Microgravity triggers ferroptosis and accelerates senescence in the MG-63 cell model of osteoblastic cells.

In space, cells sustain strong modifications of their mechanical environment. Mechanosensitive molecules at the cell membrane regulate mechanotransduction pathways that induce adaptive responses through the regulation of gene expression, post-translational modifications, protein interactions or intracellular trafficking, among others. In the current study, human osteoblastic cells were cultured on the ISS in microgravity and at 1 g in a centrifuge, as onboard controls. RNAseq analyses showed that microgravity inhibits cell proliferation and DNA repair, stimulates inflammatory pathways and induces ferroptosis and senescence, two pathways related to ageing. Morphological hallmarks of senescence, such as reduced nuclear size and changes in chromatin architecture, proliferation marker distribution, tubulin acetylation and lysosomal transport were identified by immunofluorescence microscopy, reinforcing the hypothesis of induction of cell senescence in microgravity during space flight. These processes could be attributed, at least in part, to the regulation of YAP1 and its downstream effectors NUPR1 and CKAP2L.

Acidosis-induced regulation of adipocyte G0S2 promotes crosstalk between adipocytes and breast cancer cells as well as tumor progression.

Bidirectional interactions between cancer cells and their microenvironment govern tumor progression. Among the stromal cells in this microenvironment, adipocytes have been reported to upregulate cancer cell migration and invasion by producing fatty acids. Conversely, cancer cells alter adipocyte phenotype notably via increased lipolysis. We aimed to identify the mechanisms through which cancer cells trigger adipocyte lipolysis and evaluate the functional consequences on cancer progression. Here, we show that cancer cell-induced acidification of the extracellular medium strongly promotes preadipocyte lipolysis through a mechanism that does not involve lipophagy but requires adipose triglyceride lipase (ATGL) activity. This increased lipolysis is triggered mainly by attenuation of the G0/G1 switch gene 2 (G0S2)-induced inhibition of ATGL. G0S2-mediated regulation in preadipocytes affects their communication with breast cancer cells, modifying the phenotype of the cancer cells and increasing their resistance to chemotherapeutic agents in vitro. Furthermore, we demonstrate that the adipocyte-specific overexpression of G0S2 impairs mammary tumor growth and lung metastasis formation in vivo. Our results highlight the importance of acidosis in cancer cell-adipocyte crosstalk and identify G0S2 as the main regulator of cancer-induced lipolysis, regulating tumor establishment and spreading.

The Dual Function of RhoGDI2 in Immunity and Cancer.

RhoGDI2 is a guanine nucleotide dissociation inhibitor (GDI) specific for the Rho family of small GTPases. It is highly expressed in hematopoietic cells but is also present in a large array of other cell types. RhoGDI2 has been implicated in multiple human cancers and immunity regulation, where it can display a dual role. Despite its involvement in various biological processes, we still do not have a clear understanding of its mechanistic functions. This review sheds a light on the dual opposite role of RhoGDI2 in cancer, highlights its underappreciated role in immunity and proposes ways to explain its intricate regulatory functions.

ADAMTS2 and ADAMTS14 can substitute for ADAMTS3 in adults for pro-VEGFC activation and lymphatic homeostasis.

The capacity of ADAMTS3 to cleave pro-VEGFC into active VEGFC able to bind its receptors and to stimulate lymphangiogenesis has been clearly established during embryonic life. However, this function of ADAMTS3 is unlikely to persist in adulthood because of its restricted expression pattern after birth. Because ADAMTS2 and ADAMTS14 are closely related to ADAMTS3 and are mainly expressed in connective tissues where the lymphatic network extends, we hypothesized that they could substitute for ADAMTS3 during adulthood in mammals allowing proteolytic activation of pro-VEGFC. Here, we demonstrated that ADAMTS2 and ADAMTS14 are able to process pro-VEGFC into active VEGFC as efficiently as ADAMTS3. In vivo, adult mice lacking Adamts2 developed skin lymphedema due to a reduction of the density and diameter of lymphatic vessels, leading to a decrease of lymphatic functionality, while genetic ablation of Adamts14 had no impact. In a model of thermal cauterization of cornea, lymphangiogenesis was significantly reduced in Adamts2- and Adamts14-KO mice and further repressed in Adamts2/Adamts14 double-KO mice. In summary, we have demonstrated that ADAMTS2 and ADAMTS14 are as efficient as ADAMTS3 in activation of pro-VEGFC and are involved in the homeostasis of the lymphatic vasculature in adulthood, both in physiological and pathological processes.

In vivo N-Terminomics Highlights Novel Functions of ADAMTS2 and ADAMTS14 in Skin Collagen Matrix Building.

A disintegrin and metalloproteinase with thrombospondin type I motif (ADAMTS)2 and ADAMTS14 were originally known for their ability to cleave the aminopropeptides of fibrillar collagens. Previous work using N-terminomic approach (N-TAILS) in vitro led to the identification of new substrates, including some molecules involved in TGF-ß signaling. Here, N-TAILS was used to investigate the substrates of these two enzymes in vivo, by comparing the N-terminomes of the skin of wild type mice, mice deficient in ADAMTS2, in ADAMTS14 and in both ADAMTS2 and ADAMTS14. This study identified 68 potential extracellular and cell surface proteins, with the majority of them being cleaved by both enzymes. These analyses comfort their role in collagen matrix organization and suggest their implication in inflammatory processes. Regarding fibrillar collagen, this study demonstrates that both ADAMTS2 and ADAMTS14 are involved in the processing of the aminopropeptide of alpha1 and alpha2 type V collagen. It also revealed the existence of several cleavage sites in the Col1 domain and in the C-propeptide of type I collagens. In addition to collagens and other extracellular proteins, two major components of the cell cytoskeleton, actin and vimentin, were also identified as potential substrates. The latter data were confirmed in vitro using purified enzymes and could potentially indicate other functions for ADAMTS2 and 14. This original investigation of mouse skin degradomes by N-terminomic highlights the essential role of ADAMTS2 and ADAMTS14 in collagen matrix synthesis and turnover, and gives clues to better understand their functions in skin pathophysiology. Data are available via ProteomeXchange with identifier PXD022179.

Lipin-1, a Versatile Regulator of Lipid Homeostasis, Is a Potential Target for Fighting Cancer.

The rewiring of lipid metabolism is a major adaptation observed in cancer, and it is generally associated with the increased aggressiveness of cancer cells. Targeting lipid metabolism is therefore an appealing therapeutic strategy, but it requires a better understanding of the specific roles played by the main enzymes involved in lipid biosynthesis. Lipin-1 is a central regulator of lipid homeostasis, acting either as an enzyme or as a co-regulator of transcription. In spite of its important functions it is only recently that several groups have highlighted its role in cancer. Here, we will review the most recent research describing the role of lipin-1 in tumor progression when expressed by cancer cells or cells of the tumor microenvironment. The interest of its inhibition as an adjuvant therapy to amplify the effects of anti-cancer therapies will be also illustrated.

Mechanical strain induces a pro-fibrotic phenotype in human mitral valvular interstitial cells through RhoC/ROCK/MRTF-A and Erk1/2 signaling pathways.

The mitral valve is a complex multilayered structure populated by fibroblast-like cells, valvular interstitial cells (VIC) which are embedded in an extracellular matrix (ECM) scaffold and are submitted to the mechanical deformations affecting valve at each heartbeat, for an average of 40 million times per year. Myxomatous mitral valve (MMV) is the most frequent heart valve disease characterized by disruption of several valvular structures due to alterations of their ECM preventing the complete closure of the valve resulting in symptoms of prolapse and regurgitation. VIC and their ECM exhibit reciprocal dynamic processes between the mechanical signals issued from the ECM and the modulation of VIC phenotype responsible for ECM homeostasis of the valve. Abnormal perception and responsiveness of VIC to mechanical stress may induce an inappropriate adaptative remodeling of the valve progressively leading to MMV. To investigate the response of human VIC to mechanical strain and identify the molecular mechanisms of mechano-transduction in these cells, a cyclic equibiaxial elongation of 14% at the cardiac frequency of 1.16 Hz was applied to VIC by using a Flexercell-4000 T™ apparatus for increasing time (from 1 h to 8 h). We showed that cyclic stretch induces an early (1 h) and transient over-expression of TGFß2 and αSMA. CTGF, a profibrotic growth factor promoting the synthesis of ECM components, was strongly induced after 1 and 2 h of stretching and still upregulated at 8 h. The mechanical stress-induced CTGF up-regulation was dependent on RhoC, but not RhoA, as demonstrated by siRNA-mediated silencing approaches, and further supported by evidencing RhoC activation upon cell stretching and suppression of cell response by pharmacological inhibition of the effector ROCK1/2. It was also dependent on the MEK/Erk1/2 pathway which was activated by mechanical stress independently of RhoC and ROCK. Finally, mechanical stretching induced the nuclear translocation of myocardin related transcription factor-A (MRTF-A) which forms a transcriptional complex with SRF to promote the expression of target genes, notably CTGF. Treatment of stretched cultures with inhibitors of the identified pathways (ROCK1/2, MEK/Erk1/2, MRTF-A translocation) blocked CTGF overexpression and abrogated the increased MRTF-A nuclear translocation. CTGF is up-regulated in many pathological processes involving mechanically challenged organs, promotes ECM accumulation and is considered as a hallmark of fibrotic diseases. Pharmacological targeting of MRTF-A by newly developed inhibitors may represent a relevant therapy for MMV.

Dephosphorylation of HDAC4 by PP2A-Bδ unravels a new role for the HDAC4/MEF2 axis in myoblast fusion.

Muscle formation is controlled by a number of key myogenic transcriptional regulators that govern stage-specific gene expression programs and act as terminal effectors of intracellular signaling pathways. To date, the role of phosphatases in the signaling cascades instructing muscle development remains poorly understood. Here, we show that a specific PP2A-B55δ holoenzyme is necessary for skeletal myogenesis. The primary role of PP2A-B55δ is to dephosphorylate histone deacetylase 4 (HDAC4) following myocyte differentiation and ensure repression of Myocyte enhancer factor 2D (MEF2D)-dependent gene expression programs during myogenic fusion. As a crucial HDAC4/MEF2D target gene that governs myocyte fusion, we identify ArgBP2, an upstream inhibitor of Abl, which itself is a repressor of CrkII signaling. Consequently, cells lacking PP2A-B55δ show upregulation of ArgBP2 and hyperactivation of CrkII downstream effectors, including Rac1 and FAK, precluding cytoskeletal and membrane rearrangements associated with myoblast fusion. Both in vitro and in zebrafish, loss-of-function of PP2A-B55δ severely impairs fusion of myocytes and formation of multinucleated muscle fibers, without affecting myoblast differentiation. Taken together, our results establish PP2A-B55δ as the first protein phosphatase to be involved in myoblast fusion and suggest that reversible phosphorylation of HDAC4 may coordinate differentiation and fusion events during myogenesis.

uPARAP/Endo180 receptor is a gatekeeper of VEGFR-2/VEGFR-3 heterodimerisation during pathological lymphangiogenesis.

The development of new lymphatic vessels occurs in many cancerous and inflammatory diseases through the binding of VEGF-C to its receptors, VEGFR-2 and VEGFR-3. The regulation of VEGFR-2/VEGFR-3 heterodimerisation and its downstream signaling in lymphatic endothelial cells (LECs) remain poorly understood. Here, we identify the endocytic receptor, uPARAP, as a partner of VEGFR-2 and VEGFR-3 that regulates their heterodimerisation. Genetic ablation of uPARAP leads to hyperbranched lymphatic vasculatures in pathological conditions without affecting concomitant angiogenesis. In vitro, uPARAP controls LEC migration in response to VEGF-C but not VEGF-A or VEGF-CCys156Ser. uPARAP restricts VEGFR-2/VEGFR-3 heterodimerisation and subsequent VEGFR-2-mediated phosphorylation and inactivation of Crk-II adaptor. uPARAP promotes VEGFR-3 signaling through the Crk-II/JNK/paxillin/Rac1 pathway. Pharmacological Rac1 inhibition in uPARAP knockout mice restores the wild-type phenotype. In summary, our study identifies a molecular regulator of lymphangiogenesis, and uncovers novel molecular features of VEGFR-2/VEGFR-3 crosstalk and downstream signaling during VEGF-C-driven LEC sprouting in pathological conditions.

Myoferlin controls mitochondrial structure and activity in pancreatic ductal adenocarcinoma, and affects tumor aggressiveness.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death. Therapeutic options remain very limited and are based on classical chemotherapies. Energy metabolism reprogramming appears as an emerging hallmark of cancer and is considered a therapeutic target with considerable potential. Myoferlin, a ferlin family member protein overexpressed in PDAC, is involved in plasma membrane biology and has a tumor-promoting function. In the continuity of our previous studies, we investigated the role of myoferlin in the context of energy metabolism in PDAC. We used selected PDAC tumor samples and PDAC cell lines together with small interfering RNA technology to study the role of myoferlin in energetic metabolism. In PDAC patients, we showed that myoferlin expression is negatively correlated with overall survival and with glycolytic activity evaluated by 18F-deoxyglucose positron emission tomography. We found out that myoferlin is more abundant in lipogenic pancreatic cancer cell lines and is required to maintain a branched mitochondrial structure and a high oxidative phosphorylation activity. The observed mitochondrial fission induced by myoferlin depletion led to a decrease of cell proliferation, ATP production, and autophagy induction, thus indicating an essential role of myoferlin for PDAC cell fitness. The metabolic phenotype switch generated by myoferlin silencing could open up a new perspective in the development of therapeutic strategies, especially in the context of energy metabolism.

Propranolol sensitizes prostate cancer cells to glucose metabolism inhibition and prevents cancer progression.

Propranolol, a widely used non-selective beta-adrenergic receptor blocker, was recently shown to display anticancer properties. Its potential to synergize with certain drugs has been also outlined. However, it is necessary to take into account all the properties of propranolol to select a drug that could be efficiently combined with. Propranolol was reported to block the late phase of autophagy. Hence, we hypothesized that in condition enhancing autophagy flux, cancer cells should be especially sensitive to propranolol. 2DG, a glycolysis inhibitor, is an anti-tumor agent having limited effect in monotherapy notably due to induction of pro-survival autophagy. Here, we report that treatment of cancer cells with propranolol in combination with the glycolysis inhibitor 2DG induced a massive accumulation of autophagosome due to autophagy blockade. The propranolol +2DG treatment efficiently prevents prostate cancer cell proliferation, induces cell apoptosis, alters mitochondrial morphology, inhibits mitochondrial bioenergetics and aggravates ER stress in vitro and also suppresses tumor growth in vivo. Our study underlines for the first time the interest to take advantage of the ability of propranolol to inhibit autophagy to design new anti-cancer therapies.

A Novel Physiological Glycosaminoglycan-Deficient Splice Variant of Neuropilin-1 Is Anti-Tumorigenic In Vitro and In Vivo.

Neuropilin-1 (NRP1) is a transmembrane protein acting as a co-receptor for several growth factors and interacting with other proteins such as integrins and plexins/semaphorins. It is involved in axonal development, angiogenesis and cancer progression. Its primary mRNA is subjected to alternative splicing mechanisms generating different isoforms, some of which lack the transmembrane domain and display antagonist properties to NRP1 full size (FS). NRP1 is further post-translationally modified by the addition of glycosaminoglycans (GAG) side chains through an O-glycosylation site at serine612. Here, we characterized a novel splice variant which has never been investigated, NRP1-Δ7, differing from the NRP1-FS by a deletion of 7 amino acids occurring two residues downstream of the O-glycosylation site. This short sequence contains two aspartic residues critical for efficient glycosylation. As expected, the high molecular weight products appearing as a smear in SDS-PAGE and reflecting the presence of GAG in NRP1-FS were undetectable in the NRP1-Δ7 protein. NRP1-Δ7 mRNA was found expressed at an appreciable level, between 10 and 30% of the total NRP1, by various cells lines and tissues from human and murine origin. To investigate the biological properties of this isoform, we generated prostatic (PC3) and breast (MDA-MB-231) cancer cells able to express recombinant NRP1-FS or NRP1-Δ7 in a doxycycline-inducible manner. Cells with increased expression of NRP1-Δ7 were characterized in vitro by a significant reduction of proliferation, migration and anchorage-independent growth, while NRP1-FS had the expected opposite "pro-tumoral" effects. Upon VEGF-A165 treatment, a lower internalization rate was observed for NRP1-Δ7 than for NRP1-FS. Finally, we showed that NRP1-Δ7 inhibited growth of prostatic tumors and their vascularization in vivo. This report identifies NRP1-Δ7 as a splice variant displaying anti-tumorigenic properties in vitro and in vivo, emphasizing the need to consider this isoform in future studies.

Dynamics of internalization and recycling of the prometastatic membrane type 4 matrix metalloproteinase (MT4-MMP) in breast cancer cells.

Membrane type 4 matrix metalloproteinase (MT4-MMP) [matrix metalloproteinase (MMP) 17] is a GPI-anchored membrane-type MMP expressed on the cell surface of human breast cancer cells. In triple-negative breast cancer cells, MT4-MMP promotes primary tumour growth and lung metastases. Although the trafficking and internalization of the transmembrane membrane type 1 MMP have been extensively investigated, little is known about the regulatory mechanisms of the GPI-anchored MT4-MMP. Here, we investigated the fate and cellular trafficking of MT4-MMP by analysing its homophilic complex interactions, internalization and recycling dynamics as compared with an inert form, MT4-MMP-E249A. Oligomeric and dimeric complexes were analysed by cotransfection of cells with FLAG-tagged or Myc-tagged MT4-MMP in reducing and nonreducing immunoblotting and coimmunoprecipitation experiments. The trafficking of MT4-MMP was studied with an antibody feeding assay and confocal microscopy analysis or cell surface protein biotinylation and western blot analysis. We demonstrate that MT4-MMP forms homophilic complexes at the cell surface, and internalizes in early endosomes, and that some of the enzyme is either autodegraded or recycled to the cell surface. Our data indicate that MT4-MMP is internalized by the clathrin-independent carriers/GPI-enriched early endosomal compartments pathway, a mechanism that differs from that responsible for the internalization of other membrane-type MMP members. Although MT4-MMP localizes with caveolin-1, MT4-MMP internalization was not affected by inhibitors of caveolin-1 or clathrin endocytosis pathways, but was reduced by CDC42 or RhoA silencing with small interfering RNA. We provide a new mechanistic insight into the regulatory mechanisms of MT4-MMP, which may have implications for the design of novel therapeutic strategies for metastatic breast cancer.

Lipin-1 regulates cancer cell phenotype and is a potential target to potentiate rapamycin treatment.

Lipogenesis inhibition was reported to induce apoptosis and repress proliferation of cancer cells while barely affecting normal cells. Lipins exhibit dual function as enzymes catalyzing the dephosphorylation of phosphatidic acid to diacylglycerol and as co-transcriptional regulators. Thus, they are able to regulate lipid homeostasis at several nodal points. Here, we show that lipin-1 is up-regulated in several cancer cell lines and overexpressed in 50 % of high grade prostate cancers. The proliferation of prostate and breast cancer cells, but not of non-tumorigenic cells, was repressed upon lipin-1 knock-down. Lipin-1 depletion also decreased cancer cell migration through RhoA activation. Lipin-1 silencing did not significantly affect global lipid synthesis but enhanced the cellular concentration of phosphatidic acid. In parallel, autophagy was induced while AKT and ribosomal protein S6 phosphorylation were repressed. We also observed a compensatory regulation between lipin-1 and lipin-2 and demonstrated that their co-silencing aggravates the phenotype induced by lipin-1 silencing alone. Most interestingly, lipin-1 depletion or lipins inhibition with propranolol sensitized cancer cells to rapamycin. These data indicate that lipin-1 controls main cellular processes involved in cancer progression and that its targeting, alone or in combination with other treatments, could open new avenues in anticancer therapy.

RhoGTPases as key players in mammalian cell adaptation to microgravity.

A growing number of studies are revealing that cells reorganize their cytoskeleton when exposed to conditions of microgravity. Most, if not all, of the structural changes observed on flown cells can be explained by modulation of RhoGTPases, which are mechanosensitive switches responsible for cytoskeletal dynamics control. This review identifies general principles defining cell sensitivity to gravitational stresses. We discuss what is known about changes in cell shape, nucleus, and focal adhesions and try to establish the relationship with specific RhoGTPase activities. We conclude by considering the potential relevance of live imaging of RhoGTPase activity or cytoskeletal structures in order to enhance our understanding of cell adaptation to microgravity-related conditions.

Rac1 GTPase silencing counteracts microgravity-induced effects on osteoblastic cells.

Bone cells exposed to real microgravity display alterations of their cytoskeleton and focal adhesions, two major mechanosensitive structures. These structures are controlled by small GTPases of the Ras homology (Rho) family. We investigated the effects of RhoA, Rac1, and Cdc42 modulation of osteoblastic cells under microgravity conditions. Human MG-63 osteoblast-like cells silenced for RhoGTPases were cultured in the automated Biobox bioreactor (European Space Agency) aboard the Foton M3 satellite and compared to replicate ground-based controls. The cells were fixed after 69 h of microgravity exposure for postflight analysis of focal contacts, F-actin polymerization, vascular endothelial growth factor (VEGF) expression, and matrix targeting. We found that RhoA silencing did not affect sensitivity to microgravity but that Rac1 and, to a lesser extent, Cdc42 abrogation was particularly efficient in counteracting the spaceflight-related reduction of the number of focal contacts [-50% in silenced, scrambled (SiScr) controls vs. -15% for SiRac1], the number of F-actin fibers (-60% in SiScr controls vs. -10% for SiRac1), and the depletion of matrix-bound VEGF (-40% in SiScr controls vs. -8% for SiRac1). Collectively, these data point out the role of the VEGF/Rho GTPase axis in mechanosensing and validate Rac1-mediated signaling pathways as potential targets for counteracting microgravity effects.

PP2A regulatory subunit Bα controls endothelial contractility and vessel lumen integrity via regulation of HDAC7.

To supply tissues with nutrients and oxygen, the cardiovascular system forms a seamless, hierarchically branched, network of lumenized tubes. Here, we show that maintenance of patent vessel lumens requires the Bα regulatory subunit of protein phosphatase 2A (PP2A). Deficiency of Bα in zebrafish precludes vascular lumen stabilization resulting in perfusion defects. Similarly, inactivation of PP2A-Bα in cultured ECs induces tubulogenesis failure due to alteration of cytoskeleton dynamics, actomyosin contractility and maturation of cell-extracellular matrix (ECM) contacts. Mechanistically, we show that PP2A-Bα controls the activity of HDAC7, an essential transcriptional regulator of vascular stability. In the absence of PP2A-Bα, transcriptional repression by HDAC7 is abrogated leading to enhanced expression of the cytoskeleton adaptor protein ArgBP2. ArgBP2 hyperactivates RhoA causing inadequate rearrangements of the EC actomyosin cytoskeleton. This study unravels the first specific role for a PP2A holoenzyme in development: the PP2A-Bα/HDAC7/ArgBP2 axis maintains vascular lumens by balancing endothelial cytoskeletal dynamics and cell-matrix adhesion.

Emerging pathogenic mechanisms in human myxomatous mitral valve: lessons from past and novel data.

INTRODUCTION: Myxomatous mitral valve is one of the most common heart valves diseases in human and has been well characterized at a functional and morphological level. Diseased valves are thickened as a result of extracellular matrix remodeling and proteoglycans accumulation accompanied by the disruption of the stratified structures of the leaflets. METHODS: Global transcriptomic analysis was used as a start-up to investigate potential pathogenic mechanisms involved in the development of the human idiopathic myxomatous mitral valve, which have been elusive for many years. RESULTS: These prospective analyses have highlighted the potential role of apparently unrelated molecules in myxomatous mitral valve such as members of the transforming growth factor-ß superfamily, aggrecanases of the "a disintegrin and metalloprotease with thrombospondin repeats I" family, and a weakening of the protection against oxidative stress. We have integrated, in this review, recent transcriptomic data from our laboratory [A. Hulin, C.F. Deroanne, C.A. Lambert, B. Dumont, V. Castronovo, J.O. Defraigne, et al. Metallothionein-dependent up-regulation of TGF-beta2 participates in the remodelling of the myxomatous mitral valve. Cardiovasc Res 2012;93:480-489] and from the publication of Sainger et al. [R. Sainger, J.B. Grau, E. Branchetti, P. Poggio, W.F. Seefried, B.C. Field, et al. Human myxomatous mitral valve prolapse: role of bone morphogenetic protein 4 in valvular interstitial cell activation. J Cell Physiol 2012;227:2595-2604] with existing literature and information issued from the study of monogenic syndromes and animal models. CONCLUSION: Understanding cellular alterations and molecular mechanisms involved in myxomatous mitral valve should help at identifying relevant targets for future effective pharmacological therapy to prevent or reduce its progression.

New prospects in the roles of the C-terminal domains of VEGF-A and their cooperation for ligand binding, cellular signaling and vessels formation.

VEGF-A is a crucial growth factor for blood vessel homeostasis and pathological angiogenesis. Due to alternative splicing of its pre-mRNA, VEGF-A is produced under several isoforms characterized by the combination of their C-terminal domains, which determines their respective structure, availability and affinity for co-receptors. As controversies still exist about the specific roles of these exon-encoded domains, we systematically compared the properties of eight natural and artificial variants containing the domains encoded by exons 1-4 and various combinations of the domains encoded by exons 5, 7 and 8a or 8b. All the variants (VEGF111a, VEGF111b, VEGF121a, VEGF121b, VEGF155a, VEGF155b, VEGF165a, VEGF165b) have a similar affinity for VEGF-R2, as determined by Surface plasmon resonance analyses. They strongly differ however in terms of binding to neuropilin-1 and heparin/heparan sulfate proteoglycans. Data indicate that the 6 amino acids encoded by exon 8a must be present and cooperate with those of exons 5 or 7 for efficient binding, which was confirmed in cell culture models. We further showed that VEGF165b has inhibitory effects in vitro, as previously reported, but that the shortest VEGF variant possessing also the 6 amino acids encoded by exon 8b (VEGF111b) is remarkably proangiogenic, demonstrating the critical importance of domain interactions for defining the VEGF properties. The number, size and localization of newly formed blood vessels in a model of tumour angiogenesis strongly depend also on the C-terminal domain composition, suggesting that association of several VEGF isoforms may be more efficient for treating ischemic diseases than the use of any single variant.