phosphorylation of p70 S6-kinase (S6K) and S6 ribosomal protein (S6), which are activated downstream of mTOR complex-1 (mTORC1). To assess the function of S6K, tumor cells with genetic knockdown of S6K were analyzed for cell proliferation and migration. The effects of topical rapamycin, an mTOR inhibitor, on mTORC1 and mTOR complex-2 (mTORC2) activities, as well as on tumor growth and migration, were determined. Vascular tumors showed increased activation of S6K and S6. Genetic knockdown of S6K resulted in reduced tumor cell proliferation and migration. Rapamycin fully inhibited mTORC1 and partially inhibited mTORC2 activities, including the phosphorylation of Akt (serine 473) and PKCα, in vascular tumor cells. Rapamycin significantly reduced vascular tumor growth in vitro and in vivo. As a potential localized therapy for cutaneous vascular tumors, topically applied rapamycin effectively reduced tumor growth with limited systemic drug absorption. These findings reveal the importance of mTOR signaling pathways in benign and malignant vascular tumors. The mTOR pathway is an important therapeutic target in vascular tumors, and topical mTOR inhibitors may provide an alternative and well-tolerated therapy for the treatment of cutaneous vascular lesions.

Priming of the vascular endothelial growth factor signaling pathway by thrombospondin-1, CD36, and spleen tyrosine kinase.

CD36 plays a critical role in the inhibition of angiogenesis through binding to the type 1 repeats of thrombospondin-1 (TSP-1) and activating Fyn tyrosine kinase and MAPK pathways. Here, we reveal a novel association of CD36 with VEGFR-2 and spleen tyrosine kinase (Syk). We also address the correlation between the expression of CD36 and Syk by demonstrating that overexpression of CD36 in HUVECs up-regulates endogenous Syk expression. We also define a new role for TSP-1 and CD36 in the activation of the VEGFR-2 signaling pathway that requires Syk. Our findings also identify a role for Syk as a stimulator of VEGF-A-induced angiogenesis by increasing phosphorylation of Y1175 in VEGFR-2, which is a major tyrosine for promoting VEGF-A-induced endothelial cell migration. Together, these studies introduce a new signaling pathway for TSP-1, CD36, and Syk, and address the role of these proteins in regulating the angiogenic switch.

Thrombospondin-1 modulates vascular endothelial growth factor activity at the receptor level.

Vascular endothelial growth factor (VEGF) is a well-established stimulator of vascular permeability and angiogenesis, whereas thrombospondin-1 (TSP-1) is a potent angiogenic inhibitor. In this study, we have found that the TSP-1 receptors CD36 and beta1 integrin associate with the VEGF receptor 2 (VEGFR2). The coclustering of receptors that regulate angiogenesis may provide the endothelial cell with a platform for integration of positive and negative signals in the plane of the membrane. Thus, this complex may represent a molecular switch that regulates angiogenesis and determines endothelial cell behavior. In this context, physiological levels of TSP-1 appear to support VEGFR2 function on both the cellular and tissue level, because phosphorylation of VEGFR2 and vascular permeability in response to VEGF are decreased in TSP-1-null mice and isolated endothelial cells. A therapeutic agent based on the antiangiogenic domain of TSP-1, designated 3TSR (for three TSP-1 type 1 repeats), has significant antiangiogenic and antitumor efficacy. Systemic treatment of wild-type mice with 3TSR significantly decreased VEGF-induced permeability. Consistent with this result, VEGF-stimulated phosphorylation of VEGFR2 was also significantly decreased in lung extracts from 3TSR-treated mice. Moreover, 3TSR significantly decreased VEGF-stimulated VEGFR2 phosphorylation in human dermal microvascular endothelial cells in culture. Taken together, the results indicate that TSP-1 and 3TSR modulate the function of VEGFR2.

VEGF-A/VEGFR Inhibition Restores Hematopoietic Homeostasis in the Bone Marrow and Attenuates Tumor Growth.

Antiangiogenesis-based cancer therapies, specifically those targeting the VEGF-A/VEGFR2 pathway, have been approved for subsets of solid tumors. However, these therapies result in an increase in hematologic adverse events. We surmised that both the bone marrow vasculature and VEGF receptor-positive hematopoietic cells could be impacted by VEGF pathway-targeted therapies. We used a mouse model of spontaneous breast cancer to decipher the mechanism by which VEGF pathway inhibition alters hematopoiesis. Tumor-bearing animals, while exhibiting increased angiogenesis at the primary tumor site, showed signs of shrinkage in the sinusoidal bone marrow vasculature accompanied by an increase in the hematopoietic stem cell-containing Lin-cKit(+)Sca1(+) (LKS) progenitor population. Therapeutic intervention by targeting VEGF-A, VEGFR2, and VEGFR3 inhibited tumor growth, consistent with observed alterations in the primary tumor vascular bed. These treatments also displayed systemic effects, including reversal of the tumor-induced shrinkage of sinusoidal vessels and altered population balance of hematopoietic stem cells in the bone marrow, manifested by the restoration of sinusoidal vessel morphology and hematopoietic homeostasis. These data indicate that tumor cells exert an aberrant systemic effect on the bone marrow microenvironment and VEGF-A/VEGFR targeting restores bone marrow function.

Akt1 and akt3 exert opposing roles in the regulation of vascular tumor growth.

Vascular tumors are endothelial cell neoplasms whose mechanisms of tumorigenesis are poorly understood. Moreover, current therapies, particularly those for malignant lesions, have little beneficial effect on clinical outcomes. In this study, we show that endothelial activation of the Akt1 kinase is sufficient to drive de novo tumor formation. Mechanistic investigations uncovered opposing functions for different Akt isoforms in this regulation, where Akt1 promotes and Akt3 inhibits vascular tumor growth. Akt3 exerted negative effects on tumor endothelial cell growth and migration by inhibiting activation of the translation regulatory kinase S6-Kinase (S6K) through modulation of Rictor expression. S6K in turn acted through a negative feedback loop to restrain Akt3 expression. Conversely, S6K signaling was increased in vascular tumor cells where Akt3 was silenced, and the growth of these tumor cells was inhibited by a novel S6K inhibitor. Overall, our findings offer a preclinical proof of concept for the therapeutic utility of treating vascular tumors, such as angiosarcomas, with S6K inhibitors.

Functional overlap and cooperativity among alphav and beta1 integrin subfamilies during skin angiogenesis.

Angiogenesis requires endothelial cell survival and proliferation, which depend upon cytokine stimulation together with integrin-mediated cell adhesion to extracellular matrix; however, the question of which specific integrins are the best targets for suppressing neovascularization is controversial and unresolved. Therefore, we designed experiments to compare contributions of individual integrins from both the alphav and beta1 integrin subfamilies. With immobilized antibodies, we determined that adhesion through integrins alpha1beta1, alpha2beta1, alphavbeta3, and alphavbeta5 each individually supported dermal microvascular endothelial cell survival. Also, substratum coated with collagen I (which binds alpha1beta1 and alpha2beta1) and vitronectin (which binds alphavbeta3 and alphavbeta5) each supported survival. Importantly, substratum coated with combinations of collagen I and vitronectin were most effective at promoting survival, and survival on three-dimensional collagen I gels was strongly enhanced by vitronectin. Vascular endothelial growth factor activation of the p44/p42 mitogen-activated protein kinase pathway, which is required for angiogenesis, was supported by adhesion through either alpha1beta1, alpha2beta1, alphavbeta3, or alphavbeta5, and pharmacologic inhibition of this pathway blocked proliferation and suppressed survival. Therefore, these studies establish that the alpha1beta1, alpha2beta1, alphavbeta3, and alphavbeta5 integrins each support dermal microvascular endothelial cell viability, and that each collaborate with vascular endothelial growth factor to support robust activation of the mitogen-activated protein kinase pathway which mediates both proliferation and survival. Moreover, survival is supported most significantly by extracellular matrices, which engage all of these integrins in combination. Consistent with important complementary and overlapping functions, combined antagonism of these integrins provided superior inhibition of angiogenesis in skin, indicating that multiplicity of integrin involvement should be considered in designing strategies for controlling neovascularization.

Inhibition of Tie-2 signaling induces endothelial cell apoptosis, decreases Akt signaling, and induces endothelial cell expression of the endogenous anti-angiogenic molecule, thrombospondin-1.

Small molecule inhibitors of endothelial cell specific tyrosine kinases are currently under investigation as potential means to block tumor angiogenesis. We have investigated the utility of blocking Tie-2 signaling in endothelial cells as a potential anti-angiogenic strategy. We have found that interruption of Tie-2 signaling either via RNAi or overexpression of a kinase-dead Tie-2 led to loss of endothelial cell viability, even in the presence of serum. Mechanistically, this is linked to a block in Akt signaling and increased thrombospondin expression. Thrombospondins are endogenous anti-angiogenic matricellular proteins known to regulate tumor growth and angiogenesis. We observed that both Tie-2 and subsequent PI3Kinase signaling regulates thrombospondin-1 expression. These data have lead to the model that Angiopoietin signaling through Tie-2 activates PI3Kinase/Akt, which represses thrombospondin expression. Thus, targeting Tie-2 with small molecules maybe efficacious as an anti-angiogenic therapy.

Overexpression of MyrAkt1 in endothelial cells leads to erythropoietin- and BMP4-independent splenic erythropoiesis in mice.

Under steady state conditions, erythropoiesis occurs in the bone marrow. However, in mice, stress or tissue hypoxia results in increased erythropoiesis in the spleen. There is increasing evidence that the hematopoietic microenvironment, including endothelial cells, plays an important role in regulating erythropoiesis. Here, we show that short-term expression of constitutively active Akt in the endothelium of mice results in non-anemic stress erythropoiesis in the spleen. The initiation of this stress response was independent of erythropoietin and BMP4, and was observed in endothelial myrAkt1 mice reconstituted with wild-type bone marrow. Together, these data suggest that endothelial cell hyperactivation is a potentially novel pathway of inducing red cell production under stress.

Correction: Overexpression of MyrAkt1 in Endothelial Cells Leads to Erythropoietin- and BMP4-Independent Splenic Erythropoiesis in Mice.

[This corrects the article on p. e55095 in vol. 8.].

RhoB controls coordination of adult angiogenesis and lymphangiogenesis following injury by regulating VEZF1-mediated transcription.

Mechanisms governing the distinct temporal dynamics that characterize post-natal angiogenesis and lymphangiogenesis elicited by cutaneous wounds and inflammation remain unclear. RhoB, a stress-induced small GTPase, modulates cellular responses to growth factors, genotoxic stress and neoplastic transformation. Here we show, using RhoB null mice, that loss of RhoB decreases pathological angiogenesis in the ischaemic retina and reduces angiogenesis in response to cutaneous wounding, but enhances lymphangiogenesis following both dermal wounding and inflammatory challenge. We link these unique and opposing roles of RhoB in blood versus lymphatic vasculatures to the RhoB-mediated differential regulation of sprouting and proliferation in primary human blood versus lymphatic endothelial cells. We demonstrate that nuclear RhoB-GTP controls expression of distinct gene sets in each endothelial lineage by regulating VEZF1-mediated transcription. Finally, we identify a small-molecule inhibitor of VEZF1-DNA interaction that recapitulates RhoB loss in ischaemic retinopathy. Our findings establish the first intra-endothelial molecular pathway governing the phased response of angiogenesis and lymphangiogenesis following injury.

RhoB differentially controls Akt function in tumor cells and stromal endothelial cells during breast tumorigenesis.

Tumors are composed of cancer cells but also a larger number of diverse stromal cells in the tumor microenvironment. Stromal cells provide essential supports to tumor pathophysiology but the distinct characteristics of their signaling networks are not usually considered in developing drugs to target tumors. This oversight potentially confounds proof-of-concept studies and increases drug development risks. Here, we show in established murine and human models of breast cancer how differential regulation of Akt by the small GTPase RhoB in cancer cells or stromal endothelial cells determines their dormancy versus outgrowth when angiogenesis becomes critical. In cancer cells in vitro or in vivo, RhoB functions as a tumor suppressor that restricts EGF receptor (EGFR) cell surface occupancy as well as Akt signaling. However, after activation of the angiogenic switch, RhoB functions as a tumor promoter by sustaining endothelial Akt signaling, growth, and survival of stromal endothelial cells that mediate tumor neoangiogenesis. Altogether, the positive impact of RhoB on angiogenesis and progression supercedes its negative impact in cancer cells themselves. Our findings elucidate the dominant positive role of RhoB in cancer. More generally, they illustrate how differential gene function effects on signaling pathways in the tumor stromal component can complicate the challenge of developing therapeutics to target cancer pathophysiology.

Thrombospondin-1 modulates VEGF-A-mediated Akt signaling and capillary survival in the developing retina.

Microvascular development is often perceived to result from a balance of positive and negative factors that impact signaling for proliferation and survival. The survival signaling that results from hypoxia-induced VEGF-A has been well established, but the factors that antagonize this signaling have been poorly studied. As endogenous inhibitors of angiogenesis, thrombospondins (TSPs) are likely candidates to affect survival signaling. Here we report that TSP1 antagonized microvascular survival to retinal hyperoxia, and Akt signaling in both the retina and in cultured endothelial cells. TSP1 expression is correlated with the association of the CD36 receptor with Src versus Fyn. In the presence of TSP1, CD36 is coprecipitated with Fyn as previously shown by others. However, in the absence of TSP1, there is a preferential association with Src. We now demonstrate that these Src family kinases play an important role in modulating microvascular survival in response to TSP1 by crossing tsp1(-/-) mice to the src(-/-) and fyn(-/-) mice and testing the survival of retinal blood vessels in hyperoxia. We find that tsp1(-/-), fyn(-/-), and double-mutant tsp1(-/-)/fyn(-/-) mice have a similar enhancement of capillary survival in oxygen, whereas in a tsp(-/-) background, the loss of only one allele of src restores the balance in survival and apoptosis to that of wild-type mice. Taken together, we hypothesize that TSP1 antagonizes VEGF-driven Akt survival signaling in part through the recruitment of Fyn to membrane domains containing CD36, but when TSP1 is absent, an opposing Src recruitment contributes to VEGF-driven Akt phosphorylation and capillary survival.

Akt promotes endocardial-mesenchyme transition.

Endothelial to mesenchyme transition (EndMT) can be observed during the formation of endocardial cushions from the endocardium, the endothelial lining of the atrioventricular canal (AVC), of the developing heart at embryonic day 9.5 (E9.5). Many regulators of the process have been identified; however, the mechanisms driving the initial commitment decision of endothelial cells to EndMT have been difficult to separate from processes required for mesenchymal proliferation and migration. We have several lines of evidence that suggest a central role for Akt signaling in committing endothelial cells to enter EndMT. Akt1 mRNA was restricted to the endocardium of endocardial cushions while they were forming. The PI3K/Akt signaling pathway is necessary for mesenchyme outgrowth, as sprouting was inhibited in AVC explant cultures treated with the PI3K inhibitor LY294002. Furthermore, endothelial marker, VE-cadherin, was downregulated and mesenchyme markers, N-cadherin and Snail, were induced in response to expression of a constitutively active form of Akt1 (myrAkt1) in endothelial cells. Finally, we isolated the function of Akt1 signaling in the commitment to the transition using a transgenic model where myrAkt1 was pulsed only in endocardial cells and turned off after EndMT initiation. In this way, we determined that increased Akt signaling in the endocardium drives EndMT and discounted its other functions in cushion mesenchymal cells.

Palomid 529, a novel small-molecule drug, is a TORC1/TORC2 inhibitor that reduces tumor growth, tumor angiogenesis, and vascular permeability.

It has become clear that the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is central for promoting both tumor and tumor stroma and is therefore a major target for anticancer drug development. First- and second-generation rapalogs (prototypical mTOR inhibitors) have shown promise but, due to the complex nature of mTOR signaling, can result in counterproductive feedback signaling to potentiate upstream Akt signaling. We present a novel PI3K/Akt/mTOR inhibitor, Palomid 529 (P529), which inhibits the TORC1 and TORC2 complexes and shows both inhibition of Akt signaling and mTOR signaling similarly in tumor and vasculature. We show that P529 inhibits tumor growth, angiogenesis, and vascular permeability. It retains the beneficial aspects of tumor vascular normalization that rapamycin boasts. However, P529 has the additional benefit of blocking pAktS473 signaling consistent with blocking TORC2 in all cells and thus bypassing feedback loops that lead to increased Akt signaling in some tumor cells.

The alpha(1)beta(1) and alpha(2)beta(1) integrins provide critical support for vascular endothelial growth factor signaling, endothelial cell migration, and tumor angiogenesis.

Angiogenesis is a complex process, involving functional cooperativity between cytokines and endothelial cell (EC) surface integrins. In this study, we investigated the mechanisms through which the alpha(1)beta(1) and alpha(2)beta(1) integrins support angiogenesis driven by vascular endothelial growth factor (VEGF). Dermal microvascular EC attachment through either alpha(1)beta(1) or alpha(2)beta(1) supported robust VEGF activation of the Erk1/Erk2 (p44/42) mitogen-activated protein kinase signal transduction pathway that drives EC proliferation. Haptotactic EC migration toward collagen I was dependent on alpha(1)beta(1) and alpha(2)beta(1) as was VEGF-stimulated chemotaxis of ECs in a uniform collagen matrix. Consistent with the functions of alpha(1)beta(1) and alpha(2)beta(1) in supporting signal transduction and EC migration, antibody antagonism of either integrin resulted in potent inhibition of VEGF-driven angiogenesis in mouse skin. Moreover, combined antagonism of alpha(1)beta(1) and alpha(2)beta(1) substantially reduced tumor growth and angiogenesis of human squamous cell carcinoma xenografts. Collectively, these studies identify critical collaborative functions for the alpha(1)beta(1) and alpha(2)beta(1) integrins in supporting VEGF signal transduction, EC migration, and tumor angiogenesis.

Molecular profiling of angiogenesis markers.

The goal of this study was to develop a sensitive, simple, and widely applicable assay to measure copy numbers of specific mRNAs using real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and identify a profile of gene expression closely associated with angiogenesis. We measured a panel of nine potential angiogenesis markers from a mouse transgenic model of prostate adenocarcinoma (TRAMP) and a mouse skin model of vascular endothelial growth factor (VEGF)-driven angiogenesis. In both models, expression of VEGF correlated with expression of mRNAs encoding other angiogenic cytokines (angiopoietin-1 and angiopoietin-2), endothelial cell receptor tyrosine kinases (Flt-1, KDR, Tie-1), and endothelial cell adhesion molecules (VE-cadherin, PECAM-1). Relative to control, in dermis highly stimulated by VEGF, the Ang-2 mRNA transcript numbers increased 35-fold, PECAM-1 and VE-cadherin increased 10-fold, Tie-1 increased 8-fold, KDR and Flt-1 each increased 4-fold, and Ang-1 increased 2-fold. All transcript numbers were correspondingly reduced in skin with less VEGF expression, indicating a relationship of each of these seven markers with VEGF. Thus, this study identifies a highly efficient method for precise quantification of a panel of seven specific mRNAs that correlate with VEGF expression and VEGF-induced neovascularization, and it provides evidence that real-time quantitative RT-PCR offers a highly sensitive strategy for monitoring angiogenesis.