Akt3 activation by R-Ras in an endothelial cell enforces quiescence and barrier stability of neighboring endothelial cells via Jagged1.

Communication between adjacent endothelial cells is important for the homeostasis of blood vessels. We show that quiescent endothelial cells use Jagged1 to instruct neighboring endothelial cells to assume a quiescent phenotype and secure the endothelial barrier. This phenotype enforcement by neighboring cells is operated by R-Ras through activation of Akt3, which results in upregulation of a Notch ligand Jagged1 and consequential upregulation of Notch target genes, such as UNC5B, and VE-cadherin accumulation in the neighboring cells. These signaling events lead to the stable interaction between neighboring endothelial cells to continue to fortify juxtacrine signaling via Jagged1-Notch. This mode of intercellular signaling provides a positive feedback regulation of endothelial cell-cell interactions and cellular quiescence required for the stabilization of the endothelium.

Partial RAG deficiency in humans induces dysregulated peripheral lymphocyte development and humoral tolerance defect with accumulation of T-bet+ B cells.

The recombination-activating genes (RAG) 1 and 2 are indispensable for diversifying the primary B cell receptor repertoire and pruning self-reactive clones via receptor editing in the bone marrow; however, the impact of RAG1/RAG2 on peripheral tolerance is unknown. Partial RAG deficiency (pRD) manifesting with late-onset immune dysregulation represents an 'experiment of nature' to explore this conundrum. By studying B cell development and subset-specific repertoires in pRD, we demonstrate that reduced RAG activity impinges on peripheral tolerance through the generation of a restricted primary B cell repertoire, persistent antigenic stimulation and an inflammatory milieu with elevated B cell-activating factor. This unique environment gradually provokes profound B cell dysregulation with widespread activation, remarkable extrafollicular maturation and persistence, expansion and somatic diversification of self-reactive clones. Through the model of pRD, we reveal a RAG-dependent 'domino effect' that impacts stringency of tolerance and B cell fate in the periphery.

Molecular Signature of Tumor-Associated High Endothelial Venules That Can Predict Breast Cancer Survival.

High endothelial venules (HEV) are specialized post-capillary venules that recruit naïve lymphocytes to lymph nodes. HEVs are essential for the development of adaptive immunity. HEVs can also develop in tumors where they are thought to be important for recruiting naïve T cells and B cells into the tumors and locally enhancing antitumor immunity by supporting the formation of tertiary lymphoid structures. Herein, we used comparative transcriptome analysis of human breast cancer to investigate genes differentially expressed between tumor-associated HEVs and the rest of the tumor vasculature. Tumor vessels highly expressing HEV-upregulated genes, such as the homeobox gene MEOX2 and the tetraspanin gene TSPAN7, were associated with extensive infiltration of T and B cells and the occurrence of tertiary lymphoid structures, which is known to predict therapeutic responses to immune-checkpoint inhibitors. Moreover, high transcript counts of these genes in clinical tumor specimens were associated with a significant survival benefit in advanced breast cancer. The molecular signature of HEVs identified herein may be useful for guiding immunotherapies and provides a new direction for investigating tumor-associated HEVs and their clinical significance. See related Spotlight by Gallimore, p. 371.

R-Ras Deficiency in Pericytes Causes Frequent Microphthalmia and Perturbs Retinal Vascular Development.

PURPOSE: The retinal vasculature is heavily invested by pericytes. Small GTPase R-Ras is highly expressed in endothelial cells and pericytes, suggesting importance of this Ras homolog for the regulation of the blood vessel wall. We investigated the specific contribution of pericyte-expressed R-Ras to the development of the retinal vasculature. METHODS: The effect of R-Ras deficiency in pericytes was analyzed in pericyte-targeted conditional Rras knockout mice at birth and during the capillary plexus formation in the neonatal retina. RESULTS: The offspring of these mice frequently exhibited unilateral microphthalmia. Analyses of the developing retinal vasculature in the eyes without microphthalmia revealed excessive endothelial cell proliferation, sprouting, and branching of the capillary plexus in these animals. These vessels were structurally defective with diminished pericyte coverage and basement membrane formation. Furthermore, these vessels showed reduced VE-cadherin staining and significantly elevated plasma leakage indicating the breakdown of the blood-retinal barrier. This defect was associated with considerable macrophage infiltration in the retina. CONCLUSIONS: The normal retinal vascular development is dependent on R-Ras expression in pericytes, and the absence of it leads to unattenuated angiogenesis and significantly weakens the blood-retinal barrier. Our findings underscore the importance of R-Ras for pericyte function during the normal eye development.

MicroRNA-211 Modulates the DUSP6-ERK5 Signaling Axis to Promote BRAFV600E-Driven Melanoma Growth In Vivo and BRAF/MEK Inhibitor Resistance.

MicroRNAs (miRs) are important posttranscriptional regulators of cell fate in both normal and disease states. miR-211 has previously been shown to be a direct regulator of metabolism in BRAFV600E-mutant melanoma cells in vitro. Here, we report that miR-211 expression promotes the aggressive growth of BRAFV600E-mutant melanoma xenografts in vivo. miR-211 promoted proliferation through the posttranscriptional activation of extracellular signal-regulated kinase (ERK) 5 signaling, which has recently been implicated in the resistance to BRAF and MAPK/ERK kinase inhibitors. We therefore examined whether miR-211 similarly modulated melanoma resistance to the BRAF inhibitor vemurafenib and the MAPK/ERK kinase inhibitor cobimetinib. Consistent with this model, miR-211 expression increased melanoma cell resistance to both the inhibitors, and this resistance was associated with an increased ERK5 phosphorylation. miR-211 mediates these effects by directly inhibiting the expression of DUSP6, an ERK5 pathway-specific phosphatase and now shown to be an miR-211 target gene. These results dissect the role of the miR-211-DUSP6-ERK5 axis in melanoma tumor growth and suggest a mechanism for the development of drug-resistant tumors and a target for overcoming resistance.

High Endothelial Venules Accelerate Naive T Cell Recruitment by Tumor Necrosis Factor-Mediated R-Ras Upregulation.

Recruitment of naive T cells to lymph nodes is essential for the development of adaptive immunity. Upon pathogen infection, lymph nodes promptly increase the influx of naive T cells from the circulation in order to screen and prime the T cells. The precise contribution of the lymph node vasculature to the regulation of this process remains unclear. Here we show a role for the Ras GTPase, R-Ras, in the functional adaptation of high endothelial venules to increase naive T cell trafficking to the lymph nodes. R-Ras is transiently up-regulated in the endothelium of high endothelial venules by the inflammatory cytokine tumor necrosis factor (TNF) within 24 hours of pathogen inoculation. TNF induces R-Ras upregulation in endothelial cells via JNK and p38 mitogen-activated protein kinase but not NF-κB. Studies of T cell trafficking found that the loss of function of endothelial R-Ras impairs the rapid acceleration of naive T cell recruitment to the lymph nodes upon inflammation. This defect diminished the ability of naive OT-1 T cells to develop antitumor activity against ovalbumin-expressing melanoma. Proteomic analyses suggest that endothelial R-Ras facilitates TNF-dependent transendothelial migration (diapedesis) of naive T cells by modulating molecular assembly the at T cell-endothelial cell interface. These findings give new mechanistic insights into the functional adaptation of high endothelial venules to accelerate naive T cell recruitment to the lymph nodes.

Prostaglandin E2 breaks down pericyte-endothelial cell interaction via EP1 and EP4-dependent downregulation of pericyte N-cadherin, connexin-43, and R-Ras.

A close association between pericytes and endothelial cells (ECs) is crucial to the stability and function of capillary blood vessels and microvessels. The loss or dysfunction of pericytes results in significant disruption of these blood vessels as observed in pathological conditions, including cancer, diabetes, stroke, and Alzheimer's disease. Prostaglandin E2 (PGE2) is a lipid mediator of inflammation, and its tissue concentration is elevated in cancer and neurological disorders. Here, we show that the exposure to PGE2 switches pericytes to a fast-migrating, loosely adhered phenotype that fails to intimately interact with ECs. N-cadherin and connexin-43 in adherens junction and gap junction between pericytes and ECs are downregulated by EP-4 and EP-1-dependent mechanisms, leading to breakdown of the pericyte-EC interaction. Furthermore, R-Ras, a small GTPase important for vascular normalization and vessel stability, is transcriptionally repressed by PGE2 in an EP4-dependent manner. Mouse dermal capillary vessels lose pericyte coverage substantially upon PGE2 injection into the skin. Our results suggest that EP-mediated direct disruption of pericytes by PGE2 is a key process for vascular destabilization. Restoring pericyte-EC interaction using inhibitors of PGE2 signaling may offer a therapeutic strategy in cancer and neurological disorders, in which pericyte dysfunction contributes to the disease progression.

Mitochondria Penetrating Peptide-Conjugated TAMRA for Live-Cell Long-Term Tracking.

Mitochondria are essential targets for treatment of diseases with mitochondrial disorders such as diabetes, cancer, and cardiovascular and neurodegenerative diseases. Mitochondria penetrating peptides (MPPs) are composed of cationic and hydrophobic amino acids that can target and permeate the mitochondrial membrane. Herein, a novel d-argine-phenylalanine-d-argine-phenylalanine-d-argine-phenylalanine-NH2 (rFrFrF) was tagged with a rhodamine-based fluorescent chromophore (TAMRA). This probe (TAMRA-rFrFrF) exhibited advantageous properties for long-term mitochondria tracking as demonstrated by fluorescence microscopy. Cell viability assays and oxygen consumption rates indicate low cytotoxicity and high biocompatibility of the new contrast agent. Colocalization studies suggest that TAMRA-rFrFrF is a promising candidate for continuous mitochondrial tracking for up to 3 days.

Prolonged activation of cAMP signaling leads to endothelial barrier disruption via transcriptional repression of RRAS.

The increase in cAMP levels in endothelial cells triggers cellular signaling to alter vascular permeability. It is generally considered that cAMP signaling stabilizes the endothelial barrier function and reduces permeability. However, previous studies have only examined the permeability shortly after cAMP elevation and thus have only investigated acute responses. Because cAMP is a key regulator of gene expression, elevated cAMP may have a delayed but profound impact on the endothelial permeability by altering the expression of the genes that are vital for the vessel wall stability. The small guanosine triphosphate hydrolase Ras-related protein (R-Ras) stabilizes VE-cadherin clustering and enhances endothelial barrier function, thereby stabilizing the integrity of blood vessel wall. Here we show that cAMP controls endothelial permeability through RRAS gene regulation. The prolonged cAMP elevation transcriptionally repressed RRAS in endothelial cells via a cAMP response element-binding protein (CREB) 3-dependent mechanism and significantly disrupted the adherens junction. These effects resulted in a marked increase of endothelial permeability that was reversed by R-Ras transduction. Furthermore, cAMP elevation in the endothelium by prostaglandin E2 or phosphodiesterase type 4 inhibition caused plasma leakage from intact microvessels in mouse skin. Our study demonstrated that, contrary to the widely accepted notion, cAMP elevation in endothelial cells ultimately increases vascular permeability, and the cAMP-dependent RRAS repression critically contributes to this effect.-Perrot, C. Y., Sawada, J., Komatsu, M. Prolonged activation of cyclic AMP signaling leads to endothelial barrier disruption via transcriptional repression of RRAS.

R-Ras-Akt axis induces endothelial lumenogenesis and regulates the patency of regenerating vasculature.

The formation of endothelial lumen is fundamental to angiogenesis and essential to the oxygenation of hypoxic tissues. The molecular mechanism underlying this important process remains obscure. Here, we show that Akt activation by a Ras homolog, R-Ras, stabilizes the microtubule cytoskeleton in endothelial cells leading to endothelial lumenogenesis. The activation of Akt by the potent angiogenic factor VEGF-A does not strongly stabilize microtubules or sufficiently promote lumen formation, hence demonstrating a distinct role for the R-Ras-Akt axis. We show in mice that this pathway is important for the lumenization of new capillaries and microvessels developing in ischemic muscles to allow sufficient tissue reperfusion after ischemic injury. Our work identifies a role for Akt in lumenogenesis and the significance of the R-Ras-Akt signaling for the patency of regenerating blood vessels.

Lack of R-Ras Leads to Increased Vascular Permeability in Ischemic Retinopathy.

PURPOSE: The role of R-Ras in retinal angiogenesis and vascular permeability was evaluated in an oxygen-induced retinopathy (OIR) model using R-Ras knockout (KO) mice and in human diabetic neovascular membranes. METHODS: Mice deficient for R-Ras and their wild-type (WT) littermates were subjected to 75% oxygen from postnatal day 7 (P7) to P12 and then returned to room air. At P17 retinal vascularization was examined from whole mounts, and retinal vascular permeability was studied using Miles assay. Real-time RT-PCR, Western blotting, and immunohistochemistry were used to assess the expression of R-Ras in retina during development or in the OIR model. The degree of pericyte coverage and vascular endothelial (VE)-cadherin expression on WT and R-Ras KO retinal blood vessels was quantified using confocal microscopy. The correlation of R-Ras with vascular endothelial growth factor receptor 2 (VEGFR2) and human serum albumin on human proliferative diabetic retinopathy membranes was assessed using immunohistochemistry. RESULTS: In retina, R-Ras expression was mostly restricted to the vasculature. Retinal vessels in the R-Ras KO mice were significantly more permeable than WT controls in the OIR model. A significant reduction in the direct physical contact between pericytes and blood vessel endothelium as well as reduced VE-cadherin immunostaining was found in R-Ras-deficient mice. In human proliferative diabetic retinopathy neovascular membranes, R-Ras expression negatively correlated with increased vascular leakage and expression of VEGFR2, a marker of blood vessel immaturity. CONCLUSIONS: Our results suggest that R-Ras has a role in controlling retinal vessel maturation and stabilization in ischemic retinopathy and provides a potential target for pharmacologic manipulation to treat diabetic retinopathy.

The Long Noncoding RNA SPRIGHTLY Regulates Cell Proliferation in Primary Human Melanocytes.

The long noncoding RNA SPRIGHTLY (formerly SPRY4-IT1), which lies within the intronic region of the SPRY4 gene, is up-regulated in human melanoma cells compared to melanocytes. SPRIGHTLY regulates a number of cancer hallmarks, including proliferation, motility, and apoptosis. To better understand its oncogenic role, SPRIGHTLY was stably transfected into human melanocytes, which resulted in increased cellular proliferation, colony formation, invasion, and development of a multinucleated dendritic-like phenotype. RNA sequencing and mass spectrometric analysis of SPRIGHTLY-expressing cells revealed changes in the expression of genes involved in cell proliferation, apoptosis, chromosome organization, regulation of DNA damage responses, and cell cycle. The proliferation marker Ki67, minichromosome maintenance genes 2-5, antiapoptotic gene X-linked inhibitor of apoptosis, and baculoviral IAP repeat-containing 7 were all up-regulated in SPRIGHTLY-expressing melanocytes, whereas the proapoptotic tumor suppressor gene DPPIV/CD26 was down-regulated, followed by an increase in extracellular signal-regulated kinase 1/2 phosphorylation, suggesting an increase in mitogen-activated protein kinase activity. Because down-regulation of DPPIV is known to be associated with malignant transformation in melanocytes, SPRIGHTLY-mediated DPPIV down-regulation may play an important role in melanoma pathobiology. Together, these findings provide important insights into how SPRIGHTLY regulates cell proliferation and anchorage-independent colony formation in primary human melanocytes.

RGD-conjugated two-photon absorbing near-IR emitting fluorescent probes for tumor vasculature imaging.

Observation of the activation and inhibition of angiogenesis processes is important in the progression of cancer. Application of targeting peptides, such as a small peptide that contains adjacent L-arginine (R), glycine (G) and L-aspartic acid (D) residues can afford high selectivity and deep penetration in vessel imaging. To facilitate deep tissue vasculature imaging, probes that can be excited via two-photon absorption (2PA) in the near-infrared (NIR) and subsequently emit in the NIR are essential. In this study, the enhancement of tissue image quality with RGD conjugates was investigated with new NIR-emitting pyranyl fluorophore derivatives in two-photon fluorescence microscopy. Linear and nonlinear photophysical properties of the new probes were comprehensively characterized; significantly the probes exhibited good 2PA over a broad spectral range from 700-1100 nm. Cell and tissue images were then acquired and examined, revealing deep penetration and high contrast with the new pyranyl RGD-conjugates up to 350 µm in tumor tissue.

Developmental origin of age-related coronary artery disease.

AIM: Age and injury cause structural and functional changes in coronary artery smooth muscle cells (caSMCs) that influence the pathogenesis of coronary artery disease. Although paracrine signalling is widely believed to drive phenotypic changes in caSMCs, here we show that developmental origin within the fetal epicardium can have a profound effect as well. METHODS AND RESULTS: Fluorescent dye and transgene pulse-labelling techniques in mice revealed that the majority of caSMCs are derived from Wt1(+), Gata5-Cre(+) cells that migrate before E12.5, whereas a minority of cells are derived from a later-emigrating, Wt1(+), Gata5-Cre(-) population. We functionally evaluated the influence of early emigrating cells on coronary artery development and disease by Gata5-Cre excision of Rbpj, which prevents their contribution to coronary artery smooth muscle cells. Ablation of the Gata5-Cre(+) population resulted in coronary arteries consisting solely of Gata5-Cre(-) caSMCs. These coronary arteries appeared normal into early adulthood; however, by 5-8 months of age, they became progressively fibrotic, lost the adventitial outer elastin layer, were dysfunctional and leaky, and animals showed early mortality. CONCLUSION: Taken together, these data reveal heterogeneity in the fetal epicardium that is linked to coronary artery integrity, and that distortion of the coronaries epicardial origin predisposes to adult onset disease.

R-Ras protein inhibits autophosphorylation of vascular endothelial growth factor receptor 2 in endothelial cells and suppresses receptor activation in tumor vasculature.

Abnormal angiogenesis is associated with a broad range of medical conditions, including cancer. The formation of neovasculature with functionally defective blood vessels significantly impacts tumor progression, metastasis, and the efficacy of anticancer therapies. Vascular endothelial growth factor (VEGF) potently induces vascular permeability and vessel growth in the tumor microenvironment, and its inhibition normalizes tumor vasculature. In contrast, the signaling of the small GTPase R-Ras inhibits excessive angiogenic growth and promotes the maturation of regenerating blood vessels. R-Ras signaling counteracts VEGF-induced vessel sprouting, permeability, and invasive activities of endothelial cells. In this study, we investigated the effect of R-Ras on VEGF receptor 2 (VEGFR2) activation by VEGF, the key mechanism for angiogenic stimulation. We show that tyrosine phosphorylation of VEGFR2 is significantly elevated in the tumor vasculature and dermal microvessels of VEGF-injected skin in R-Ras knockout mice. In cultured endothelial cells, R-Ras suppressed the internalization of VEGFR2, which is required for full activation of the receptor by VEGF. Consequently, R-Ras strongly suppressed autophosphorylation of the receptor at all five major tyrosine phosphorylation sites. Conversely, silencing of R-Ras resulted in increased VEGFR2 phosphorylation. This effect of R-Ras on VEGFR2 was, at least in part, dependent on vascular endothelial cadherin. These findings identify a novel function of R-Ras to control the response of endothelial cells to VEGF and suggest an underlying mechanism by which R-Ras regulates angiogenesis.

R-Ras Inhibits VEGF-Induced p38MAPK Activation and HSP27 Phosphorylation in Endothelial Cells.

R-Ras is a Ras family small GTPase that is highly expressed in mature functional blood vessels in normal tissues. It inhibits pathological angiogenesis and promotes vessel maturation and stabilization. Previous studies suggest that R-Ras affects cellular signaling in endothelial cells, pericytes and smooth-muscle cells to regulate vessel formation and remodeling in adult tissues. R-Ras suppresses VEGF-induced endothelial permeability and vessel sprouting while promoting normalization of pathologically developing vessels in mice. It attenuates VEGF receptor-2 (VEGFR2) activation by inhibiting internalization of the receptor upon VEGF ligand binding, leading to significant reduction of VEGFR2 autophosphorylation. Here, we show that R-Ras strongly suppresses the VEGF-dependent activation of stress-activated protein kinase-2/p38 mitogen-activated protein kinase (SAPK2/p38MAPK) and the phosphorylation of downstream heat-shock protein 27 (HSP27), a regulator of actin cytoskeleton organization, in endothelial cells. The suppression of p38MAPK activation and HSP27 phosphorylation by R-Ras concurred with altered actin cytoskeleton architecture, reduced membrane protrusion and inhibition of endothelial cell migration toward VEGF. Silencing of endogenous R-Ras by RNA interference increased membrane protrusion and cell migration stimulated by VEGF, and these effects were offset by p38MAPK inhibitor SB203580. These results suggest that R-Ras regulates angiogenic activities of endothelial cells in part via inhibition of the p38MAPK-HSP27 axis of VEGF signaling.

Peptide-coated liposomal fasudil enhances site specific vasodilation in pulmonary arterial hypertension.

This study sought to develop a liposomal delivery system of fasudil--an investigational drug for the treatment of pulmonary arterial hypertension (PAH)--that will preferentially accumulate in the PAH lungs. Liposomal fasudil was prepared by film-hydration method, and the drug was encapsulated by active loading. The liposome surface was coated with a targeting moiety, CARSKNKDC, a cyclic peptide; the liposomes were characterized for size, polydispersity index, zeta potential, and storage and nebulization stability. The in vitro drug release profiles and uptake by TGF-ß activated pulmonary arterial smooth muscle cells (PASMC) and alveolar macrophages were evaluated. The pharmacokinetics were monitored in male Sprague-Dawley rats, and the pulmonary hemodynamics were studied in acute and chronic PAH rats. The size, polydispersity index (PDI), and zeta potential of the liposomes were 206-216 nm, 0.058-0.084, and -20-42.7 mV, respectively. The formulations showed minimal changes in structural integrity when nebulized with a commercial microsprayer. The optimized formulation was stable for >4 weeks when stored at 4 °C. Fasudil was released in a continuous fashion over 120 h with a cumulative release of 76%. Peptide-linked liposomes were taken up at a higher degree by TGF-ß activated PASMCs; but alveolar macrophages could not engulf peptide-coated liposomes. The formulations did not injure the lungs; the half-life of liposomal fasudil was 34-fold higher than that of plain fasudil after intravenous administration. Peptide-linked liposomal fasudil, as opposed to plain liposomes, reduced the mean pulmonary arterial pressure by 35-40%, without influencing the mean systemic arterial pressure. This study establishes that CAR-conjugated inhalable liposomal fasudil offers favorable pharmacokinetics and produces pulmonary vasculature specific dilatation.

A novel vascular homing peptide strategy to selectively enhance pulmonary drug efficacy in pulmonary arterial hypertension.

A major limitation in the pharmacological treatment of pulmonary arterial hypertension (PAH) is the lack of pulmonary vascular selectivity. Recent studies have identified a tissue-penetrating homing peptide, CARSKNKDC (CAR), which specifically homes to hypertensive pulmonary arteries but not to normal pulmonary vessels or other tissues. Some tissue-penetrating vascular homing peptides have a unique ability to facilitate transport of co-administered drugs into the targeted cells/tissues without requiring physical conjugation of the drug to the peptide (bystander effect). We tested the hypothesis that co-administered CAR would selectively enhance the pulmonary vascular effects of i.v. vasodilators in Sugen5416/hypoxia/normoxia-exposed PAH rats. Systemically administered CAR was predominantly detected in cells of remodeled pulmonary arteries. Intravenously co-administered CAR enhanced pulmonary, but not systemic, effects of the vasodilators, fasudil and imatinib, in PAH rats. CAR increased lung tissue imatinib concentration in isolated PAH lungs without increasing pulmonary vascular permeability. Sublingual CAR was also effective in selectively enhancing the pulmonary vasodilation by imatinib and sildenafil. Our results suggest a new paradigm in the treatment of PAH, using an i.v./sublingual tissue-penetrating homing peptide to selectively augment pulmonary vascular effects of nonselective drugs without the potentially problematic conjugation process. CAR may be particularly useful as an add-on therapy to selectively enhance the pulmonary vascular efficacy of any ongoing drug treatment in patients with PAH.

Correction: Deep Vascular Imaging in Wounds by Two-Photon Fluorescence Microscopy.

[This corrects the article DOI: 10.1371/journal.pone.0067559.].