Class II α-isoform of phosphatidylinositol 3-kinases (PI3K-C2α) is localized in endosomes, the trans-Golgi network and clathrin-coated vesicles, however, its functional role is little understood. Global or endothelial cell (EC)-specific targeted disruption of PI3K-C2α resulted in embryonic lethality due to defects in sprouting angiogenesis and vascular maturation. PI3K-C2α knockdown in ECs induced decreased phospatidylinositol 3-phosphate-enriched endosomes, impaired endosomal trafficking, and defective delivery of VE-cadherin to EC junctions and its assembly. PI3K-C2α knockdown also impeded cell signaling including vascular endothelial growth factor receptor internalization and endosomal RhoA activation. These together led to defective EC migration, proliferation, tube formation and barrier integrity. Endothelial PI3K-C2α deletion suppressed post-ischemic and tumor angiogenesis, and diminished vascular barrier function, with greatly augmented susceptibility to anaphylaxis and a higher incidence of dissecting aortic aneurysm formation in response to angiotensin II infusion. Thus, PI3K-C2α plays a crucial role in vascular formation and barrier integrity, and represents a new therapeutic target for vascular diseases. 3Formation of the vascular network by vasculogenesis and angiogenesis is essential for embryonic development, repair and remodeling of tissues in adults, as well as tumor growth. The angiogenic response to vascular endothelial growth factor (VEGF) and other factors begins with vascular leakage and dissolution of the subendothelial basement membrane, followed by proliferation and migration of vascular EC 1,2 . Then, formation of the intercellular junctions results in initial sprouts from existing vessels. The newly formed endothelial tubes are associated with mural cells, i.e. smooth muscle cells (SMC) and pericytes, thus becoming mature and stabilized 3 . Tightness of the intercellular junctions, particularly adherens junctions composed of VE-cadherin, controls vascular permeability 4,5 . Quiescent, stabilized vasculature with intact barrier integrity dominates in the healthy condition. In contrast, in pathological conditions, such as tumors, the vasculature is generally inmaturate and leaky. In the case of vascular insult such as excessive angiotensin II (Ang II) activity, increased vascular permeability is asssociated with leukocyte infiltration in the vascular wall and vascular disruption 6,7 . Therefore, stabilization of the vasculature and maintenance of vascular integrity is essential for vascular and tissue homeostasis 8,9 .PI3Ks are an enzyme family that phosphorylates membrane inositol lipids at the 3' position of the inositol ring. The lipid products of PI3Ks serve as important intracellular messengers by interacting with effector proteins, which include protein kinases, guanine nucleotide exchangers for G proteins, and actin cytoskeleton-regulating proteins. Through these actions, PI3Ks regulate a diverse array of cellular processes 10-12 .PI3Ks comprise three classes. Class I PI...

These results suggest that chronic activation of SPHK1-S1P signalling results in both pathological cardiac remodelling through ROS mediated by S1P3 and favourable cardioprotective effects.

Sphingosine-1-phosphate (S1P) has been implicated in tumor angiogenesis by acting through the G i -coupled chemotactic receptor S1P 1 . Here, we report that the distinct receptor S1P 2 is responsible for mediating the G 12/13 /Rho-dependent inhibitory effects of S1P on Akt, Rac, and cell migration, thereby negatively regulating tumor angiogenesis and tumor growth. By using S1P 2 LacZ/+ mice, we found that S1P 2 was expressed in both tumor and normal blood vessels in many organs, in both endothelial cells (EC) and vascular smooth muscle cells, as well as in tumor-associated, CD11b-positive bone marrow-derived cells (BMDC). Lewis lung carcinoma or B16 melanoma cells implanted in S1P 2 -deficient (S1P 2 −/− ) mice displayed accelerated tumor growth and angiogenesis with enhanced association of vascular smooth muscle cells and pericytes. S1P 2 −/− ECs exhibited enhanced Rac activity, Akt phosphorylation, cell migration, proliferation, and tube formation in vitro. Coinjection of S1P 2 −/− ECs and tumor cells into wild-type mice also produced a relative enhancement of tumor growth and angiogenesis in vivo. S1P 2 −/− mice were also more efficient at recruiting CD11b-positive BMDCs into tumors compared with wild-type siblings. Bone marrow chimera experiments revealed that S1P 2 acted in BMDCs to promote tumor growth and angiogenesis. Our results indicate that, in contrast to endothelial S1P 1 , which stimulates tumor angiogenesis, S1P 2 on ECs and BMDCs mediates a potent inhibition of tumor angiogenesis, suggesting a novel therapeutic tactic for anticancer treatment.Cancer Res; 70(2); 772-81. ©2010 AACR.

Aortic aneurysm, including thoracic aortic aneurysm and abdominal aortic aneurysm, is the second most prevalent aortic disease following atherosclerosis, representing the ninth‐leading cause of death globally. Open surgery and endovascular procedures are the major treatments for aortic aneurysm. Typically, thoracic aortic aneurysm has a more robust genetic background than abdominal aortic aneurysm. Abdominal aortic aneurysm shares many features with thoracic aortic aneurysm, including loss of vascular smooth muscle cells (VSMCs), extracellular matrix degradation and inflammation. Although there are limitations to perfectly recapitulating all features of human aortic aneurysm, experimental models provide valuable tools to understand the molecular mechanisms and test novel therapies before human clinical trials. Among the cell types involved in aortic aneurysm development, VSMC dysfunction correlates with loss of aortic wall structural integrity. Here, we discuss the role of VSMCs in aortic aneurysm development. The loss of VSMCs, VSMC phenotypic switching, secretion of inflammatory cytokines, increased matrix metalloproteinase activity, elevated reactive oxygen species, defective autophagy, and increased senescence contribute to aortic aneurysm development. Further studies on aortic aneurysm pathogenesis and elucidation of the underlying signaling pathways are necessary to identify more novel targets for treating this prevalent and clinical impactful disease.

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. Cancer Res; 75(1); 40-50. Ó2014 AACR.

Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that has pleiotropic effects in a variety of cell types including ECs, SMCs, and macrophages, all of which are central to the development of atherosclerosis. It may therefore exert stimulatory and inhibitory effects on atherosclerosis. Here, we investigated the role of the S1P receptor S1PR2 in atherosclerosis by analyzing S1pr2 -/-mice with an Apoe -/-background. S1PR2 was expressed in macrophages, ECs, and SMCs in atherosclerotic aortas. In S1pr2 -/-Apoe -/-mice fed a high-cholesterol diet for 4 months, the area of the atherosclerotic plaque was markedly decreased, with reduced macrophage density, increased SMC density, increased eNOS phosphorylation, and downregulation of proinflammatory cytokines compared with S1pr2 +/+ Apoe -/-mice. Bone marrow chimera experiments indicated a major role for macrophage S1PR2 in atherogenesis. S1pr2 -/-Apoe -/-macrophages showed diminished Rho/Rho kinase/NF-κB (ROCK/NF-κB) activity. Consequently, they also displayed reduced cytokine expression, reduced oxidized LDL uptake, and stimulated cholesterol efflux associated with decreased scavenger receptor expression and increased cholesterol efflux transporter expression. S1pr2 -/-Apoe -/-ECs also showed reduced ROCK and NF-κB activities, with decreased MCP-1 expression and elevated eNOS phosphorylation. Pharmacologic S1PR2 blockade in S1pr2 +/+ Apoe -/-mice diminished the atherosclerotic plaque area in aortas and modified LDL accumulation in macrophages. We conclude therefore that S1PR2 plays a critical role in atherogenesis and may serve as a novel therapeutic target for atherosclerosis.

Background: Sphingosine-1-phosphate receptor-2, S1P 2 , is expressed in vascular

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 PKCa, 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.