Wa Du

Endothelial PI3K-C2α, a class II PI3K, has an essential role in angiogenesis and vascular barrier function

The class II α-isoform of phosphatidylinositol 3-kinase (PI3K-C2α) is localized in endosomes, the trans-Golgi network and clathrin-coated vesicles; however, its functional role is not well understood. Global or endothelial-cell–specific deficiency of PI3K-C2α resulted in embryonic lethality caused by defects in sprouting angiogenesis and vascular maturation. PI3K-C2α knockdown in endothelial cells resulted in a decrease in the number of PI3-phosphate–enriched endosomes, impaired endosomal trafficking, defective delivery of VE-cadherin to endothelial cell junctions and defective junction assembly. PI3K-C2α knockdown also impaired endothelial cell signaling, including vascular endothelial growth factor receptor internalization and endosomal RhoA activation. Together, the effects of PI3K-C2α knockdown led to defective endothelial cell migration, proliferation, tube formation and barrier integrity. Endothelial PI3K-C2α deficiency in vivo suppressed postischemic and tumor angiogenesis and diminished vascular barrier function with a greatly augmented susceptibility to anaphylaxis and a higher incidence of dissecting aortic aneurysm formation in response to angiotensin II infusion. Thus, PI3K-C2α has a crucial role in vascular formation and barrier integrity and represents a new therapeutic target for vascular disease.

S1P3-mediated cardiac fibrosis in sphingosine kinase 1 transgenic mice involves reactive oxygen species

AIMS Sphingosine kinase 1 (SPHK1), its product sphingosine-1-phosphate (S1P), and S1P receptor subtypes have been suggested to play protective roles for cardiomyocytes in animal models of ischaemic preconditioning and cardiac ischaemia/reperfusion injury. To get more insight into roles for SPHK1 in vivo, we have generated SPHK1-transgenic (TG) mice and analysed the cardiac phenotype. METHODS AND RESULTS SPHK1-TG mice overexpressed SPHK1 in diverse tissues, with a nearly 20-fold increase in enzymatic activity. The TG mice grew normally with normal blood chemistry, cell counts, heart rate, and blood pressure. Unexpectedly, TG mice with high but not low expression levels of SPHK1 developed progressive myocardial degeneration and fibrosis, with upregulation of embryonic genes, elevated RhoA and Rac1 activity, stimulation of Smad3 phosphorylation, and increased levels of oxidative stress markers. Treatment of juvenile TG mice with pitavastatin, an established inhibitor of the Rho family G proteins, or deletion of S1P3, a major myocardial S1P receptor subtype that couples to Rho GTPases and transactivates Smad signalling, both inhibited cardiac fibrosis with concomitant inhibition of SPHK1-dependent Smad-3 phosphorylation. In addition, the anti-oxidant N-2-mercaptopropyonylglycine, which reduces reactive oxygen species (ROS), also inhibited cardiac fibrosis. In in vivo ischaemia/reperfusion injury, the size of myocardial infarct was 30% decreased in SPHK1-TG mice compared with wild-type mice. CONCLUSION 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.

S1P2, the G Protein–Coupled Receptor for Sphingosine-1-Phosphate, Negatively Regulates Tumor Angiogenesis and Tumor Growth In vivo in Mice

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.

Sphingosine-1-phosphate receptor-2 deficiency leads to inhibition of macrophage proinflammatory activities and atherosclerosis in apoE-deficient mice.

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.

Roles of sphingosine-1-phosphate signaling in angiogenesis

Sphingosine-1-phosphate (S1P) is a blood-borne lipid mediator with pleiotropic biological activities. S1P acts via the specific cell surface G-protein-coupled receptors, S1P(1-5). S1P(1) and S1P(2) were originally identified from vascular endothelial cells (ECs) and smooth muscle cells, respectively. Emerging evidence shows that S1P plays crucial roles in the regulation of vascular functions, including vascular formation, barrier protection and vascular tone via S1P(1), S1P(2) and S1P(3). In particular, S1P regulates vascular formation through multiple mechanisms; S1P exerts both positive and negative effects on angiogenesis and vascular maturation. The positive and negative effects of S1P are mediated by S1P(1) and S1P(2), respectively. These effects of S1P(1) and S1P(2) are probably mediated by the S1P receptors expressed in multiple cell types including ECs and bone-marrow-derived cells. The receptor-subtype-specific, distinct effects of S1P favor the development of novel therapeutic tactics for antitumor angiogenesis in cancer and therapeutic angiogenesis in ischemic diseases.

Sustained delivery of sphingosine-1-phosphate using poly(lactic-co-glycolic acid)-based microparticles stimulates Akt/ERK-eNOS mediated angiogenesis and vascular maturation restoring blood flow in ischemic limbs of mice

Therapeutic angiogenesis is a promising strategy for treating ischemia. The lysophospholipid mediator sphingosine-1-phosphate (S1P) acts on vascular endothelial cells to stimulate migration and tube formation, and plays the critical role in developmental angiogenesis. We developed poly(lactic-co-glycolic-acid) (PLGA)-based S1P-containing microparticles (PLGA-S1P), which are biodegradable and continuously release S1P, and studied the effects of PLGA-S1P on neovascularization in murine ischemic hindlimbs. Intramuscular injections of PLGA-S1P stimulated blood flow in C57BL/6 mice dose-dependently, with repeated administrations at a 3-day interval, rather than a single bolus or 6-day interval, over 28 days conferring the optimal stimulating effect. In Balb/c mice that exhibit limb necrosis and dysfunction due to retarded blood flow recovery, injections of PLGA-S1P stimulated blood flow with alleviation of limb necrosis and dysfunction. PLGA-S1P alone did not induce edema in ischemic limbs, and rather blocked vascular endothelial growth factor-induced edema. PLGA-S1P not only increased the microvessel densities in ischemic muscle, but promoted coverage of vessels with smooth muscle cells and pericytes, thus stabilizing vessels. PLGA-S1P stimulated Akt and ERK with increased phosphorylation of endothelial nitric oxide synthase in ischemic muscle. The effects of the nitric oxide synthase inhibitor, Nomega-nitro-L-arginine methylester, showed that PLGA-S1P-induced blood flow stimulation was partially dependent on nitric oxide. Injections of PLGA-S1P also increased the expression of angiogenic factors and the recruitment of CD45-, CD11b- and Gr-1-positive myeloid cells, which are implicated in post-ischemic angiogenesis, into ischemic muscle. These results indicate that PLGA-based, sustained local delivery of S1P is a potentially useful therapeutic modality for stimulating post-ischemic angiogenesis.

Sphingosine-1-phosphate receptor 2 protects against anaphylactic shock through suppression of endothelial nitric oxide synthase in mice.

BACKGROUND Sphingosine-1-phosphate receptor 2 (S1P(2)) is expressed in vascular endothelial cells (ECs). However, the role of S1P(2) in vascular barrier integrity and anaphylaxis is not well understood. Endothelial nitric oxide synthase (eNOS) generates nitric oxide to mediate vascular leakage, compromising survival in patients with anaphylaxis. We recently observed that endothelial S1P(2) inhibits Akt, an activating kinase of eNOS. OBJECTIVE We tested the hypothesis that endothelial S1P(2) might suppress eNOS, exerting a protective effect against endothelial barrier disruption and anaphylaxis. METHODS Mice deficient in S1P(2) and eNOS underwent antigen challenge or platelet-activating factor (PAF) injection. Analyses were performed to examine vascular permeability and the underlying mechanisms. RESULTS S1pr2 deletion augmented vascular leakage and lethality after either antigen challenge or PAF injection. PAF injection induced activation of Akt and eNOS in the aortas and lungs of S1pr2-null mice, which were augmented compared with values seen in wild-type mice. Consistently, PAF-induced increase in cyclic guanosine monophosphate levels in the aorta was enhanced in S1pr-null mice. Genetic Nos3 deletion or pharmacologic eNOS blockade protected S1pr2-null mice from aggravation of barrier disruption after antigen challenge and PAF injection. ECs isolated from S1pr2-null mice exhibited greater stimulation of Akt and eNOS, with enhanced nitric oxide production in response to sphingosine-1-phosphate or PAF, compared with that seen in wild-type ECs. Moreover, S1pr2-deficient ECs showed more severe disassembly of adherens junctions with augmented S-nitrosylation of β-catenin in response to PAF, which was restored by pharmacologic eNOS blockade. CONCLUSION S1P(2) diminishes harmful robust eNOS stimulation and thereby attenuates vascular barrier disruption, suggesting potential usefulness of S1P(2) agonists as novel therapeutic agents for anaphylaxis.