Vascular senescence and leak are features of the early breakdown of the blood-brain barrier in Alzheimer's disease models.

Alzheimer's disease (AD) is an age-related disease, with loss of integrity of the blood-brain barrier (BBB) being an early feature. Cellular senescence is one of the reported nine hallmarks of aging. Here, we show for the first time the presence of senescent cells in the vasculature in AD patients and mouse models of AD. Senescent endothelial cells and pericytes are present in APP/PS1 transgenic mice but not in wild-type littermates at the time of amyloid deposition. In vitro, senescent endothelial cells display altered VE-cadherin expression and loss of cell junction formation and increased permeability. Consistent with this, senescent endothelial cells in APP/PS1 mice are present at areas of vascular leak that have decreased claudin-5 and VE-cadherin expression confirming BBB breakdown. Furthermore, single cell sequencing of endothelial cells from APP/PS1 transgenic mice confirms that adhesion molecule pathways are among the most highly altered pathways in these cells. At the pre-plaque stage, the vasculature shows significant signs of breakdown, with a general loss of VE-cadherin, leakage within the microcirculation, and obvious pericyte perturbation. Although senescent vascular cells were not directly observed at sites of vascular leak, senescent cells were close to the leak area. Thus, we would suggest in AD that there is a progressive induction of senescence in constituents of the neurovascular unit contributing to an increasing loss of vascular integrity. Targeting the vasculature early in AD, either with senolytics or with drugs that improve the integrity of the BBB may be valid therapeutic strategies.

Novel miRNA-based drug CD5-2 reduces liver tumor growth in diethylnitrosamine-treated mice by normalizing tumor vasculature and altering immune infiltrate.

Introduction: Liver cancers exhibit abnormal (leaky) vasculature, hypoxia and an immunosuppressive microenvironment. Normalization of tumor vasculature is an emerging approach to treat many cancers. Blockmir CD5-2 is a novel oligonucleotide-based inhibitor of the miR-27a interaction with VE-Cadherin, the endothelial-specific cadherin. The combination of a vasoactive medication with inhibition of immune checkpoints such as programmed cell death protein 1 (PD1) has been shown to be effective in treating liver cancer in humans. We aimed to study the effect of CD5-2 combined with checkpoint inhibition (using an antibody against PD1) on liver tumor growth, vasculature and immune infiltrate in the diethylnitrosamine (DEN)-induced liver tumor mouse model. Methods: We first analyzed human miR-27a and VE-Cadherin expression data from The Cancer Genome Atlas for hepatocellular carcinoma. CD5-2 and/or anti-PD1 antibody were given to the DEN-treated mice from age 7-months until harvest at age 9-months. Tumor and non-tumor liver tissues were analyzed using histology, immunohistochemistry, immunofluorescence and scanning electron microscopy. Results: Human data showed high miR-27a and low VE-Cadherin were both significantly associated with poorer prognosis. Mice treated with CD5-2 plus anti-PD1 antibody had significantly smaller liver tumors (50% reduction) compared to mice treated with either agent alone, controls, or untreated mice. There was no difference in tumor number. Histologically, tumors in CD5-2-treated mice had less leaky vessels with higher VE-Cadherin expression and less tumor hypoxia compared to non-CD5-2-treated mice. Only tumors in the combination CD5-2 plus anti-PD1 antibody group exhibited a more favorable immune infiltrate (significantly higher CD3+ and CD8+ T cells and lower Ly6G+ neutrophils) compared to tumors from other groups. Discussion: CD5-2 normalized tumor vasculature and reduced hypoxia in DEN-induced liver tumors. CD5-2 plus anti-PD1 antibody reduced liver tumor size possibly by altering the immune infiltrate to a more immunosupportive one.

Liver-specific deletion of miR-181ab1 reduces liver tumour progression via upregulation of CBX7.

MiR-181 expression levels increased in hepatocellular carcinoma (HCC) compared to non-cancerous tissues. MiR-181 has been widely reported as a possible driver of tumourigenesis but also acts as a tumour suppressor. In addition, the miR-181 family regulates the development and function of immune and vascular cells, which play vital roles in the progression of tumours. More complicatedly, many genes have been identified as miR-181 targets to mediate the effects of miR-181. However, the role of miR-181 in the development of primary tumours remains largely unexplored. We aimed to examine the function of miR-181 and its vital mediators in the progression of diethylnitrosamine-induced primary liver cancers in mice. The size of liver tumours was significantly reduced by 90% in global (GKO) or liver-specific (LKO) 181ab1 knockout mice but not in hematopoietic and endothelial lineage-specific knockout mice, compared to WT mice. In addition, the number of tumours was significantly reduced by 50% in GKO mice. Whole-genome RNA-seq analysis and immunohistochemistry showed that epithelial-mesenchymal transition was partially reversed in GKO tumours compared to WT tumours. The expression of CBX7, a confirmed miR-181 target, was up-regulated in GKO compared to WT tumours. Stable CBX7 expression was achieved with an AAV/Transposase Hybrid-Vector System and up-regulated CBX7 expression inhibited liver tumour progression in WT mice. Hepatic CBX7 deletion restored the progression of LKO liver tumours. MiR-181a expression was the lowest and CBX7 expression the highest in iClust2 and 3 subclasses of human HCC compared to iClust1. Gene expression profiles of GKO tumours overlapped with low-proliferative peri-portal-type HCCs. Liver-specific loss of miR-181ab1 inhibited primary liver tumour progression via up-regulating CBX7 expression, but tumour induction requires both hepatic and non-hepatic miR-181. Also, miR-181ab1-deficient liver tumours may resemble low-proliferative periportal-type human HCC. miR-181 was increased with liver tumour growth. More miR-181, darker colour and higher shape. CBX7 was very low in pericentral hepatocytes, increased in early liver tumours, but reduced in advanced liver tumours. Its levels were maintained in miR-181 KO liver tumours. In tumours (T), brown (darker is more) represents miR-181, the blue circle (thicker is more) represents CBX7.

Identification of circulating sphingosine kinase-related metabolites for prediction of type 2 diabetes.

BACKGROUND: Sphingosine Kinase (SphK) that catalyzes sphingosine (Sph) to sphingosine 1-phosphate (S1P), plays a key role in both sphingolipid metabolism and cellular signaling. While SphK has been implicated in type 2 diabetes mellitus (T2DM), it is unexplored in humans. Herein, we investigated whether circulating SphK-related metabolites are associated with T2DM incidence in an established prospective cohort. METHODS: Levels of SphK-related sphingolipid metabolites, including Sph, S1P, dihydrosphingosine (dhSph) and dihydro-S1P (dhS1P) in serum were measured by targeted-lipidomic analyses. By accessing to an established prospective cohort that involves a total of 2486 non-diabetic adults at baseline, 100 subjects who developed T2DM after a mean follow-up of 4.2-years, along with 100 control subjects matched strictly with age, sex, BMI and fasting glucose, were randomly enrolled for the present study. RESULTS: Comparison with the control group, medians of serum dhS1P and dhS1P/dhSph ratio at baseline were elevated significantly prior to the onset of T2DM. Each SD increment of dhS1P and dhS1P/dhSph ratio was associated with 53.5% and 54.1% increased risk of incident diabetes, respectively. The predictive effect of circulating dhS1P and dhS1P/dhSph ratio on T2DM incidence was independent of conventional risk factors in multivariate regression models. Furthermore, combination of serum dhS1P and dhS1P/dhSph ratio with conventional clinical indices significantly improved the accuracy of T2DM prediction (AUROC, 0.726), especially for normoglycemic subjects (AUROC, 0.859). CONCLUSION: Circulating levels of dhS1P and dhS1P/dhSph ratio are strongly associated with increased risk of T2DM, and could serve as a useful biomarker for prediction of incident T2DM in normoglycemic populations.

The aging endothelium.

Cellular senescence is now recognized as one of the hallmarks of aging. Herein, we examine current findings on senescence of the vascular endothelium and its impacts on age-related vascular diseases. Endothelial senescence can result in systemic metabolic changes, implicating senescence in chronic diseases such as diabetes, obesity and atherosclerosis. Senolytics, drugs that eliminate senescent cells, afford new therapeutic strategies for control of these chronic diseases.

The VE-Cadherin/ß-catenin signalling axis regulates immune cell infiltration into tumours.

Vascular normalisation, the process that reverses the structural and functional abnormalities seen in tumour-associated vessels, is also accompanied by changes in leucocyte trafficking. Our previous studies have shown the normalisation effects of the agent CD5-2 which acts to stabilise VE-Cadherin leading to increased penetration of CD8+ T cells but decreased infiltration of neutrophils (CD11b+Gr1hi) into tumour parenchyma. In the present study, we demonstrate that VE-Cadherin stabilisation through CD5-2 treatment of purified endothelial cells (ECs) results in a similar leucocyte-selective regulation of transmigration, suggesting the existence of an endothelial specific intrinsic mechanism. Further, we show by RNA sequencing (RNA-seq)-based transcriptomic analysis, that treatment of ECs with CD5-2 regulates chemokines known to be involved in leucocyte transmigration, including upregulation of CCL2 and CXCL10 that facilitate CD8+ T cell transmigration. Both in vitro and in vivo mechanistic studies revealed that the increased CCL2 expression was dependent on expression of VE-Cadherin and downstream activation of the AKT/GSK3ß/ß-catenin/TCF4 signalling pathway. CD5-2 treatment also contributed to the reorganisation of the cytoskeleton, inducing reorganisation of stress fibres to circumferential actin, which previously has been described as associated with the stabilisation of the endothelial barrier, and amplification of the transcellular migration of CD8+ T cells. Thus, we propose that promotion of endothelial junctional integrity during vascular normalisation not only inhibits vascular leak but also resets the endothelial dependent regulation of immune cell infiltration.

Regulation of hepatic insulin signaling and glucose homeostasis by sphingosine kinase 2.

Sphingolipid dysregulation is often associated with insulin resistance, while the enzymes controlling sphingolipid metabolism are emerging as therapeutic targets for improving insulin sensitivity. We report herein that sphingosine kinase 2 (SphK2), a key enzyme in sphingolipid catabolism, plays a critical role in the regulation of hepatic insulin signaling and glucose homeostasis both in vitro and in vivo. Hepatocyte-specific Sphk2 knockout mice exhibit pronounced insulin resistance and glucose intolerance. Likewise, SphK2-deficient hepatocytes are resistant to insulin-induced activation of the phosphoinositide 3-kinase (PI3K)-Akt-FoxO1 pathway and elevated hepatic glucose production. Mechanistically, SphK2 deficiency leads to the accumulation of sphingosine that, in turn, suppresses hepatic insulin signaling by inhibiting PI3K activation in hepatocytes. Either reexpressing functional SphK2 or pharmacologically inhibiting sphingosine production restores insulin sensitivity in SphK2-deficient hepatocytes. In conclusion, the current study provides both experimental findings and mechanistic data showing that SphK2 and sphingosine in the liver are critical regulators of insulin sensitivity and glucose homeostasis.

Targeting miR-27a/VE-cadherin interactions rescues cerebral cavernous malformations in mice.

Cerebral cavernous malformations (CCMs) are vascular lesions predominantly developing in the central nervous system (CNS), with no effective treatments other than surgery. Loss-of-function mutation in CCM1/krev interaction trapped 1 (KRIT1), CCM2, or CCM3/programmed cell death 10 (PDCD10) causes lesions that are characterized by abnormal vascular integrity. Vascular endothelial cadherin (VE-cadherin), a major regulator of endothelial cell (EC) junctional integrity is strongly disorganized in ECs lining the CCM lesions. We report here that microRNA-27a (miR-27a), a negative regulator of VE-cadherin, is elevated in ECs isolated from mouse brains developing early CCM lesions and in cultured ECs with CCM1 or CCM2 depletion. Furthermore, we show miR-27a acts downstream of kruppel-like factor (KLF)2 and KLF4, two known key transcription factors involved in CCM lesion development. Using CD5-2 (a target site blocker [TSB]) to prevent the miR-27a/VE-cadherin mRNA interaction, we present a potential therapy to increase VE-cadherin expression and thus rescue the abnormal vascular integrity. In CCM1- or CCM2-depleted ECs, CD5-2 reduces monolayer permeability, and in Ccm1 heterozygous mice, it restores dermal vessel barrier function. In a neonatal mouse model of CCM disease, CD5-2 normalizes vasculature and reduces vascular leakage in the lesions, inhibits the development of large lesions, and significantly reduces the size of established lesions in the hindbrain. Furthermore, CD5-2 limits the accumulation of inflammatory cells in the lesion area. Our work has established that VE-cadherin is a potential therapeutic target for normalization of the vasculature and highlights that targeting miR-27a/VE-cadherin interaction by CD5-2 is a potential novel therapy for the devastating disease, CCM.

YAP and the RhoC regulator ARHGAP18, are required to mediate flow-dependent endothelial cell alignment.

BACKGROUND: Vascular endothelial cell alignment in the direction of flow is an adaptive response that protects against aortic diseases such as atherosclerosis. The RhoGTPases are known to regulate this alignment. We have shown previously that ARHGAP18 in endothelial cells is a negative regulator of RhoC and its expression is essential in flow-mediated alignment. Depletion of ARHGAP18 inhibits alignment and results in the induction of a pro-inflammatory phenotype. In embryogenesis, ARHGAP18 was identified as a downstream effector of the Yes-associated protein, YAP, which regulates cell shape and size. METHODS: We have used siRNA technology to deplete either ARHGAP18 or YAP in human endothelial cells. The in vitro studies were performed under athero-protective, laminar flow conditions. The analysis of YAP activity was also investigated, using high performance confocal imaging, in our ARHGAP18 knockout mutant mice. RESULTS: We show here that loss of ARHGAP18, although decreasing the expression of YAP results in its nuclear localisation consistent with activation. We further show that depletion of YAP itself results in its activation as defined by an in increase in its nuclear localisation and an increase in the YAP target gene, CyR61. Depletion of YAP, similar to that observed for ARHGAP18 depletion, results in loss of endothelial cell alignment under high shear stress mediated flow and also in the activation of NFkB, as determined by p65 nuclear localisation. In contrast, ARHGAP18 overexpression results in upregulation of YAP, its phosphorylation, and a decrease in the YAP target gene Cyr61, consistent with YAP inactivation. Finally, in ARHGAP18 deleted mice, in regions where there is a loss of endothelial cell alignment, a situation associated with a priming of the cells to a pro-inflammatory phenotype, YAP shows nuclear localisation. CONCLUSION: Our results show that YAP is downstream of ARHGAP18 in mature endothelial cells and that this pathway is involved in the athero-protective alignment of endothelial cells under laminar shear stress. ARHGAP18 depletion leads to a disruption of the junctions as seen by loss of VE-Cadherin localisation to these regions and a concomitant localisation of YAP to the nucleus.

Low fluid shear stress conditions contribute to activation of cerebral cavernous malformation signalling pathways.

Cerebral cavernous malformations (CCMs) are vascular malformations that cause hemorrhagic stroke. CCMs can arise from loss-of-function mutations in any one of CCM1 (KRIT1), CCM2 or CCM3 (PDCD10). Despite the mutation being in all endothelial cells the CCM lesions develop primarily in the regions with low fluid shear stress (FSS). Here we investigated the role of FSS in the signalling pathways associated with loss of function of CCM genes. We performed transcriptomic analysis on CCM1 or CCM2-silenced endothelial cells subjected to various FSS. The results showed 1382 genes were deregulated under low FSS, whereas only 29 genes were deregulated under high FSS. Key CCM downstream signalling pathways, including increased KLF2/4 expression, actin cytoskeleton reorganization, TGF-ß and toll-like receptor signalling pathways and also oxidative stress pathways, were all highly upregulated but only under low FSS. We also show that the key known phenotypes of CCM lesions such as disrupted endothelial cell junction, increased inflammatory response/oxidative stress and elevated RhoA-ROCK activity, are only exhibited in monolayers of CCM-silenced endothelial cells subjected to low FSS. Our data establishes that shear stress acts as a previously unappreciated but important regulator for CCM gene function and may determine the site of CCM lesion development.

ARHGAP18: A Flow-Responsive Gene That Regulates Endothelial Cell Alignment and Protects Against Atherosclerosis.

Background Vascular endothelial cell (EC) alignment in the direction of flow is an adaptive response that protects against aortic diseases, such as atherosclerosis. The Rho GTP ases are known to regulate this alignment. Herein, we analyze the effect of ARHGAP 18 on the regulation of EC alignment and examine the effect of ARHGAP 18 deficiency on the development of atherosclerosis in mice. Methods and Results We used in vitro analysis of ECs under flow conditions together with apolipoprotein E-/- Arhgap 18-/- double-mutant mice to study the function of ARHGAP 18 in a high-fat diet-induced model of atherosclerosis. Depletion of ARHGAP 18 inhibited the alignment of ECs in the direction of flow and promoted inflammatory phenotype, as evidenced by disrupted junctions and increased expression of nuclear factor-κB and intercellular adhesion molecule-1 and decreased endothelial nitric oxide synthase. Mice with double deletion in ARHGAP 18 and apolipoprotein E and fed a high-fat diet show early onset of atherosclerosis, with lesions developing in atheroprotective regions. Conclusions ARHGAP 18 is a protective gene that maintains EC alignments in the direction of flow. Deletion of ARHGAP 18 led to loss of EC ability to align and promoted atherosclerosis development.

Therapeutic regulation of VE-cadherin with a novel oligonucleotide drug for diabetic eye complications using retinopathy mouse models.

AIMS/HYPOTHESIS: A major feature of diabetic retinopathy is breakdown of the blood-retinal barrier, resulting in macular oedema. We have developed a novel oligonucleotide-based drug, CD5-2, that specifically increases expression of the key junctional protein involved in barrier integrity in endothelial cells, vascular-endothelial-specific cadherin (VE-cadherin). CD5-2 prevents the mRNA silencing by the pro-angiogenic microRNA, miR-27a. CD5-2 was evaluated in animal models of ocular neovascularisation and vascular leak to determine its potential efficacy for diabetic retinopathy. METHODS: CD5-2 was tested in three mouse models of retinal dysfunction: conditional Müller cell depletion, streptozotocin-induced diabetes and oxygen-induced retinopathy. Vascular permeability in the Müller cell-knockout model was assessed by fluorescein angiography. The Evans Blue leakage method was used to determine vascular permeability in streptozotocin- and oxygen-induced retinopathy models. The effects of CD5-2 on retinal neovascularisation, inter-endothelial junctions and pericyte coverage in streptozotocin- and oxygen-induced retinopathy models were determined by staining for isolectin-B4, VE-cadherin and neural/glial antigen 2 (NG2). Blockmir CD5-2 localisation in diseased retina was determined using fluorescent in situ hybridisation. The effects of CD5-2 on VE-cadherin expression and in diabetic retinopathy-associated pathways, such as the transforming growth factor beta (TGF-ß) and wingless/integrated (WNT) pathway, were confirmed using western blot of lysates from HUVECs, a mouse brain endothelial cell line and a VE-cadherin null mouse endothelial cell line. RESULTS: CD5-2 penetrated the vasculature of the eye in the oxygen-induced retinopathy model. Treatment of diseased mice with CD5-2 resulted in reduced vascular leak in all three animal models, enhanced expression of VE-cadherin in the microvessels of the eye and improved pericyte coverage of the retinal vasculature in streptozotocin-induced diabetic models and oxygen-induced retinopathy models. Further, CD5-2 reduced the activation of retinal microglial cells in the streptozotocin-induced diabetic model. The positive effects of CD5-2 seen in vivo were further confirmed in vitro by increased protein expression of VE-cadherin, SMAD2/3 activity, and platelet-derived growth factor B (PDGF-B). CONCLUSIONS/INTERPRETATION: CD5-2 has therapeutic potential for individuals with vascular-leak-associated retinal diseases based on its ease of delivery and its ability to reverse vascular dysfunction and inflammatory aspects in three animal models of retinopathy.

Deletion of sphingosine kinase 1 inhibits liver tumorigenesis in diethylnitrosamine-treated mice.

Primary liver cancer is the 3rd leading cause of cancer deaths worldwide with very few effective treatments. Sphingosine kinase 1 (SphK1), a key regulator of sphingolipid metabolites, is over-expressed in human hepatocellular carcinoma (HCC) and our previous studies have shown that SphK1 is important in liver injury. We aimed to explore the role of SphK1 specifically in liver tumorigenesis using the SphK1 knockout (SphK1-/-) mouse. SphK1 deletion significantly reduced the number and the size of DEN-induced liver cancers in mice. Mechanistically, fewer proliferating but more apoptotic and senescent cells were detected in SphK1 deficient tumors compared to WT tumors. There was an increase in sphingosine rather than a decrease in sphingosine 1-phosphate (S1P) in SphK1 deficient tumors. Furthermore, the STAT3-S1PR pathway that has been reported previously to mediate the effect of SphK1 on colorectal cancers was not altered by SphK1 deletion in liver cancer. Instead, c-Myc protein expression was down-regulated by SphK1 deletion. In conclusion, this is the first in vivo evidence that SphK1 contributes to hepatocarcinogenesis. However, the downstream signaling pathways impacting on the development of HCC via SphK1 are organ specific providing further evidence that simply transferring known oncogenic molecular pathway targeting into HCC is not always valid.

ARHGAP18 Protects Against Thoracic Aortic Aneurysm Formation by Mitigating the Synthetic and Proinflammatory Smooth Muscle Cell Phenotype.

RATIONALE: Thoracic aortic aneurysm (TAA) is a potentially lethal condition, which can affect individuals of all ages. TAA may be complicated by the sudden onset of life-threatening dissection or rupture. The underlying mechanisms leading to TAA formation, particularly in the nonsyndromal idiopathic group of patients, are not well understood. Thus, identification of new genes and targets that are involved in TAA pathogenesis are required to help prevent and reverse the disease phenotype. OBJECTIVE: Here we explore the role of ARHGAP18, a novel Rho GAP expressed by smooth muscle cells (SMCs), in the pathogenesis of TAA. METHODS AND RESULTS: Using human and mouse aortic samples, we report that ARHGAP18 levels were significantly reduced in the SMC layer of aortic aneurysms. Arhgap18 global knockout (Arhgap18-/-) mice exhibited a highly synthetic, proteolytic, and proinflammatory smooth muscle phenotype under basal conditions and when challenged with angiotensin II, developed TAA with increased frequency and severity compared with littermate controls. Chromatin immunoprecipitation studies revealed this phenotype is partly associated with strong enrichment of H3K4me3 and depletion of H3K27me3 at the MMP2 and TNF-α promoters in Arhgap18-deficient SMC. We further show that TAA formation in the Arhgap18-/- mice is associated with loss of Akt activation. The abnormal SMC phenotype observed in the Arhgap18-/- mice can be partially rescued by pharmacological treatment with the mTORC1 inhibitor rapamycin, which reduces the synthetic and proinflammatory phenotype of Arhgap18-deficient SMC. CONCLUSION: We have identified ARHGAP18 as a novel protective gene against TAA formation and define an additional target for the future development of treatments to limit TAA pathogenesis.

Targeting Vascular Endothelial-Cadherin in Tumor-Associated Blood Vessels Promotes T-cell-Mediated Immunotherapy.

T-cell infiltration of solid tumors is associated with improved prognosis and favorable responses to immunotherapy. Mechanisms that enable tumor infiltration of CD8+ T cells have not been defined, nor have drugs that assist this process been discovered. Here we address these issues with a focus on VE-cadherin, a major endothelial cell-specific junctional protein that controls vascular integrity. A decrease in VE-cadherin expression is associated with tumor pathology. We developed an oligonucleotide-based inhibitor (CD5-2), which disrupted the interaction of VE-cadherin with its regulator miR-27a, resulting in increased VE-cadherin expression. Administration of CD5-2 in tumor-bearing mice enhanced expression of VE-cadherin in tumor endothelium, activating TIE-2 and tight junction pathways and normalizing vessel structure and function. CD5-2 administration also enhanced tumor-specific T-cell infiltration and spatially redistributed CD8+ T cells within the tumor parenchyma. Finally, CD5-2 treatment enhanced the efficacy of anti-PD-1 blocking antibody. Our work establishes a role for VE-cadherin in T-cell infiltration in tumors and offers a preclinical proof of concept for CD5-2 as a therapeutic modifier of cancer immunotherapy via effects on the tumor vasculature. Cancer Res; 77(16); 4434-47. ©2017 AACR.

The RhoGAP protein ARHGAP18/SENEX localizes to microtubules and regulates their stability in endothelial cells.

RhoGTPases are important regulators of the cell cytoskeleton, controlling cell shape, migration and proliferation. Previously we showed that ARHGAP18 in endothelial cells is important in cell junctions. Here we show, using structured illumination microscopy (SIM), ground-state depletion (GSD), and total internal reflection fluorescence microscopy (TIRF) that a proportion of ARHGAP18 localizes to microtubules in endothelial cells, as well as in nonendothelial cells, an association confirmed biochemically. In endothelial cells, some ARHGAP18 puncta also colocalized to Weibel-Palade bodies on the microtubules. Depletion of ARHGAP18 by small interfering RNA or analysis of endothelial cells isolated from ARHGAP18-knockout mice showed microtubule destabilization, as evidenced by altered morphology and decreased acetylated α-tubulin and glu-tubulin. The destabilization was rescued by inhibition of ROCK and histone deacetylase 6 but not by a GAP-mutant form of ARHGAP18. Depletion of ARHGAP18 resulted in a failure to secrete endothelin-1 and a reduction in neutrophil transmigration, both known to be microtubule dependent. Thrombin, a critical regulator of the Rho-mediated barrier function of endothelial cells through microtubule destabilization, enhanced the plasma membrane-bound fraction of ARHGAP18. Thus, in endothelial cells, ARHGAP18 may act as a significant regulator of vascular homeostasis.

The Poly-cistronic miR-23-27-24 Complexes Target Endothelial Cell Junctions: Differential Functional and Molecular Effects of miR-23a and miR-23b.

The regulation of function of endothelial cell-cell junctions is fundamental in sustaining vascular integrity. The polycistronic microRNA (miR) complexes containing miR-23a-27a-24-2, and 23b-27b-24-1 are predicted to target the majority of major endothelial junctional proteins. We focus on miR-23a and miR-23b, and investigate the functional effects of these miRs on junctions. While miR-23a and 23b only differ by 1 nucleotide (g19) outside the seed region and thus are predicted to have the same targets, they function differently with miR-23a inhibiting permeability and miR-23b inhibiting angiogenesis. Both miRs target the junctional attractive molecule (tight junction protein 2) ZO-2 and the repulsive molecule junctional adhesion molecule C (JAM-C), although the inhibition of JAM-C by miR-23a is more profound than by miR-23b. The difference in potency is attributable to differences at g19 since a mutation of the t17, the g19 binding site of miR-23b in the 3'UTR of JAM-C restores identity. We also show that the pattern of expression of miR-23a and miR-23b and their targets are different. Thus, the paralogues miR-23a and miR-23b can have profoundly different effects on endothelial cell function due at least partially to selective effects on target proteins and differences in expression patterns of the miRs. This work exposes a hitherto unappreciated complexity in therapeutically targeting miRs.

Deletion of sphingosine kinase 1 ameliorates hepatic steatosis in diet-induced obese mice: Role of PPARγ.

Sphingolipid metabolites have emerged playing important roles in the pathogenesis of nonalcoholic fatty liver disease, whereas the underlying mechanism remains largely unknown. In the present study, we provide both in vitro and in vivo evidence showing a pathogenic role of sphingosine kinase 1 (SphK1) in hepatocellular steatosis. We found that levels of SphK1 expression were significantly increased in steatotic hepatocytes. Enforced overexpression of SphK1 or treatment with sphingosine 1-phosphate (S1P) markedly enhanced hepatic lipid accumulation. In contrast, the siRNA-mediated knockdown of SphK1 or S1P receptors, S1P2 and S1P3, profoundly inhibited lipid accumulation in hepatocytes. Moreover, Sphk1(-/-) mice exhibited a significant amelioration of hepatosteatosis under diet-induced obese (DIO) conditions, compared to wild-type littermates. In addition, DIO-induced up-regulation of PPARγ and its target genes were significantly reduced by SphK1 deficiency. Furthermore, treatment of hepatocytes with S1P induces a dose-dependent increase in PPARγ expression at the transcriptional level. Blockage of S1P receptors and the Akt-mTOR signaling profoundly inhibited S1P-induced PPARγ expression. Notably, down-regulation of PPARγ by using its siRNA significantly diminished the pro-steatotic effect of SphK1/S1P. Thus, the study demonstrates a new pathway connecting SphK1 and PPARγ involved in the pathogenesis of hepatocellular steatosis.

Caveolae control the anti-inflammatory phenotype of senescent endothelial cells.

Senescent endothelial cells (EC) have been identified in cardiovascular disease, in angiogenic tumour associated vessels and in aged individuals. We have previously identified a novel anti-inflammatory senescent phenotype of EC. We show here that caveolae are critical in the induction of this anti-inflammatory senescent state. Senescent EC induced by either the overexpression of ARHGAP18/SENEX or by H2O2 showed significantly increased numbers of caveolae and associated proteins Caveolin-1, cavin-1 and cavin-2. Depletion of these proteins by RNA interference decreased senescence induced by ARHGAP18 and by H2O2. ARHGAP18 overexpression induced a predominantly anti-inflammatory senescent population and depletion of the caveolae-associated proteins resulted in the preferential reduction in this senescent population as measured by neutrophil adhesion and adhesion protein expression after TNFα treatment. In confirmation, EC isolated from the aortas of CAV-1(-/-) mice failed to induce this anti-inflammatory senescent cell population upon expression of ARHGAP18, whereas EC from wild-type mice showed a significant increase. NF-κB is one of the major transcription factors mediating the induction of E-selectin and VCAM-1 expression, adhesion molecules responsible for leucocyte attachment to EC. TNFα-induced activation of NF-κB was suppressed in ARHGAP18-induced senescent EC, and this inhibition was reversed by Caveolin-1 knock-down. Thus, out results demonstrate that an increase in caveolae and its component proteins in senescent ECs is associated with inhibition of the NF-kB signalling pathway and promotion of the anti-inflammatory senescent pathway.

ARHGAP18: an endogenous inhibitor of angiogenesis, limiting tip formation and stabilizing junctions.

The formation of the vascular network requires a tightly controlled balance of pro-angiogenic and stabilizing signals. Perturbation of this balance can result in dysregulated blood vessel morphogenesis and drive pathologies including cancer. Here, we have identified a novel gene, ARHGAP18, as an endogenous negative regulator of angiogenesis, limiting pro-angiogenic signaling and promoting vascular stability. Loss of ARHGAP18 promotes EC hypersprouting during zebrafish and murine retinal vessel development and enhances tumor vascularization and growth. Endogenous ARHGAP18 acts specifically on RhoC and relocalizes to the angiogenic and destabilized EC junctions in a ROCK dependent manner, where it is important in reaffirming stable EC junctions and suppressing tip cell behavior, at least partially through regulation of tip cell genes, Dll4, Flk-1 and Flt-4. These findings highlight ARHGAP18 as a specific RhoGAP to fine tune vascular morphogenesis, limiting tip cell formation and promoting junctional integrity to stabilize the angiogenic architecture.