Murine endothelial serine palmitoyltransferase 1 (SPTLC1) is required for vascular development and systemic sphingolipid homeostasis.

Serine palmitoyl transferase (SPT), the rate-limiting enzyme in the de novo synthesis of sphingolipids (SL), is needed for embryonic development, physiological homeostasis, and response to stress. The functions of de novo SL synthesis in vascular endothelial cells (EC), which line the entire circulatory system, are not well understood. Here, we show that the de novo SL synthesis in EC not only regulates vascular development but also maintains circulatory and peripheral organ SL levels. Mice with an endothelial-specific gene knockout of SPTLC1 (Sptlc1 ECKO), an essential subunit of the SPT complex, exhibited reduced EC proliferation and tip/stalk cell differentiation, resulting in delayed retinal vascular development. In addition, Sptlc1 ECKO mice had reduced retinal neovascularization in the oxygen-induced retinopathy model. Mechanistic studies suggest that EC SL produced from the de novo pathway are needed for lipid raft formation and efficient VEGF signaling. Post-natal deletion of the EC Sptlc1 also showed rapid reduction of several SL metabolites in plasma, red blood cells, and peripheral organs (lung and liver) but not in the retina, part of the central nervous system (CNS). In the liver, EC de novo SL synthesis was important for acetaminophen-induced rapid ceramide elevation and hepatotoxicity. These results suggest that EC-derived SL metabolites are in constant flux between the vasculature, circulatory elements, and parenchymal cells of non-CNS organs. Taken together, our data point to the central role of the endothelial SL biosynthesis in maintaining vascular development, neovascular proliferation, non-CNS tissue metabolic homeostasis, and hepatocyte response to stress.

Sphingosine 1-Phosphate Receptor Signaling Establishes AP-1 Gradients to Allow for Retinal Endothelial Cell Specialization.

Transcriptional mechanisms that drive angiogenesis and organotypic vascular endothelial cell specialization are poorly understood. Here, we show that retinal endothelial sphingosine 1-phosphate receptors (S1PRs), which restrain vascular endothelial growth factor (VEGF)-induced angiogenesis, spatially restrict expression of JunB, a member of the activator protein 1 (AP-1) family of transcription factors (TFs). Mechanistically, VEGF induces JunB expression at the sprouting vascular front while S1PR-dependent vascular endothelial (VE)-cadherin assembly suppresses JunB expression in the nascent vascular network, thus creating a gradient of this TF. Endothelial-specific JunB knockout mice showed diminished expression of neurovascular guidance genes and attenuated retinal vascular network progression. In addition, endothelial S1PR signaling is required for normal expression of ß-catenin-dependent genes such as TCF/LEF1 and ZIC3 TFs, transporters, and junctional proteins. These results show that S1PR signaling restricts JunB function to the expanding vascular front, thus creating an AP-1 gradient and enabling organotypic endothelial cell specialization of the vascular network.

Excessive vascular sprouting underlies cerebral hemorrhage in mice lacking αVß8-TGFß signaling in the brain.

Vascular development of the central nervous system and blood-brain barrier (BBB) induction are closely linked processes. The role of factors that promote endothelial sprouting and vascular leak, such as vascular endothelial growth factor A, are well described, but the factors that suppress angiogenic sprouting and their impact on the BBB are poorly understood. Here, we show that integrin αVß8 activates angiosuppressive TGFß gradients in the brain, which inhibit endothelial cell sprouting. Loss of αVß8 in the brain or downstream TGFß1-TGFBR2-ALK5-Smad3 signaling in endothelial cells increases vascular sprouting, branching and proliferation, leading to vascular dysplasia and hemorrhage. Importantly, BBB function in Itgb8 mutants is intact during early stages of vascular dysgenesis before hemorrhage. By contrast, Pdgfb(ret/ret) mice, which exhibit severe BBB disruption and vascular leak due to pericyte deficiency, have comparatively normal vascular morphogenesis and do not exhibit brain hemorrhage. Our data therefore suggest that abnormal vascular sprouting and patterning, not BBB dysfunction, underlie developmental cerebral hemorrhage.

Lim domain binding 2: a key driver of transendothelial migration of leukocytes and atherosclerosis.

OBJECTIVE: Using a multi-tissue, genome-wide gene expression approach, we recently identified a gene module linked to the extent of human atherosclerosis. This atherosclerosis module was enriched with inherited risk for coronary and carotid artery disease (CAD) and overlapped with genes in the transendothelial migration of leukocyte (TEML) pathway. Among the atherosclerosis module genes, the transcription cofactor Lim domain binding 2 (LDB2) was the most connected in a CAD vascular wall regulatory gene network. Here, we used human genomics and atherosclerosis-prone mice to evaluate the possible role of LDB2 in TEML and atherosclerosis. APPROACH AND RESULTS: mRNA profiles generated from blood macrophages in patients with CAD were used to infer transcription factor regulatory gene networks; Ldlr(-/-)Apob(100/100) mice were used to study the effects of Ldb2 deficiency on TEML activity and atherogenesis. LDB2 was the most connected gene in a transcription factor regulatory network inferred from TEML and atherosclerosis module genes in CAD macrophages. In Ldlr(-/-)Apob(100/100) mice, loss of Ldb2 increased atherosclerotic lesion size ≈2-fold and decreased plaque stability. The exacerbated atherosclerosis was caused by increased TEML activity, as demonstrated in air-pouch and retinal vasculature models in vivo, by ex vivo perfusion of primary leukocytes, and by leukocyte migration in vitro. In THP1 cells, migration was increased by overexpression and decreased by small interfering RNA inhibition of LDB2. A functional LDB2 variant (rs10939673) was associated with the risk and extent of CAD across several cohorts. CONCLUSIONS: As a key driver of the TEML pathway in CAD macrophages, LDB2 is a novel candidate to target CAD by inhibiting the overall activity of TEML.

Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment.

Lack of robust methods for establishment and expansion of pluripotent human embryonic stem (hES) cells still hampers development of cell therapy. Laminins (LN) are a family of highly cell-type specific basement membrane proteins important for cell adhesion, differentiation, migration and phenotype stability. Here we produce and isolate a human recombinant LN-521 isoform and develop a cell culture matrix containing LN-521 and E-cadherin, which both localize to stem cell niches in vivo. This matrix allows clonal derivation, clonal survival and long-term self-renewal of hES cells under completely chemically defined and xeno-free conditions without ROCK inhibitors. Neither LN-521 nor E-cadherin alone enable clonal survival of hES cells. The LN-521/E-cadherin matrix allows hES cell line derivation from blastocyst inner cell mass and single blastomere cells without a need to destroy the embryo. This method can facilitate the generation of hES cell lines for development of different cell types for regenerative medicine purposes.

Sphingosine-1-phosphate activates the AKT pathway to protect small intestines from radiation-induced endothelial apoptosis.

A previous in vitro study showed that sphingosine-1-phosphate (S1P), a ceramide antagonist, preserved endothelial cells in culture from radiation-induced apoptosis. We proposed to validate the role of S1P in tissue radioprotection by inhibiting acute gastrointestinal (GI) syndrome induced by endothelial cell apoptosis after high dose of radiation. Retro-orbital S1P was injected in mice exposed to 15 Gy, a dose-inducing GI syndrome within 10 days. Overall survival and apoptosis on intestines sections were studied. Intestinal cell type targeted by S1P and early molecular survival pathways were researched using irradiated in vitro cell models and in vivo mouse models. We showed that retro-orbital S1P injection before irradiation prevented GI syndrome by inhibiting endothelium collapse. We defined endothelium as a specific therapeutic target because only these cells and not intestinal epithelial cells, or B and T lymphocytes, were protected. Pharmacologic approaches using AKT inhibitor and pertussis toxin established that S1P affords endothelial cell protection in vitro and in vivo through a mechanism involving AKT and 7-pass transmembrane receptors coupled to Gi proteins. Our results provide strong pharmacologic and mechanistic proofs that S1P protects endothelial cells against acute radiation enteropathy.

Comparative toxicity and efficacy of combined radioimmunotherapy and antiangiogenic therapy in carcinoembryonic antigen-expressing medullary thyroid cancer xenograft.

UNLABELLED: A significant antitumor effect was previously observed with radioimmunotherapy using anti-carcinoembryonic antigen (131)I-F6 monoclonal antibody in medullary thyroid cancer-bearing nude mice. Nevertheless, no complete response was observed. As seen with chemotherapy, drugs targeting the tumor microenvironment might improve radioimmunotherapy efficacy. This study evaluated the toxicity and efficacy of combining radioimmunotherapy with thalidomide or a cyclopeptidic vascular endothelial growth inhibitor (CBOP11) in mice grafted with the TT human medullary thyroid cancer cell line. METHODS: Six to 10 nude mice treated with 92.5 MBq of (131)I-F6 in association with 200 mg/kg/d of oral thalidomide during 20 d by force-feeding or 0.45 mg/kg/d of CBOP11 during 25 d using subcutaneous minipumps were compared with control mice receiving either treatment or naked F6 or nonspecific (131)I-734. Combined therapies included (131)I-F6 at day 0 followed by thalidomide between days 20 and 40, thalidomide between days 0 and 20 followed by (131)I-F6 at day 25, (131)I-F6 at day 0 and CBOP11 between days 0 and 25, CBOP11 between days 0 and 25 followed by (131)I-F6 at day 25, and (131)I-F6 at day 0 followed by CBOP11 between days 20 and 45. Animal weight, hematologic toxicity, tumor volume, and serum calcitonin were monitored for the following 3 mo. Improvement of (125)I-F6 tumor biodistribution by antiangiogenic drug was studied after pretreatment by thalidomide. Follow-up of the tumor after combined antiangiogenic and radioimmunotherapy therapies was performed by histology studies. RESULTS: Combined associations, as compared with radioimmunotherapy alone, increased leukopenia but not thrombocytopenia. Tumor volume-quadrupling time (TVQT) was 22.8 +/- 3.3 d in the control group, 29.9 +/- 3.6 d in the group treated with thalidomide, 34.6 +/- 4.4 d in the group treated with CBOP11, and 51.0 +/- 2.8 d after radioimmunotherapy alone. As compared with radioimmunotherapy, TVQT was significantly longer (P < 0.01) after thalidomide followed by radioimmunotherapy (69.83 +/- 3.9), CBOP11 followed by radioimmunotherapy (71.3 +/- 6.1), and CBOP11-radioimmunotherapy in concomitance (64.2 +/- 6.1). Nevertheless, TVQT was not increased after radioimmunotherapy followed by thalidomide (48.8 +/- 4) and radioimmunotherapy followed by CBOP11 (56.8 +/- 4.8). Surprisingly, pretreatment by CBOP11 or thalidomide sensitized larger tumors (>300 mm(3)) to radioimmunotherapy. Change in calcitonin levels confirmed morphologic tumor response. Tumor uptake 24 h after injection of (125)I-F6 was 4.5 +/- 0.6 percentage injected dose per gram (%ID/g) without pretreatment and 8.7 +/- 1.3 %ID/g with pretreatment by thalidomide. An increase of the antitumor effect observed using the antiangiogenic drug combined with radioimmunotherapy was correlated with a decrease of blood vessels shown by von Willebrand immunostaining. CONCLUSION: Pretreatment with antiangiogenic therapies improved radioimmunotherapy efficacy, with acceptable toxicity. Future investigations will be performed to understand how antiangiogenic agents sensitize large tumors to radioimmunotherapy.

Plasma membrane signaling induced by ionizing radiation.

For decades, DNA has been considered as the main cellular target of deleterious effects of ionizing radiation (IR). Nevertheless, molecular signals initiated at cellular membranes are now identified as critical events in a large spectrum of radiation-induced cellular processes. If IR provokes DNA damage directly by energy deposit on the DNA double helix and indirectly by reactive species, origin of IR-induced molecular events initiated at the plasma membrane remains more obscure. Generation of reactive oxygen/nitrogen species (ROS/RNS) inducing proteins and lipids modifications seems to be the prevalent hypothesis. However, spatial and temporal relocalization of proteins and/or lipids represents also potential mechanisms of cell signaling generation. In the context of an oxidative stress such as IR, the best example is the translocation of the enzyme acid sphingomyelinase (ASMase) from lysosomes to the outer layer of cell membrane, which then induces sphingomyelin hydrolysis and ceramide formation. Ceramide coalescence with cholesterol forms lipids microdomains in the plasma membrane, enhancing clustering of signaling receptors (death receptors like FAS, TNF, CD40, TRAIL or G protein-coupled receptors). In this manuscript, we propose to overview the different key molecular mechanisms induced at the plasma membrane after IR in perspective with their linked molecular actors.

Sphingosine-1-phosphate protects proliferating endothelial cells from ceramide-induced apoptosis but not from DNA damage-induced mitotic death.

Because of the central role of the endothelium in tissue homeostasis, protecting the vasculature from radiation-induced death is a major concern in tissue radioprotection. Premitotic apoptosis and mitotic death are two prevalent cell death pathways induced by ionizing radiation. Endothelial cells undergo apoptosis after radiation through generation of the sphingolipid ceramide. However, if mitotic death is known as the established radiation-induced death pathway for cycling eukaryotic cells, direct involvement of mitotic death in proliferating endothelial radiosensitivity has not been clearly shown. In this study, we proved that proliferating human microvascular endothelial cells (HMEC-1) undergo two waves of death after exposure to 15 Gy radiation: an early premitotic apoptosis dependent on ceramide generation and a delayed DNA damage-induced mitotic death. The fact that sphingosine-1-phosphate (S1P), a ceramide antagonist, protects HMEC-1 only from membrane-dependent apoptosis but not from DNA damage-induced mitotic death proves the independence of the two pathways. Furthermore, adding nocodazole, a mitotic inhibitor, to S1P affected both cell death mechanisms and fully prevented radiation-induced death. If our results fit with the standard model in which S1P signaling inhibits ceramide-mediated apoptosis induced by antitumor treatments, such as radiotherapy, they exclude, for the first time, a significant role of S1P-induced molecular survival pathway against mitotic death. Discrimination between ceramide-mediated apoptosis and DNA damage-induced mitotic death may give the opportunity to define a new class of radioprotectors for normal tissues in which quiescent endothelium represents the most sensitive target, while excluding malignant tumor containing pro-proliferating angiogenic endothelial cells that are sensitive to mitotic death.