An application of mass spectrometry for quality control of biologicals: Highly sensitive profiling of plasma residuals in human plasma-derived immunoglobulin.

Thromboembolic events (TEE) associated to trace amounts of plasmatic activated coagulation factor XI (FXIa) in administrated immunoglobulin (Ig) have recently raised concerns and hence there is a need for highly sensitive profiling of residual plasma source proteins. This study aims to consider LC-ESI-QTOF data-dependent acquisition in combination with sample fractionation for this purpose. Sample fractionation proved mandatory to enable identification of plasma residuals. Two approaches were compared: Ig depletion with protein G - protein A affinity chromatography and low-abundant protein enrichment with a combinatorial peptide ligand library (ProteoMiner™, Bio-Rad). The latter allowed a higher number of identifications. Highly sensitive detection of prothrombotic FXIa was assessed with confident identification of a 1ng/mg spike. Moreover, different residuals compositions were profiled for various commercial Ig products. Using a quantitative label free analysis, a TEE-positive Ig batch was distinguished from other regular Ig products, with increased levels of FXIa but also other unique proteins. This could have prevented the recently observed TEE problems with Ig. The method is a convenient tool to better characterize Ig products after any plasma pool or manufacture process change, gaining insights in the product quality profile without any prior information required. BIOLOGICAL SIGNIFICANCE: This study characterized residual plasma proteins in Ig products, using bottom-up LC-MS/MS with conventional data-dependent acquisition, preceded by sample fractionation. Without any prior information or target-specific development, >30 proteins were identified in a commercial Ig product. Quality control relevance was demonstrated with the identification of FXIa spiked at 1ng/mg in Ig, which is below the minimal thrombotic dose of 3ng/mg observed in an in vivo model. Relative label-free quantitation highlighted significant differences in normalized abundances of residual proteins between Ig products. A TEE-positive batch was distinguished by unique profile of residual proteins, including FXIa but also various blood stream-regulator proteins (fibrinogen, angiotensinogen, antithrombin-III, complement component C8, …). Those results emphasize that MS screening is a relevant first-line test to prevent any undesired concentration of plasma impurities after a plasma pool or manufacturing process change.

Aberrant activation of fatty acid synthesis suppresses primary cilium formation and distorts tissue development.

Aberrant activation of fatty acid synthesis is a key feature of many advanced human cancers. Unlike in classical lipogenic tissues, this process has been implicated in membrane production required for rapid cell proliferation. Here, to gain further insight into the consequences of tumor-associated fatty acid synthesis, we have mimicked the lipogenic phenotype of cancer cells in Xenopus embryos by microinjection of RNA encoding the lipogenic transcription factor sterol regulatory element binding protein 1c (SREBP1c). Dramatic morphologic changes were observed that could be linked to alterations in Wnt and Hedgehog signaling, and ultimately to a distortion of the primary cilium. This is a sophisticated microtubular sensory organelle that is expressed on the surface of nearly every cell type and that is lost in many cancers. SREBP1c-induced loss of the primary cilium could be confirmed in mammalian Madin-Darby canine kidney (MDCK) cells and was mediated by changes in the supply of fatty acids. Conversely, inhibition of fatty acid synthesis in highly lipogenic human prostate cancer cells restored the formation of the primary cilium. Lipid-induced ciliary loss was associated with mislocalization of apical proteins, distortion of cell polarization, and aberrant epithelial tissue development as revealed in three-dimensional cultures of MDCK cells and in the developing mouse prostate. These data imply that tumor-associated lipogenesis, in addition to rendering cells more autonomous in terms of lipid supply, disturbs cilium formation and contributes to impaired environmental sensing, aberrant signaling, and distortion of polarized tissue architecture, which are all hallmarks of cancer.

De novo lipogenesis protects cancer cells from free radicals and chemotherapeutics by promoting membrane lipid saturation.

Activation of de novo lipogenesis in cancer cells is increasingly recognized as a hallmark of aggressive cancers and has been implicated in the production of membranes for rapid cell proliferation. In the current report, we provide evidence that this activation has a more profound role. Using a mass spectrometry-based phospholipid analysis approach, we show that clinical tumor tissues that display the lipogenic phenotype show an increase in the degree of lipid saturation compared with nonlipogenic tumors. Reversal of the lipogenic switch in cancer cells by treatment with the lipogenesis inhibitor soraphen A or by targeting lipogenic enzymes with small interfering RNA leads to a marked decrease in saturated and mono-unsaturated phospholipid species and increases the relative degree of polyunsaturation. Because polyunsaturated acyl chains are more susceptible to peroxidation, inhibition of lipogenesis increases the levels of peroxidation end products and renders cells more susceptible to oxidative stress-induced cell death. As saturated lipids pack more densely, modulation of lipogenesis also alters lateral and transversal membrane dynamics as revealed by diffusion of membrane-targeted green fluorescent protein and by the uptake and response to doxorubicin. These data show that shifting lipid acquisition from lipid uptake toward de novo lipogenesis dramatically changes membrane properties and protects cells from both endogenous and exogenous insults. These findings provide important new insights into the role of de novo lipogenesis in cancer cells, and they provide a rationale for the use of lipogenesis inhibitors as antineoplastic agents and as chemotherapeutic sensitizers.

Chemical inhibition of acetyl-CoA carboxylase induces growth arrest and cytotoxicity selectively in cancer cells.

Development and progression of cancer is accompanied by marked changes in the expression and activity of enzymes involved in the cellular homeostasis of fatty acids. One class of enzymes that play a particularly important role in this process are the acetyl-CoA carboxylases (ACC). ACCs produce malonyl-CoA, an intermediate metabolite that functions as substrate for fatty acid synthesis and as negative regulator of fatty acid oxidation. Here, using the potent ACC inhibitor soraphen A, a macrocyclic polyketide from myxobacteria, we show that ACC activity in cancer cells is essential for proliferation and survival. Even at nanomolar concentrations, soraphen A can block fatty acid synthesis and stimulate fatty acid oxidation in LNCaP and PC-3M prostate cancer cells. As a result, the phospholipid content of cancer cells decreased, and cells stopped proliferating and ultimately died. LNCaP cells predominantly died through apoptosis, whereas PC-3M cells showed signs of autophagy. Supplementation of the culture medium with exogenous palmitic acid completely abolished the effects of soraphen A and rescued the cells from cell death. Interestingly, when added to cultures of premalignant BPH-1 cells, soraphen A only slightly affected cell proliferation and did not induce cell death. Together, these findings indicate that cancer cells have become dependent on ACC activity to provide the cell with a sufficient supply of fatty acids to permit proliferation and survival, introducing the concept of using small-molecule ACC inhibitors as therapeutic agents for cancer.

Squalene synthase, a determinant of Raft-associated cholesterol and modulator of cancer cell proliferation.

Several cues for cell proliferation, migration, and survival are transmitted through lipid rafts, membrane microdomains enriched in sphingolipids and cholesterol. Cells obtain cholesterol from the circulation but can also synthesize cholesterol de novo through the mevalonate/isoprenoid pathway. This pathway, however, has several branches and also produces non-sterol isoprenoids. Squalene synthase (SQS) is the enzyme that determines the switch toward sterol biosynthesis. Here we demonstrate that in prostate cancer cells SQS expression is enhanced by androgens, channeling intermediates of the mevalonate/isoprenoid pathway toward cholesterol synthesis. Interestingly, the resulting increase in de novo synthesis of cholesterol mainly affects the cholesterol content of lipid rafts, while leaving non-raft cholesterol levels unaffected. Conversely, RNA interference-mediated SQS inhibition results in a decrease of raft-associated cholesterol. These data show that SQS activity and de novo cholesterol synthesis are determinants of membrane microdomain-associated cholesterol in cancer cells. Remarkably, SQS knock down also attenuates proliferation and induces death of prostate cancer cells. Similar effects are observed when cancer cells are treated with the chemical SQS inhibitor zaragozic acid A. Importantly, although the anti-tumor effect of statins has previously been attributed to inhibition of protein isoprenylation, the present study shows that specific inhibition of the cholesterol biosynthesis branch of the mevalonate/isoprenoid pathway also induces cancer cell death. These findings significantly underscore the importance of de novo cholesterol synthesis for cancer cell biology and suggest that SQS is a potential novel target for antineoplastic intervention.

Methotrexate enhances the antianabolic and antiproliferative effects of 5-aminoimidazole-4-carboxamide riboside.

Because of its ability to mimic a low energy status of the cell, the cell-permeable nucleoside 5-aminoimidazole-4-carboxamide (AICA) riboside was proposed as an antineoplastic agent switching off major energy-consuming processes associated with the malignant phenotype (lipid production, DNA synthesis, cell proliferation, cell migration, etc.). Key to the antineoplastic action of AICA riboside is its conversion to ZMP, an AMP mimetic that at high concentrations activates the AMP-activated protein kinase (AMPK). Here, in an attempt to increase the efficacy of AICA riboside, we pretreated cancer cells with methotrexate, an antimetabolite blocking the metabolism of ZMP. Methotrexate enhanced the AICA riboside-induced accumulation of ZMP and led to a decrease in the levels of ATP, which functions as an intrasteric inhibitor of AMPK. Consequently, methotrexate markedly sensitized AMPK for activation by AICA riboside and potentiated the inhibitory effects of AICA riboside on tumor-associated processes. As cotreatment elicited antiproliferative effects already at concentrations of compounds that were only marginally effective when used alone, our findings on the cooperation between methotrexate and AICA riboside provide new opportunities both for the application of classic antimetabolic chemotherapeutics, such as methotrexate, and for the exploitation of the energy-sensing machinery as a target for cancer intervention.

Increased lipogenesis in cancer cells: new players, novel targets.

PURPOSE OF REVIEW: This review evaluates recent findings on the mechanisms by which lipogenic enzymes are upregulated or activated in cancer cells, the implications of increased lipogenesis for cancer cell biology and the feasibility of exploiting this pathway and its regulators as targets for antineoplastic intervention. RECENT FINDINGS: The list of cancer types showing increased lipogenic enzyme expression keeps growing and further evidence is accumulating that growth factor signaling and particularly activation of the phosphatidylinositol 3'-kinase/protein kinase B pathway plays a role in this process. This signaling pathway stimulates lipogenic gene transcription through activation of the lipogenic transcription factor sterol regulatory element-binding protein-1 and directly activates lipogenic enzymes such as ATP-citrate lyase, linking the upregulation of lipogenesis in cancer cells to the well known tumor-associated increase in glycolysis. Steroid hormones, overexpression of the ubiquitin-specific protease-2a and mutations in breast cancer susceptibility gene 1 may further enhance lipid synthesis. While fatty acid synthase is further established as a target for antineoplastic intervention, recent findings show that interference with acetyl-CoA carboxylase-alpha, ATP citrate lyase or the AMP-activated protein kinase limits cancer cell proliferation and survival. SUMMARY: The same disturbances in signaling pathways responsible for oncogenic transformation may also contribute to the increased lipogenesis observed in tumor cells. Increased lipogenesis involves modulation of multiple lipogenic enzymes at both transcriptional and posttranscriptional level and is linked to other cancer-associated metabolic changes. Not only fatty acid synthase, but in fact all key enzymes involved in fatty acid synthesis as well as key metabolic regulators are potential targets for antineoplastic intervention.

Genetic evidence for a tumor suppressor role of HIF-2alpha.

The hypoxia-inducible transcription factors HIF-1alpha and HIF-2alpha are activated in hypoxic tumor regions. However, their role in tumorigenesis remains controversial, as tumor growth promoter and suppressor activities have been ascribed to HIF-1alpha, while the role of HIF-2alpha remains largely unknown. Here, we show that overexpression of HIF-2alpha in rat glioma tumors enhances angiogenesis but reduces growth of these tumors, in part by increasing tumor cell apoptosis. Moreover, siRNA knockdown of HIF-2alpha reduced apoptosis in hypoxic human malignant glioblastoma cells. Furthermore, inhibition of HIF by overexpression of a dominant-negative HIF transgene in glioma cells or HIF-2alpha deficiency in teratomas reduced vascularization but accelerated growth of these tumor types. These findings urge careful consideration of using HIF inhibitors as cancer therapeutic strategies.

RNA interference-mediated silencing of the acetyl-CoA-carboxylase-alpha gene induces growth inhibition and apoptosis of prostate cancer cells.

Overexpression of lipogenic enzymes is a common characteristic of many cancers. Thus far, studies aimed at the exploration of lipogenic enzymes as targets for cancer intervention have focused on fatty acid synthase (FAS), the enzyme catalyzing the terminal steps in fatty acid synthesis. Chemical inhibition or RNA interference (RNAi)-mediated knockdown of FAS consistently inhibits the growth and induces death of cancer cells. Accumulation of the FAS substrate malonyl-CoA has been implicated in the mechanism of cytotoxicity of FAS inhibition. Here, using RNAi technology, we have knocked down the expression of acetyl-CoA carboxylase-alpha (ACC-alpha), the enzyme providing the malonyl-CoA substrate. Silencing of the ACC-alpha gene resulted in a similar inhibition of cell proliferation and induction of caspase-mediated apoptosis of highly lipogenic LNCaP prostate cancer cells as observed after FAS RNAi. In nonmalignant cells with low lipogenic activity, no cytotoxic effects of knockdown of ACC-alpha or FAS were observed. These findings indicate that accumulation of malonyl-CoA is not a prerequisite for cytotoxicity induced by inhibition of tumor-associated lipogenesis and suggest that in addition to FAS, ACC-alpha is a potential target for cancer intervention.

Mimicry of a cellular low energy status blocks tumor cell anabolism and suppresses the malignant phenotype.

Aggressive cancer cells typically show a high rate of energy-consuming anabolic processes driving the synthesis of lipids, proteins, and DNA. Here, we took advantage of the ability of the cell-permeable nucleoside 5-aminoimidazole-4-carboxamide (AICA) riboside to increase the intracellular levels of AICA ribotide, an AMP analogue, mimicking a low energy status of the cell. Treatment of cancer cells with AICA riboside impeded lipogenesis, decreased protein translation, and blocked DNA synthesis. Cells treated with AICA riboside stopped proliferating and lost their invasive properties and their ability to form colonies. When administered in vivo, AICA riboside attenuated the growth of MDA-MB-231 tumors in nude mice. These findings point toward a central tie between energy, anabolism, and cancer and suggest that the cellular energy sensing machinery in cancer cells is an exploitable target for cancer prevention and/or therapy.

Induction of cancer cell apoptosis by flavonoids is associated with their ability to inhibit fatty acid synthase activity.

The consumption of food products containing high amounts of flavonoids has been reported to lower the risk of various cancers. The mechanisms underlying the cancer-protective effects of these naturally occurring polyphenolic compounds, however, remain elusive. Based on our previous finding that the cytotoxic effect of the flavanol epigallocatechin-3-gallate on prostate cancer cells correlates with its ability to inhibit fatty acid synthase (FAS, a key lipogenic enzyme overexpressed in many human cancers), we examined the anti-lipogenic effects of a panel of 18 naturally occurring polyphenolic compounds. In addition to epigallocatechin-3-gallate, five other flavonoids, more particularly luteolin, quercetin, kaempferol, apigenin, and taxifolin, also markedly inhibited cancer cell lipogenesis. Interestingly, in both prostate and breast cancer cells, a remarkable dose-response parallelism was observed between flavonoid-induced inhibition of fatty acid synthesis, inhibition of cell growth, and induction of apoptosis. In support for a role of fatty acid synthesis in these effects, the addition of exogenous palmitate, the end product of FAS, markedly suppressed the cytotoxic effects of flavonoids. Taken together, these findings indicate that the potential of flavonoids to induce apoptosis in cancer cells is strongly associated with their FAS inhibitory properties, thereby providing a new mechanism by which polyphenolic compounds may exert their cancer-preventive and antineoplastic effects.

A novel role for vascular endothelial growth factor as an autocrine survival factor for embryonic stem cells during hypoxia.

Vascular endothelial growth factor (VEGF) is best known for its angiogenic activity on endothelial cells, but it also affects neurons, pneumocytes, and other mature cell types as well as endothelial, neural, and hematopoietic progenitors. Here, we examined its effect on pluripotential embryonic stem (ES) cells under hypoxic stress. ES cells were found to produce VEGF and to express VEGF receptor-2 and neuropilin-1 (Nrp-1), a VEGF165 isoform-specific receptor. During hypoxia, expression levels of VEGF, Flk-1, and Nrp-1 were elevated. Inhibition or targeted gene inactivation of VEGF increased ES cell apoptosis during prolonged hypoxia (48 h) by about 10-fold. The survival activity of VEGF was specific since inhibition of other growth factors (including basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor, platelet-derived growth factor, and placental growth factor) had no effect. Neuropilin-1 was involved in the VEGF-survival activity since overexpression of Nrp-1 decreased hypoxia-induced apoptosis about 3-fold. The hypoxia-response element, via which hypoxia-inducible transcription factors up-regulate VEGF expression under hypoxic conditions, was critical since targeted deletion of this element in the VEGF promoter enhanced hypoxia-induced ES cell apoptosis to the same extent as VEGF inhibition or gene inactivation. Thus, VEGF plays a critical role in survival of ES cells during prolonged hypoxia.

Androgens, lipogenesis and prostate cancer.

Both experimental and epidemiological data indicate that androgens are among the main factors controlling the development, maintenance and progression of prostate cancer. Identifying the genes that are regulated by androgens represents a major step towards the elucidation of the mechanisms underlying the impact of androgens on prostate cancer cell biology and is an attractive approach to find novel targets for prostate cancer therapy. Among the genes that have been identified thus far, several genes encode lipogenic enzymes. Studies aimed at the elucidation of the mechanisms underlying androgen regulation of lipogenic genes revealed that androgens coordinately stimulate the expression of these genes through interference with the molecular mechanism controlling activation of sterol regulatory element-binding proteins (SREBPs), lipogenic transcription factors governing cellular lipid homeostasis. The resulting increase in lipogenesis serves the synthesis of key membrane components (phospholipids, cholesterol) and is a major hallmark of cancer cells. Pharmacologic inhibition of lipogenesis or RNA-interference-mediated down-regulation of key lipogenic genes induces apoptosis in cancer cell lines and reduces tumor growth in xenograft models. While increased lipogenesis is already found in the earliest stages of cancer development (PIN) and initially is androgen-responsive it persists or re-emerges with the development of androgen-independent cancer, indicating that lipogenesis is a fundamental aspect of prostate cancer cell biology and is a potential target for chemoprevention and for antineoplastic therapy in advanced prostate cancer.

Cardia bifida, defective heart development and abnormal neural crest migration in embryos lacking hypoxia-inducible factor-1alpha.

OBJECTIVES: Previous studies have revealed the essential role of hypoxia-inducible factor-1alpha (HIF-1alpha), a basic helix-loop-helix transcription factor, in cardiovascular development. We attempted to further characterize the underlying mechanisms resulting in abnormal cardiogenesis and defective angiogenesis in mice deficient for HIF-1alpha (HIF-1alpha(-/-)). METHODS: We analyzed cardiovascular development in HIF-1alpha(-/-) embryos at both the macroscopic and microscopic level. Gene expression was determined by RT-PCR, in situ hybridization and immunohistochemistry. Embryonic survival was studied using whole embryo culture. RESULTS: HIF-1alpha deficiency caused cardia bifida in some embryos, while cardiac looping was disturbed in others. These defects did not result from abnormal cardiomyocyte commitment or differentiation, but may relate to defective ventricle formation caused by reduced expression of myocyte enhancer factor 2C (MEF2C) and eHAND. In addition, remodeling of the aortic outflow tract and cephalic blood vessels was abnormal in HIF-1alpha(-/-) embryos. These malformations, together with the hypoplastic pharyngeal arches, are presumably induced by defective neural crest cell (NCC) migration. Impaired migration might be related to insufficient levels of semaphorin-3A (Sema3A). Hyperoxia prolonged survival but only partially rescued the developmental program of cultured HIF-1alpha(-/-) embryos. CONCLUSION: HIF-1alpha is essential for proper cardiac development by modulating both neural crest migration and ventricle formation.

Epigallocatechin-3-gallate is a potent natural inhibitor of fatty acid synthase in intact cells and selectively induces apoptosis in prostate cancer cells.

Chemical inhibitors of fatty acid synthase (FAS) inhibit growth and induce apoptosis in several cancer cell lines in vitro and in tumor xenografts in vivo. Recently the green tea component epigallocatechin-3-gallate (EGCG) was shown to act as a natural inhibitor of FAS in chicken liver extracts. Here we investigated whether EGCG inhibits FAS activity in cultured prostate cancer cells and how this inhibition affects endogenous lipid synthesis, cell proliferation and cell viability. The high levels of FAS activity in LNCaP cells were dose-dependently inhibited by EGCG and this inhibition was paralleled by decreased endogenous lipid synthesis, inhibition of cell growth and induction of apoptosis. In contrast, epicatechin (EC), another closely related green tea polyphenolic compound, which does not inhibit FAS, had no effect on LNCaP cell growth or viability. Treatment of nonmalignant cells with low levels of FAS activity (fibroblasts) with EGCG led to a decrease in growth rate but not to induction of apoptosis. These data indicate that EGCG inhibits FAS activity as efficiently as presently known synthetic inhibitors and selectively causes apoptosis in LNCaP cells but not in nontumoral fibroblasts. These findings establish EGCG as a potent natural inhibitor of FAS in intact cells and strengthen the molecular basis for the use of EGCG as a chemopreventive and therapeutic antineoplastic agent.

RNA interference-mediated silencing of the fatty acid synthase gene attenuates growth and induces morphological changes and apoptosis of LNCaP prostate cancer cells.

Fatty acid synthase (FASE), a key enzyme in the biosynthesis of fatty acids, is markedly overexpressed in many human epithelial cancers, rendering it an interesting target for antineoplastic therapy. Here, using the potent and highly sequence-specific mechanism of RNA interference (RNAi), we have silenced the expression of FASE in lymph node carcinoma of the prostate (LNCaP) cells. RNAi-mediated down-regulation of FASE expression resulted in a major decrease in the synthesis of triglycerides and phospholipids and induced marked morphological changes, including a reduction in cell volume, a loss of cell-cell contacts, and the formation of spider-like extrusions. Furthermore, silencing of the FASE gene by RNAi significantly inhibited LNCaP cell growth and ultimately resulted in induction of apoptosis. Importantly and in striking contrast with LNCaP cells, RNAi-mediated inhibition of FASE did not influence growth rate or viability of nonmalignant cultured skin fibroblasts. These data indicate that RNAi opens new avenues toward the study of the role of FASE overexpression in tumor cells and provides an interesting and selective alternative to chemical FASE inhibitors in the development of antineoplastic therapy.

Heterozygous deficiency of hypoxia-inducible factor-2alpha protects mice against pulmonary hypertension and right ventricular dysfunction during prolonged hypoxia.

Chronic hypoxia induces pulmonary vascular remodeling, leading to pulmonary hypertension, right ventricular hypertrophy, and heart failure. Heterozygous deficiency of hypoxia-inducible factor-1alpha (HIF-1alpha), which mediates the cellular response to hypoxia by increasing expression of genes involved in erythropoiesis and angiogenesis, has been previously shown to delay hypoxia-induced pulmonary hypertension. HIF-2alpha is a homologue of HIF-1alpha and is abundantly expressed in the lung, but its role in pulmonary hypertension remains unknown. Therefore, we analyzed the pulmonary response of WT and viable heterozygous HIF-2alpha-deficient (Hif2alpha(+/-)) mice after exposure to 10% O(2) for 4 weeks. In contrast to WT mice, Hif2alpha(+/-) mice were fully protected against pulmonary hypertension and right ventricular hypertrophy, unveiling a critical role of HIF-2alpha in hypoxia-induced pulmonary vascular remodeling. Pulmonary expression levels of endothelin-1 and plasma catecholamine levels were increased threefold and 12-fold respectively in WT but not in Hif2alpha(+/-) mice after hypoxia, suggesting that HIF-2alpha-mediated upregulation of these vasoconstrictors contributes to the development of hypoxic pulmonary vascular remodeling.

Fatty acid synthase drives the synthesis of phospholipids partitioning into detergent-resistant membrane microdomains.

Fatty acid synthase (FAS) is a key metabolic enzyme catalyzing the synthesis of long-chain saturated fatty acids. It plays a central role in the production of surfactant in fetal lungs, in the supply of fatty components of milk, and in the conversion and storage of energy in liver and adipose tissue. Remarkably high levels of FAS expression are found in the majority of human epithelial cancers. As the role of FAS in cancer cells remains largely unknown, we have initiated studies to assess the fate of newly synthesized lipids in cancer cells and have estimated the contribution of FAS to the synthesis of specific lipid classes by treating the cells with small interfering RNAs targeting FAS. Here, we show that in cancer cells FAS plays a major role in the synthesis of phospholipids partitioning into detergent-resistant membrane microdomains. These are raft-aggregates implicated in key cellular processes including signal transduction, intracellular trafficking, cell polarization, and cell migration. These findings reveal a novel role for FAS, provide important new insights into the otherwise poorly understood mechanisms underlying the control of lipid composition of membrane microdomains, and point to a link between FAS overexpression and dysregulation of membrane composition and functioning in tumor cells.

Loss of placental growth factor protects mice against vascular permeability in pathological conditions.

Vascular leakage contributes to numerous disorders but only a limited number of molecules have been demonstrated to modulate permeability of the vessel wall. The vascular endothelial growth factor (VEGF) is a potent inducer of vascular leakage. Previous studies demonstrated that exogenous administration of placental growth factor (PlGF), a homologue of VEGF, stimulates vascular permeability but the role of endogenous PlGF in plasma extravasation during pathological conditions remains unknown. We recently generated PlGF deficient (PlGF(-/-)) mice and demonstrated that loss of PlGF impaired pathological angiogenesis by attenuating the response to VEGF. Here, we demonstrate that absence of PlGF reduces vascular leakage induced by skin wounding, allergens, and neurogenic inflammation. These findings suggest that inhibition of PlGF might be an attractive tool to reduce vascular leakage in various diseases.

Loss of HIF-2alpha and inhibition of VEGF impair fetal lung maturation, whereas treatment with VEGF prevents fatal respiratory distress in premature mice.

Respiratory distress syndrome (RDS) due to insufficient production of surfactant is a common and severe complication of preterm delivery. Here, we report that loss of the hypoxia-inducible transcription factor-2alpha (HIF-2alpha) caused fatal RDS in neonatal mice due to insufficient surfactant production by alveolar type 2 cells. VEGF, a target of HIF-2alpha, regulates fetal lung maturation: because VEGF levels in alveolar cells were reduced in HIF-2alpha-deficient fetuses; mice with a deficiency of the VEGF(164) and VEGF(188) isoforms or of the HIF-binding site in the VEGF promotor died of RDS; intrauterine delivery of anti-VEGF-receptor-2 antibodies caused RDS and VEGF stimulated production of surfactant proteins by cultured type 2 pneumocytes. Intrauterine delivery or postnatal intratracheal instillation of VEGF stimulated conversion of glycogen to surfactant and protected preterm mice against RDS. The pneumotrophic effect of VEGF may have therapeutic potential for lung maturation in preterm infants.