Deletion of vascular thromboxane A2 receptors and its impact on angiotensin II-induced hypertension and atherosclerotic lesion formation in the aorta of Ldlr-deficient mice.

The thromboxane A2 receptor (TP) has been shown to play a role in angiotensin II (Ang II)-mediated hypertension and pathological vascular remodeling. To assess the impact of vascular TP on Ang II-induced hypertension, atherogenesis, and pathological aortic alterations, i.e. aneurysms, we analysed Western-type diet-fed and Ang II-infused TPVSMC KO/Ldlr KO, TPEC KO/Ldlr KO mice and their respective wild-type littermates (TPWT/Ldlr KO). These analyses showed that neither EC- nor VSMC-specific deletion of the TP significantly affected basal or Ang II-induced blood pressure or aortic atherosclerotic lesion area. In contrast, VSMC-specific TP deletion abolished and EC-specific TP deletion surprisingly reduced the ex vivo reactivity of aortic rings to the TP agonist U-46619, whereas VSMC-specific TP knockout also diminished the ex vivo response of aortic rings to Ang II. Furthermore, despite similar systemic blood pressure, there was a trend towards less atherogenesis in the aortic arch and a trend towards fewer pathological aortic alterations in Ang II-treated female TPVSMC KO/Ldlr KO mice. Survival was impaired in male mice after Ang II infusion and tended to be higher in TPVSMC KO/Ldlr KO mice than in TPWT/Ldlr KO littermates. Thus, our data may suggest a deleterious role of the TP expressed in VSMC in the pathogenesis of Ang II-induced aortic atherosclerosis in female mice, and a surprising role of the endothelial TP in TP-mediated aortic contraction. However, future studies are needed to substantiate and further elucidate the role of the vascular TP in the pathogenesis of Ang II-induced hypertension, aortic atherosclerosis and aneurysm formation.

A current overview of RhoA, RhoB, and RhoC functions in vascular biology and pathology.

The Rho subfamily members of Rho GTPases, RhoA, RhoB, and RhoC, are key regulators of signal transduction in a variety of cellular processes, including regulation of actomyosin and microtubule dynamics, cell shape, cell adhesion, cell division, cell migration, vesicle/membrane trafficking, and cell proliferation. Traditionally, the focus of research on RhoA/B/C has been on tumor biology, as dysregulation of expression or function of these proteins plays an important role in the pathogenesis of various cancer entities. However, RhoA, RhoB, and RhoC are also important in the context of vascular biology and pathology because they influence endothelial barrier function, vascular smooth muscle contractility and proliferation, vascular function and remodelling as well as angiogenesis. In this context, RhoA/B/C exploit numerous effector molecules to transmit their signals, and their activity is regulated by a variety of guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs) that enable precise spatiotemporal activation often in concert with other Rho GTPases. Although their protein structure is very similar, different mechanisms of regulation of gene expression, different localization, and to some extent different interaction with RhoGAPs and RhoGEFs have been observed for RhoA/B/C. In this review, we aim to provide a current overview of the Rho subfamily as regulators of vascular biology and pathology, analyzing database information and existing literature on expression, protein structure, and interaction with effectors and regulatory proteins. In this setting, we will also discuss recent findings on Rho effectors, RhoGEFs, RhoGAPs, as well as guanine nucleotide dissociation inhibitors (RhoGDIs).

Active RhoA Exerts an Inhibitory Effect on the Homeostasis and Angiogenic Capacity of Human Endothelial Cells.

Background The small GTPase RhoA (Ras homolog gene family, member A) regulates a variety of cellular processes, including cell motility, proliferation, survival, and permeability. In addition, there are reports indicating that RhoA-ROCK (rho associated coiled-coil containing protein kinase) activation is essential for VEGF (vascular endothelial growth factor)-mediated angiogenesis, whereas other work suggests VEGF-antagonistic effects of the RhoA-ROCK axis. Methods and Results To elucidate this issue, we examined human umbilical vein endothelial cells and human coronary artery endothelial cells after stable overexpression (lentiviral transduction) of constitutively active (G14V/Q63L), dominant-negative (T19N), or wild-type RhoA using a series of in vitro angiogenesis assays (proliferation, migration, tube formation, angiogenic sprouting, endothelial cell viability) and a human umbilical vein endothelial cells xenograft assay in immune-incompetent NOD scid gamma mice in vivo. Here, we report that expression of active and wild-type RhoA but not dominant-negative RhoA significantly inhibited endothelial cell proliferation, migration, tube formation, and angiogenic sprouting in vitro. Moreover, active RhoA increased endothelial cell death in vitro and decreased human umbilical vein endothelial cell-related angiogenesis in vivo. Inhibition of RhoA by C3 transferase antagonized the inhibitory effects of RhoA and strongly enhanced VEGF-induced angiogenic sprouting in control-treated cells. In contrast, inhibition of RhoA effectors ROCK1/2 and LIMK1/2 (LIM domain kinase 1/2) did not significantly affect RhoA-related effects, but increased angiogenic sprouting and migration of control-treated cells. In agreement with these data, VEGF did not activate RhoA in human umbilical vein endothelial cells as measured by a Förster resonance energy transfer-based biosensor. Furthermore, global transcriptome and subsequent bioinformatic gene ontology enrichment analyses revealed that constitutively active RhoA induced a differentially expressed gene pattern that was enriched for gene ontology biological process terms associated with mitotic nuclear division, cell proliferation, cell motility, and cell adhesion, which included a significant decrease in VEGFR-2 (vascular endothelial growth factor receptor 2) and NOS3 (nitric oxide synthase 3) expression. Conclusions Our data demonstrate that increased RhoA activity has the potential to trigger endothelial dysfunction and antiangiogenic effects independently of its well-characterized downstream effectors ROCK and LIMK.

Thromboxane A2 receptor activation via Gα13-RhoA/C-ROCK-LIMK2-dependent signal transduction inhibits angiogenic sprouting of human endothelial cells.

We could previously show that thromboxane A2 receptor (TP) activation inhibits the angiogenic capacity of human endothelial cells, but the underlying mechanisms remained unclear. Therefore, the aim of this study was to elucidate TP signal transduction pathways relevant to angiogenic sprouting of human endothelial cells. To clarify this matter, we used RNAi-mediated gene silencing as well as pharmacological inhibition of potential TP downstream targets in human umbilical vein endothelial cells (HUVEC) and VEGF-induced angiogenic sprouting of HUVEC spheroids in vitro as a functional read-out. In this experimental set-up, the TP agonist U-46619 completely blocked VEGF-induced angiogenic sprouting of HUVEC spheroids. Moreover, in live-cell analyses TP activation induced endothelial cell contraction, sprout retraction as well as endothelial cell tension and focal adhesion dysregulation of HUVEC. These effects were reversed by pharmacological TP inhibition or TP knockdown. Moreover, we identified a TP-Gα13-RhoA/C-ROCK-LIMK2-dependent signal transduction pathway to be relevant for U-46619-induced inhibition of VEGF-mediated HUVEC sprouting. In line with these results, U-46619-mediated TP activation potently induced RhoA and RhoC activity in live HUVEC as measured by FRET biosensors. Interestingly, pharmacological inhibition of ROCK and LIMK2 also normalized U-46619-induced endothelial cell tension and focal adhesion dysregulation of HUVEC. In summary, our work reveals mechanisms by which the TP may disturb angiogenic endothelial function in disease states associated with sustained endothelial TP activation.

The F2-isoprostane 8-iso-PGF2α attenuates atherosclerotic lesion formation in Ldlr-deficient mice - Potential role of vascular thromboxane A2 receptors.

The F2-isoprostane 8-iso-PGF2α (also known as 15-F2t-isoprostane, iPF2α-III, 8-epi PGF2α, 15(S)-8-iso-PGF2α, or 8-Isoprostane), a thromboxane A2 receptor (TP) agonist, stable biomarker of oxidative stress, and risk marker of cardiovascular disease, has been proposed to aggravate atherogenesis in genetic mouse models of atherosclerotic vascular disease. Moreover, the TP plays an eminent role in the pathophysiology of endothelial dysfunction, atherogenesis, and cardiovascular disease. Yet it is unknown, how the TP expressed by vascular cells affects atherogenesis or 8-iso-PGF2α-related effects in mouse models of atherosclerosis. We studied Ldlr-deficient vascular endothelial-specific (EC) and vascular smooth muscle cell (VSMC)-specific TP knockout mice (TPECKO/Ldlr KO; TPVSMCKO/Ldlr KO) and corresponding wild-type littermates (TPWT/Ldlr KO). The mice were fed a Western-type diet for eight weeks and received either 8-iso-PGF2α or vehicle infusions via osmotic pumps. Subsequently, arterial blood pressure, atherosclerotic lesion formation, and lipid profiles were analyzed. We found that VSMC-, but not EC-specific TP deletion, attenuated atherogenesis without affecting blood pressure or plasma lipid profiles of the mice. In contrast to a previous report, 8-iso-PGF2α tended to reduce atherogenesis in TPWT/Ldlr KO and TPEC KO/Ldlr KO mice, again without significantly affecting blood pressure or lipid profiles of these mice. However, no further reduction in atherogenesis was observed in 8-iso-PGF2α-treated TPVSMC KO/Ldlr KO mice. Our work suggests that the TP expressed in VSMC but not the TP expressed in EC is involved in atherosclerotic lesion formation in Ldlr-deficient mice. Furthermore, we report an inhibitory effect of 8-iso-PGF2α on atherogenesis in this experimental atherosclerosis model, which paradoxically appears to be related to the presence of the TP in VSMC.

A Thromboxane A2 Receptor-Driven COX-2-Dependent Feedback Loop That Affects Endothelial Homeostasis and Angiogenesis.

BACKGROUND: TP (thromboxane A2 receptor) plays an eminent role in the pathophysiology of endothelial dysfunction and cardiovascular disease. Moreover, its expression is reported to increase in the intimal layer of blood vessels of cardiovascular high-risk individuals. Yet it is unknown, whether TP upregulation per se has the potential to affect the homeostasis of the vascular endothelium. METHODS: We combined global transcriptome analysis, lipid mediator profiling, functional cell analyses, and in vivo angiogenesis assays to study the effects of endothelial TP overexpression or knockdown/knockout on the angiogenic capacity of endothelial cells in vitro and in vivo. RESULTS: Here we report that endothelial TP expression induces COX-2 (cyclooxygenase-2) in a Gi/o- and Gq/11-dependent manner, thereby promoting its own activation via the auto/paracrine release of TP agonists, such as PGH2 (prostaglandin H2) or prostaglandin F2 but not TxA2 (thromboxane A2). TP overexpression induces endothelial cell tension and aberrant cell morphology, affects focal adhesion dynamics, and inhibits the angiogenic capacity of human endothelial cells in vitro and in vivo, whereas TP knockdown or endothelial-specific TP knockout exerts opposing effects. Consequently, this TP-dependent feedback loop is disrupted by pharmacological TP or COX-2 inhibition and by genetic reconstitution of PGH2-metabolizing prostacyclin synthase even in the absence of functional prostacyclin receptor expression. CONCLUSIONS: Our work uncovers a TP-driven COX-2-dependent feedback loop and important effector mechanisms that directly link TP upregulation to angiostatic TP signaling in endothelial cells. By these previously unrecognized mechanisms, pathological endothelial upregulation of the TP could directly foster endothelial dysfunction, microvascular rarefaction, and systemic hypertension even in the absence of exogenous sources of TP agonists.

Impact of DICER1 and DROSHA on the Angiogenic Capacity of Human Endothelial Cells.

RNAi-mediated knockdown of DICER1 and DROSHA, enzymes critically involved in miRNA biogenesis, has been postulated to affect the homeostasis and the angiogenic capacity of human endothelial cells. To re-evaluate this issue, we reduced the expression of DICER1 or DROSHA by RNAi-mediated knockdown and subsequently investigated the effect of these interventions on the angiogenic capacity of human umbilical vein endothelial cells (HUVEC) in vitro (proliferation, migration, tube formation, endothelial cell spheroid sprouting) and in a HUVEC xenograft assay in immune incompetent NSGTM mice in vivo. In contrast to previous reports, neither knockdown of DICER1 nor knockdown of DROSHA profoundly affected migration or tube formation of HUVEC or the angiogenic capacity of HUVEC in vivo. Furthermore, knockdown of DICER1 and the combined knockdown of DICER1 and DROSHA tended to increase VEGF-induced BrdU incorporation and induced angiogenic sprouting from HUVEC spheroids. Consistent with these observations, global proteomic analyses showed that knockdown of DICER1 or DROSHA only moderately altered HUVEC protein expression profiles but additively reduced, for example, expression of the angiogenesis inhibitor thrombospondin-1. In conclusion, global reduction of miRNA biogenesis by knockdown of DICER1 or DROSHA does not inhibit the angiogenic capacity of HUVEC. Further studies are therefore needed to elucidate the influence of these enzymes in the context of human endothelial cell-related angiogenesis.

Siglec-7 restores ß-cell function and survival and reduces inflammation in pancreatic islets from patients with diabetes.

Chronic inflammation plays a key role in both type 1 and type 2 diabetes. Cytokine and chemokine production within the islets in a diabetic milieu results in ß-cell failure and diabetes progression. Identification of targets, which both prevent macrophage activation and infiltration into islets and restore ß-cell functionality is essential for effective diabetes therapy. We report that certain Sialic-acid-binding immunoglobulin-like-lectins (siglecs) are expressed in human pancreatic islets in a cell-type specific manner. Siglec-7 was expressed on ß-cells and down-regulated in type 1 and type 2 diabetes and in infiltrating activated immune cells. Over-expression of Siglec-7 in diabetic islets reduced cytokines, prevented ß-cell dysfunction and apoptosis and reduced recruiting of migrating monocytes. Our data suggest that restoration of human Siglec-7 expression may be a novel therapeutic strategy targeted to both inhibition of immune activation and preservation of ß-cell function and survival.