Zonation, ligand and dose dependence of S1PR1 signalling in blood and lymphatic vasculature.

AIMS: Circulating levels of sphingosine 1-phosphate (S1P), an HDL-associated ligand for endothelial cell (EC) protective S1P receptor-1 (S1PR1), are reduced in disease states associated with endothelial dysfunction. Yet as S1PR1 has high affinity for S1P and can be activated by ligand-independent mechanisms and EC-autonomous S1P production, it is unclear if relative reductions in circulating S1P impact endothelial function. It is also unclear how EC S1PR1 insufficiency, whether induced by ligand deficiency or by S1PR1-directed immunosuppressive therapy, affects different vascular subsets. METHODS AND RESULTS: We here fine-map the zonation of S1PR1 signalling in the murine blood and lymphatic vasculature, superimpose cell type-specific and relative deficiencies in S1P production to define ligand source- and dose-dependence, and correlate receptor engagement to essential functions. In naïve blood vessels, despite broad expression, EC S1PR1 engagement was restricted to resistance-size arteries, lung capillaries and high-endothelial venules (HEV). Similar zonation was observed for albumin extravasation in EC S1PR1 deficient mice, and brain extravasation was reproduced with arterial EC-selective S1pr1 deletion. In lymphatic EC, S1PR1 engagement was high in collecting vessels and lymph nodes and low in terminal capillaries that drain tissue fluids. While EC S1P production sustained S1PR1 signaling in lymphatics and HEV, hematopoietic cells provided ∼90% of plasma S1P and sustained signaling in resistance arteries and lung capillaries. S1PR1 signaling and endothelial function were both surprisingly sensitive to reductions in plasma S1P with apparent saturation around 50% of normal levels. S1PR1 engagement did not depend on sex or age, but modestly increased in arteries in hypertension and diabetes. Sphingosine kinase (Sphk)-2 deficiency also increased S1PR1 engagement selectively in arteries, which could be attributed to Sphk1-dependent S1P release from perivascular macrophages. CONCLUSIONS: This study highlights vessel subtype-specific S1PR1 functions and mechanisms of engagement and supports the relevance of S1P as circulating biomarker for endothelial function.

CD13 expression affects glioma patient survival and influences key functions of human glioblastoma cell lines in vitro.

CD13 (APN) is an Alanyl-Aminopeptidase with diverse functions. The role of CD13 for gliomas is still unknown. In this study, data of glioma patients obtained by TCGA and CGGA databases were used to evaluate the survival rate and prognostic value of CD13 expression level. Protein expression of CD13 was confirmed by immunofluorescence staining of fresh patient tissues. Eight human glioblastoma cell lines were studied by RT-PCR, Western Blot, immunofluorescence staining and flow cytometry to define CD13 expression. Cell lines with different CD13 expression status were treated with a CD13 inhibitor, bestatin, and examined by MTT, scratch and colony formation assaysas well as by apoptosis assay and Western Blots. Bioinformatics analysis indicated that patients with high expression of CD13 had poor survival and prognosis. Additionally, CD13 protein expression was positively associated with clinical malignant characteristics. Investigated glioblastoma cell lines showed distinct expression levels and subcellular localization of CD13 with intracellular enrichment. Bestatin treatment reduced proliferation, migration and colony formation of glioma cells in a CD13-dependent manner while apoptosis was increased. In summary, CD13 has an impact on glioma patient survival and is important for the main function of specific glioma cells.

Murine platelet production is suppressed by S1P release in the hematopoietic niche, not facilitated by blood S1P sensing.

The bioactive lipid mediator sphingosine 1-phosphate (S1P) was recently assigned critical roles in platelet biology: whereas S1P1 receptor-mediated S1P gradient sensing was reported to be essential for directing proplatelet extensions from megakaryocytes (MKs) toward bone marrow sinusoids, MK sphingosine kinase 2 (Sphk2)-derived S1P was reported to further promote platelet shedding through receptor-independent intracellular actions, and platelet aggregation through S1P1 Yet clinical use of S1P pathway modulators including fingolimod has not been associated with risk of bleeding or thrombosis. We therefore revisited the role of S1P in platelet biology in mice. Surprisingly, no reduction in platelet counts was observed when the vascular S1P gradient was ablated by impairing S1P provision to plasma or S1P degradation in interstitial fluids, nor when gradient sensing was impaired by S1pr1 deletion selectively in MKs. Moreover, S1P1 expression and signaling were both undetectable in mature MKs in situ, and MK S1pr1 deletion did not affect platelet aggregation or spreading. When S1pr1 deletion was induced in hematopoietic progenitor cells, platelet counts were instead significantly elevated. Isolated global Sphk2 deficiency was associated with thrombocytopenia, but this was not replicated by MK-restricted Sphk2 deletion and was reversed by compound deletion of either Sphk1 or S1pr2, suggesting that this phenotype arises from increased S1P export and S1P2 activation secondary to redistribution of sphingosine to Sphk1. Consistent with clinical observations, we thus observe no essential role for S1P1 in facilitating platelet production or activation. Instead, S1P restricts megakaryopoiesis through S1P1, and can further suppress thrombopoiesis through S1P2 when aberrantly secreted in the hematopoietic niche.

Paladin (X99384) is expressed in the vasculature and shifts from endothelial to vascular smooth muscle cells during mouse development.

BACKGROUND: Angiogenesis is implicated in many pathological conditions. The role of the proteins involved remains largely unknown, and few vascular-specific drug targets have been discovered. Previously, in a screen for angiogenesis regulators, we identified Paladin (mouse: X99384, human: KIAA1274), a protein containing predicted S/T/Y phosphatase domains. RESULTS: We present a mouse knockout allele for Paladin with a ß-galactosidase reporter, which in combination with Paladin antibodies demonstrate that Paladin is expressed in the vasculature. During mouse embryogenesis, Paladin is primarily expressed in capillary and venous endothelial cells. In adult mice Paladin is predominantly expressed in arterial pericytes and vascular smooth muscle cells. Paladin also displays vascular-restricted expression in human brain, astrocytomas, and glioblastomas. CONCLUSIONS: Paladin, a novel putative phosphatase, displays a dynamic expression pattern in the vasculature. During embryonic stages it is broadly expressed in endothelial cells, while in the adult it is selectively expressed in arterial smooth muscle cells.

Gamma-secretase inhibitor treatment promotes VEGF-A-driven blood vessel growth and vascular leakage but disrupts neovascular perfusion.

The Notch signaling pathway is essential for normal development due to its role in control of cell differentiation, proliferation and survival. It is also critically involved in tumorigenesis and cancer progression. A key enzyme in the activation of Notch signaling is the gamma-secretase protein complex and therefore, gamma-secretase inhibitors (GSIs)--originally developed for Alzheimer's disease--are now being evaluated in clinical trials for human malignancies. It is also clear that Notch plays an important role in angiogenesis driven by Vascular Endothelial Growth Factor A (VEGF-A)--a process instrumental for tumor growth and metastasis. The effect of GSIs on tumor vasculature has not been conclusively determined. Here we report that Compound X (CX), a GSI previously reported to potently inhibit Notch signaling in vitro and in vivo, promotes angiogenic sprouting in vitro and during developmental angiogenesis in mice. Furthermore, CX treatment suppresses tumor growth in a mouse model of renal carcinoma, leads to the formation of abnormal vessels and an increased tumor vascular density. Using a rabbit model of VEGF-A-driven angiogenesis in skeletal muscle, we demonstrate that CX treatment promotes abnormal blood vessel growth characterized by vessel occlusion, disrupted blood flow, and increased vascular leakage. Based on these findings, we propose a model for how GSIs and other Notch inhibitors disrupt tumor blood vessel perfusion, which might be useful for understanding this new class of anti-cancer agents.

RNase H-mediated retrovirus destruction in vivo triggered by oligodeoxynucleotides.

The HIV-1 RNase H can be prematurely activated by oligodeoxynucleotides targeting the highly conserved polypurine tract required for second strand DNA synthesis. This inhibits retroviral replication in cell-free HIV particles and newly infected cells. Here we extend these studies to an in vivo model of retroviral replication. Mice that are chronically infected with the spleen focus-forming virus and treated with oligodeoxynucleotides that target the polypurine tract, exhibit either transient or long-term reductions in plasma virus titer, depending on the therapeutic regimen. Treatment prior to, during or shortly after infection can delay disease progression, increase survival rates and prevent viral infection. This strategy destroys viral RNA template in virus particles in serum as well as early retroviral replication intermediates in infected cells. As it targets events common to the replication cycle of all retroviruses, this approach may be broadly applicable to retroviruses of medical and agricultural importance.