Athanasia Skoura

MicroRNAs Are Necessary for Vascular Smooth Muscle Growth, Differentiation, and Function

Objective—Regulation of vascular smooth muscle (VSM) proliferation and contractile differentiation is an important factor in vascular development and subsequent cardiovascular diseases. Recently, microRNAs (miRNAs) have been shown to regulate fundamental cellular processes in a number of cell types, but the integrated role of miRNAs in VSM in blood vessels is unknown. Here, we investigated the role of miRNAs in VSM by deleting the rate-limiting enzyme in miRNA synthesis, Dicer. Methods and Results—Deletion of Dicer in VSM results in late embryonic lethality at embryonic day 16 to 17, associated with extensive internal hemorrhage. The loss of VSM Dicer results in dilated, thin-walled blood vessels caused by a reduction in cellular proliferation. In addition, blood vessels from VSM-deleted Dicer mice exhibited impaired contractility because of a loss of contractile protein markers. We found this effect to be associated with a loss of actin stress fibers and partly rescued by overexpression of microRNA (miR)-145 or myocardin. Conclusion—Dicer-dependent miRNAs are important for VSM development and function by regulating proliferation and contractile differentiation.

Induction of Vascular Permeability by the Sphingosine-1-Phosphate Receptor–2 (S1P2R) and its Downstream Effectors ROCK and PTEN

Objectives—S1P acts via the S1PR family of G protein–coupled receptors to regulate a variety of physiological responses. Whereas S1P1R activates Gi- and PI-3-kinase–dependent signals to inhibit vascular permeability, the related S1P2R inhibits the PI-3-kinase pathway by coupling to the Rho-dependent activation of the PTEN phosphatase. However, cellular consequences of S1P2R signaling in the vascular cells are not well understood. Methods and Results—Selective signaling of the S1P2R was achieved by adenoviral-mediated expression in endothelial cells. Secondly, endogenously expressed S1P2R was blocked by the specific pharmacological antagonist JTE013. Activation of S1P2R in endothelial cells resulted in Rho-ROCK– and PTEN-dependent disruption of adherens junctions, stimulation of stress fibers, and increased paracellular permeability. JTE013 treatment of naive endothelial cells potentiated the S1P1R-dependent effects such as formation of cortical actin, blockade of stress fibers, stimulation of adherens junction assembly, and improved barrier integrity. This observation was extended to the in vivo model of vascular permeability in the rat lung: the S1P2R antagonist JTE013 significantly inhibited H2O2-induced permeability in the rat lung perfused model. Conclusions—S1P2R activation in endothelial cells increases vascular permeability. The balance of S1P1 and S1P2 receptors in the endothelium may determine the regulation of vascular permeability by S1P.

Essential role of sphingosine 1–phosphate receptor 2 in pathological angiogenesis of the mouse retina

Sphingosine 1-phosphate (S1P), a multifunctional lipid mediator that signals via the S1P family of G protein-coupled receptors (S1PR), regulates vascular maturation, permeability, and angiogenesis. In this study, we explored the role of S1P 2 receptor (S1P2R) in normal vascularization and hypoxia-triggered pathological angiogenesis of the mouse retina. S1P2R is strongly induced in ECs during hypoxic stress. When neonatal mice were subjected to ischemia-driven retinopathy, pathologic neovascularization in the vitreous chamber was suppressed in S1p2-/- mice concomitant with reduction in endothelial gaps and inflammatory cell infiltration. In addition, EC patterning and normal revascularization into the avascular zones of the retina were augmented. Reduced expression of the proinflammatory enzyme cyclooxygenase-2 (COX-2) and increased expression of eNOS were observed in the S1p2-/- mouse retina. S1P2R activation in ECs induced COX-2 expression and suppressed the expression of eNOS. These data identify the S1P2R-driven inflammatory process as an important molecular event in pathological retinal angiogenesis. We propose that antagonism of the S1P2R may be a novel therapeutic approach for the prevention and/or treatment of pathologic ocular neovascularization.

Deafness and Stria Vascularis Defects in S1P2 Receptor-null Mice

The S1P2 receptor is a member of a family of G protein-coupled receptors that bind the extracellular sphingolipid metabolite sphingosine 1-phosphate with high affinity. The receptor is widely expressed and linked to multiple G protein signaling pathways, but its physiological function has remained elusive. Here we have demonstrated that S1P2 receptor expression is essential for proper functioning of the auditory and vestibular systems. Auditory brainstem response analysis revealed that S1P2 receptor-null mice were deaf by one month of age. These null mice exhibited multiple inner ear pathologies. However, some of the earliest cellular lesions in the cochlea were found within the stria vascularis, a barrier epithelium containing the primary vasculature of the inner ear. Between 2 and 4 weeks after birth, the basal and marginal epithelial cell barriers and the capillary bed within the stria vascularis of the S1P2 receptor-null mice showed markedly disturbed structures. JTE013, an S1P2 receptor-specific antagonist, blocked the S1P-induced vasoconstriction of the spiral modiolar artery, which supplies blood directly to the stria vascularis and protects its capillary bed from high perfusion pressure. Vascular disturbance within the stria vascularis is a potential mechanism that leads to deafness in the S1P2 receptor-null mice.

Flow-Regulated Endothelial S1P Receptor-1 Signaling Sustains Vascular Development

During angiogenesis, nascent vascular sprouts fuse to form vascular networks, enabling efficient circulation. Mechanisms that stabilize the vascular plexus are not well understood. Sphingosine 1-phosphate (S1P) is a blood-borne lipid mediator implicated in the regulation of vascular and immune systems. Here we describe a mechanism by which the G protein-coupled S1P receptor-1 (S1P1) stabilizes the primary vascular network. A gradient of S1P1 expression from the mature regions of the vascular network to the growing vascular front was observed. In the absence of endothelial S1P1, adherens junctions are destabilized, barrier function is breached, and flow is perturbed, resulting in abnormal vascular hypersprouting. Interestingly, S1P1 responds to S1P as well as laminar shear stress to transduce flow-mediated signaling in endothelial cells both in vitro and in vivo. These data demonstrate that blood flow and circulating S1P activate endothelial S1P1 to stabilize blood vessels in development and homeostasis.

Smooth Muscle miRNAs Are Critical for Post-Natal Regulation of Blood Pressure and Vascular Function

Phenotypic modulation of smooth muscle cells (SMCs) plays a key role in vascular disease, including atherosclerosis. Several transcription factors have been suggested to regulate phenotypic modulation of SMCs but the decisive mechanisms remain unknown. Recent reports suggest that specific microRNAs (miRNAs) are involved in SMC differentiation and vascular disease but the global role of miRNAs in postnatal vascular SMC has not been elucidated. Thus, the objective of this study was to identify the role of Dicer-dependent miRNAs for blood pressure regulation and vascular SMC contractile function and differentiation in vivo. Tamoxifen-inducible and SMC specific deletion of Dicer was achieved by Cre-Lox recombination. Deletion of Dicer resulted in a global loss of miRNAs in aortic SMC. Furthermore, Dicer-deficient mice exhibited a dramatic reduction in blood pressure due to significant loss of vascular contractile function and SMC contractile differentiation as well as vascular remodeling. Several of these results are consistent with our previous observations in SM-Dicer deficient embryos. Therefore, miRNAs are essential for maintaining blood pressure and contractile function in resistance vessels. Although the phenotype of miR-143/145 deficient mice resembles the loss of Dicer, the phenotypes of SM-Dicer KO mice were far more severe suggesting that additional miRNAs are involved in maintaining postnatal SMC differentiation.

Sphingosine-1-Phosphate Receptor-2 Function in Myeloid Cells Regulates Vascular Inflammation and Atherosclerosis

Objective—Sphingomyelin deposition and metabolism occurs in the atherosclerotic plaque, leading to the formation of sphingosine-1-phosphate (S1P), which activates G protein–coupled receptors to regulate vascular and immune cells. The role of S1P receptors in atherosclerosis has not been examined. Methods and Results—We tested the hypothesis that S1P receptor-2 (S1PR2) regulates atherosclerosis. Apoe−/− S1pr2−/− mice showed greatly attenuated atherosclerosis compared with the Apoe−/− mice. Bone marrow transplant experiments indicate that S1PR2 function in the hematopoietic compartment is critical. S1PR2 is expressed in bone marrow–derived macrophages and in macrophage-like foam cells in atherosclerotic plaques. Reduced macrophage-like foam cells were found in the atherosclerotic plaques of Apoe−/−S1pr2−/− mice, suggesting that S1PR2 retains macrophages in atherosclerotic plaques. Lipoprotein profiles, plasma lipids, and oxidized low-density lipoprotein uptake by bone marrow–derived macrophages were not altered by the S1pr2 genotype. In contrast, endotoxin-induced inflammatory cytokine (interleukin [IL]-1&bgr;, IL-18) levels in the serum of S1PR2 knockout mice were significantly reduced. Furthermore, treatment of wild-type mice with S1PR2 antagonist JTE-013 suppressed IL-1&bgr; and IL-18 levels in plasma. Conclusion—These data suggest that S1PR2 signaling in the plaque macrophage regulates macrophage retention and inflammatory cytokine secretion, thereby promoting atherosclerosis.

Regulation of vascular physiology and pathology by the S1P2 receptor subtype.

Sphingosine-1-phosphate (S1P) is now recognized as a lipid mediator that acts via G-protein-coupled receptors. S1P receptors couple to various heterotrimeric G-proteins and regulate downstream targets and ultimately cell behaviour. The prototypical S1P1 receptor is known to couple to Gi and regulates angiogenesis, vascular development, and immune cell trafficking. In this review, we focus our attention on the S1P2 receptor, which has a unique G-protein-coupling property in that it preferentially activates the G(12/13) pathway. Recent studies indicate that the S1P2 receptor regulates critical intracellular signalling pathways, such as Rho GTPase, the phosphatase PTEN, and VE-cadherin-based adherens junctions. Analysis of mutant mice has revealed the critical role of this receptor in inner ear physiology, heart and vascular development, vascular remodelling, and vascular tone, permeability, and angiogenesis in vertebrates. These studies suggest that selective modulation of S1P2 receptor function by pharmacological tools may be useful in a variety of pathological conditions.

Lysophospholipid receptors in vertebrate development, physiology, and pathology

Lysophospholipid (LP) research has experienced a period of renaissance with the discovery of the lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) receptors in the late 1990s. Vertebrate LP receptors regulate embryogenesis, vascular development, neurogenesis, uterine development, oocyte survival, immune cell trafficking and inflammatory reactions. LP signaling is important in cancer, autoimmunity and inflammatory diseases. Research on LP biology has contributed to the development of a first-generation S1P receptor modulator that has entered phase III clinical trials for the treatment of multiple sclerosis. Further basic research on LP signaling is anticipated to lead to novel therapeutic tools to combat various human diseases.

Dynamin 2 regulation of integrin endocytosis, but not VEGF signaling, is crucial for developmental angiogenesis

Here we show that dynamin 2 (Dnm2) is essential for angiogenesis in vitro and in vivo. In cultured endothelial cells lacking Dnm2, vascular endothelial growth factor (VEGF) signaling and receptor levels are augmented whereas cell migration and morphogenesis are impaired. Mechanistically, the loss of Dnm2 increases focal adhesion size and the surface levels of multiple integrins and reduces the activation state of β1 integrin. In vivo, the constitutive or inducible loss of Dnm2 in endothelium impairs branching morphogenesis and promotes the accumulation of β1 integrin at sites of failed angiogenic sprouting. Collectively, our data show that Dnm2 uncouples VEGF signaling from function and coordinates the endocytic turnover of integrins in a manner that is crucially important for angiogenesis in vitro and in vivo.