Dual topologies of myotomal collagen XV and Tenascin C act in concert to guide and shape developing motor axons.

During development, motor axons are guided toward muscle target by various extrinsic cues including extracellular matrix (ECM) proteins whose identities and cellular source remain poorly characterized. Here, using single-cell RNAseq of sorted GFP+ cells from smyhc1:gfp-injected zebrafish embryos, we unravel the slow muscle progenitors (SMP) pseudotemporal trajectory at the single-cell level and show that differentiating SMPs are a major source of ECM proteins. The SMP core-matrisome was characterized and computationally predicted to form a basement membrane-like structure tailored for motor axon guidance, including basement membrane-associated ECM proteins, as collagen XV-B, one of the earliest core-matrisome gene transcribed in differentiating SMPs and the glycoprotein Tenascin C. To investigate how contact-mediated guidance cues are organized along the motor path to exert their function in vivo, we used microscopy-based methods to analyze and quantify motor axon navigation in tnc and col15a1b knock-out fish. We show that motor axon shape and growth rely on the timely expression of the attractive cue Collagen XV-B that locally provides axons with a permissive soft microenvironment and separately organizes the repulsive cue Tenascin C into a unique functional dual topology. Importantly, bioprinted micropatterns that mimic this in vivo ECM topology were sufficient to drive directional motor axon growth. Our study offers evidence that not only the composition of ECM cues but their topology critically influences motor axon navigation in vertebrates with potential applications in regenerative medicine for peripheral nerve injury as regenerating nerves follow their original path.

Role of KCNK3 Dysfunction in Dasatinib-associated Pulmonary Arterial Hypertension and Endothelial Cell Dysfunction.

Pulmonary arterial (PA) hypertension (PAH) is a severe cardiopulmonary disease that may be triggered by exposure to drugs such as dasatinib or facilitated by genetic predispositions. The incidence of dasatinib-associated PAH is estimated at 0.45%, suggesting individual predispositions. The mechanisms of dasatinib-associated PAH are still incomplete. We discovered a KCNK3 gene (Potassium channel subfamily K member 3; coding for outward K+ channel) variant in a patient with dasatinib-associated PAH and investigated the impact of this variant on KCNK3 function. Additionally, we assessed the effects of dasatinib exposure on KCNK3 expression. In control human PA smooth muscle cells (hPASMCs) and human pulmonary endothelial cells (hPECs), we evaluated the consequences of KCNK3 knockdown on cell migration, mitochondrial membrane potential, ATP production, and in vitro tube formation. Using mass spectrometry, we determined the KCNK3 interactome. Patch-clamp experiments revealed that the KCNK3 variant represents a loss-of-function variant. Dasatinib contributed to PA constriction by decreasing KCNK3 function and expression. In control hPASMCs, KCNK3 knockdown promotes mitochondrial membrane depolarization and glycolytic shift. Dasatinib exposure or KCNK3 knockdown reduced the number of caveolae in hPECs. Moreover, KCNK3 knockdown in control hPECs reduced migration, proliferation, and in vitro tubulogenesis. Using proximity labeling and mass spectrometry, we identified the KCNK3 interactome, revealing that KCNK3 interacts with various proteins across different cellular compartments. We identified a novel pathogenic variant in KCNK3 and showed that dasatinib downregulates KCNK3, emphasizing the relationship between dasatinib-associated PAH and KCNK3 dysfunction. We demonstrated that a loss of KCNK3-dependent signaling contributes to endothelial dysfunction in PAH and glycolytic switch of hPASMCs.

Weibel-Palade Bodies Orchestrate Pericytes During Angiogenesis.

Objective Weibel-Palade bodies (WPBs) are endothelial cell (EC)-specific organelles formed by vWF (von Willebrand factor) polymerization and that contain the proangiogenic factor Ang-2 (angiopoietin-2). WPB exocytosis has been shown to be implicated for vascular repair and inflammatory responses. Here, we investigate the role of WPBs during angiogenesis and vessel stabilization. Approach and Results WPB density in ECs decreased at the angiogenic front of retinal vascular network during development and neovascularization compared with stable vessels. In vitro, VEGF (vascular endothelial growth factor) induced a VEGFR-2 (vascular endothelial growth factor receptor-2)-dependent exocytosis of WPBs that contain Ang-2 and consequently the secretion of vWF and Ang-2. Blocking VEGF-dependant WPB exocytosis and Ang-2 secretion promoted pericyte migration toward ECs. Pericyte migration was inhibited by adding recombinant Ang-2 or by silencing Ang-1 (angiopoietin-1) or Tie2 (angiopoietin-1 receptor) in pericytes. Consistently, in vivo anti-VEGF treatment induced accumulation of WPBs in retinal vessels because of the inhibition of WPB exocytosis and promoted the increase of pericyte coverage of retinal vessels during angiogenesis. In tumor angiogenesis, depletion of WPBs in vWF knockout tumor-bearing mice promoted an increase of tumor angiogenesis and a decrease of pericyte coverage of tumor vessels. By another approach, normalized tumor vessels had higher WPB density. Conclusions We demonstrate that WPB exocytosis and Ang-2 secretion are regulated during angiogenesis to limit pericyte coverage of remodeling vessels by disrupting Ang-1/Tie2 autocrine signaling in pericytes.

NF-κB and Angiogenesis Inhibitors from the Aerial Parts of Chresta martii.

The ethyl acetate extract of the aerial parts of Chresta martii showed significant in vitro NF-κB inhibition. Bioactivity-guided isolation was undertaken using HPLC microfractionation to localize the active compounds. Different zones of the HPLC chromatogram were linked to NF-κB inhibition. In parallel to this HPLC-based activity profiling, HPLC-PDA-ESI-MS and UHPLC-TOF-HRMS were used for the early identification of some of the compounds present in the extract and to get a complete phytochemical overview. The isolation of the compounds was performed by high-speed counter-current chromatography and further semipreparative HPLC. Using this approach, 14 compounds were isolated, two of them being new sesquiterpene lactones. The structures of the isolated compounds were elucidated by spectroscopic methods including UV, ECD, NMR, and HRMS. All isolated compounds were evaluated for their inhibitory activity of NF-κB and angiogenesis, and compound 2 showed promising NF-κB inhibition activity with an IC50 of 0.7 µM. The isolated compounds 1, 2, 5, 7, and 8 caused a significant reduction in angiogenesis when evaluated by an original 3D in vitro angiogenesis assay.

A Bivalent Role of Genistein in Sprouting Angiogenesis.

The effects of genistein on angiogenesis remain poorly understood. Some studies claim an antiangiogenic effect and others claim a pro-angiogenic one. Thus, the aim of this study was to determine if genistein may exhibit bivalent angiogenic effects. To address this question, genistein angiogenic modulatory effects were examined using an in vitro 3D angiogenesis model using human umbilical vein endothelial cells. In this model, a bivalent effect of genistein was demonstrated on sprouting angiogenesis, with angiogenic stimulation at low concentrations (0.001 - 1 µM) and inhibition at higher ones (25 - 100 µM). Enhancement of the endothelial tube formation correlated with an increase in human umbilical vein endothelial cell metabolic activity and proliferation. Inhibition of angiogenesis correlated with a decreased metabolic activity, proliferation, and migration. Moreover, high concentrations of genistein influenced human umbilical vein endothelial cell morphology. Expression of genes involved in the angiogenic process in response to genistein was measured to study the mechanism of action. Secretome profiling revealed that angiogenic regulators were modulated with genistein treatment. These results suggested a bivalent effect of genistein on human umbilical vein endothelial cell growth and angiogenesis, and further investigations on the benefit of genistein for cancer chemoprevention, cancer treatment, or pro-angiogenic therapies have to be carefully considered.

Neuroligin 1 induces blood vessel maturation by cooperating with the α6 integrin.

The synaptic protein Neuroligin 1 (NLGN1), a cell adhesion molecule, is critical for the formation and consolidation of synaptic connectivity and is involved in vascular development. The mechanism through which NLGN1 acts, especially in vascular cells, is unknown. Here, we aimed at deepening our knowledge on the cellular activities and molecular pathways exploited by endothelial NLGN1 both in vitro and in vivo. We analyzed the phenotypic consequences of NLGN1 expression modulation in endothelial cells through in vitro angiogenesis assays and the mouse postnatal retinal angiogenesis model. We demonstrate that NLGN1, whereas not affecting endothelial cell proliferation or migration, modulates cell adhesion to the vessel stabilizing protein laminin through cooperation with the α6 integrin, a specific laminin receptor. Finally, we show that in vivo, NLGN1 and α6 integrin preferentially colocalize in the mature retinal vessels, whereas NLGN1 deletion causes an aberrant VE-cadherin, laminin and α6 integrin distribution in vessels, along with significant structural defects in the vascular tree.

Greater glycosaminoglycan content in human patellar tendon biopsies is associated with more pain and a lower VISA score.

BACKGROUND: People with patellar tendinopathy experience chronic pain and activity limitation, but a pertinent biochemical marker correlated with these clinical features has not been identified. The Victoria Institute of Sport Assessment (VISA) questionnaire is a condition-specific patient-rated outcome measure. Since the quantity of glycosaminoglycans (GAGs) increases with advancing tendon pathology, we hypothesised that there would be a correlation between the quantity of GAGs in the patellar tendon and the VISA score. METHODS: Tissue biopsies from athletes with chronic patellar tendinopathy (confirmed by clinical examination and MRI) were recruited (n=7), as well as controls with no history of knee pain (n=4). The quantity of sulphated GAGs in the human patellar tendons was determined with a dimethyl methylene blue (DMMB) assay; this method was first validated with rat tendon tissue. The extent and distribution of GAG species and proteoglycans (decorin, versican and aggrecan) in the human tendon biopsies were examined using immunohistochemistry. RESULTS: Greater sulphated GAG content of the patellar tendon was correlated with the greater tendon dysfunction (R(2)=0.798). The quantity of aggrecan in the tendon, a chondroitin sulphate-rich proteoglycan, also increased with advancing tendon pathology. CONCLUSIONS: Increased GAGs in the pathological human patellar tendon are related to a worse clinical status. These findings indicate that the VISA score reflects the extent of tendon tissue pathology.

Multivalent pseudopeptides targeting cell surface nucleoproteins inhibit cancer cell invasion through tissue inhibitor of metalloproteinases 3 (TIMP-3) release.

Blockage of the metastasis process remains a significant clinical challenge, requiring innovative therapeutic approaches. For this purpose, molecules that inhibit matrix metalloproteinases activity or induce the expression of their natural inhibitor, the tissue inhibitor of metalloproteinases (TIMPs), are potentially interesting. In a previous study, we have shown that synthetic ligands binding to cell surface nucleolin/nucleophosmin and known as HB 19 for the lead compound and NucAnt 6L (N6L) for the most potent analog, inhibit both tumor growth and angiogenesis. Furthermore, they prevent metastasis in a RET transgenic mice model which develops melanoma. Here, we investigated the effect of N6L on the invasion capacity of MDA-MB-435 melanoma cells. Our results show that the multivalent pseudopeptide N6L inhibited Matrigel invasion of MDA-MB-435 cells in a modified Boyden chamber model. This was associated with an increase in TIMP-3 in the cell culture medium without a change in TIMP-3 mRNA expression suggesting its release from cell surface and/or extracellular matrix. This may be explained by our demonstrated N6L interaction with sulfated glycosaminoglycans and consequently the controlled bioavailability of glycosaminoglycan-bound TIMP-3. The implication of TIMP-3 in N6L-induced inhibition of cell invasion was evidenced by siRNA silencing experiments showing that the loss of TIMP-3 expression abrogated the effect of N6L. The inhibition of tumor cell invasion by N6L demonstrated in this study, in addition to its previously established inhibitory effect on tumor growth and angiogenesis, suggests that N6L represents a promising anticancer drug candidate warranting further investigation.

Age-related changes in rat myocardium involve altered capacities of glycosaminoglycans to potentiate growth factor functions and heparan sulfate-altered sulfation.

Glycosaminoglycans (GAGs) are essential components of the extracellular matrix, the natural environment from which cell behavior is regulated by a number or tissue homeostasis guarantors including growth factors. Because most heparin-binding growth factor activities are regulated by GAGs, structural and functional alterations of these polysaccharides may consequently affect the integrity of tissues during critical physiological and pathological processes. Here, we investigated whether the aging process can induce changes in the myocardial GAG composition in rats and whether these changes can affect the activities of particular heparin-binding growth factors known to sustain cardiac tissue integrity. Our results showed an age-dependent increase of GAG levels in the left ventricle. Biochemical and immunohistological studies pointed out heparan sulfates (HS) as the GAG species that increased with age. ELISA-based competition assays showed altered capacities of the aged myocardial GAGs to bind FGF-1, FGF-2, and VEGF but not HB EGF. Mitogenic assays in cultured cells showed an age-dependent decrease of the elderly GAG capacities to potentiate FGF-2 whereas the potentiating effect on VEGF(165) was increased, as confirmed by augmented angiogenic cell proliferation in Matrigel plugs. Moreover, HS disaccharide analysis showed considerably altered 6-O-sulfation with modest changes in N- and 2-O-sulfations. Together, these findings suggest a physiological significance of HS structural and functional alterations during aging. This can be associated with an age-dependent decline of the extracellular matrix capacity to efficiently modulate not only the activity of resident or therapeutic growth factors but also the homing of resident or therapeutic cells.

Specific Features of Stromal Cells Isolated from the Two Layers of Subcutaneous Adipose Tissue: Roles of Their Secretion on Angiogenesis and Neurogenesis.

Human-adipose-tissue-derived mesenchymal stromal cells (AD-MSCs) are currently being tested as autologous-cell-based therapies for use in tissue healing and regeneration. Recent studies have also demonstrated that AD-MSC-derived exosomes contribute to tissue repair and peripheral nerve regeneration. Subcutaneous abdominal adipose tissue (AAT) is divided into two layers: the superficial layer (sAAT) and the deep layer (dAAT). However, it is unclear whether there are particular characteristics of each layer in terms of AD-MSC regenerative potential. Using AD-MSCs purified and characterized from three abdominoplasties, we compared their secretomes and exosome functions to identify which layer may be most suitable as a source for cell therapy. Phenotypical analysis of the AD-MSCs containing stromal vascular fraction did not reveal any difference between the two layers. The AD-MSC secretomes showed a very similar pattern of cytokine content and both layers were able to release exosomes with identical characteristics. However, compared to the secretome, the released exosomes showed better biological properties. Interestingly, dAAT exosomes appeared to be more effective on neuromodulation, whereas neither sAAT nor dAAT-derived exosomes had significant effects on endothelial function. It thus appears that AD-MSC-derived exosomes from the two abdominal adipose tissue layers possess different features for cell therapy.

Modulation of inflammation by Cicaderma ointment accelerates skin wound healing.

Skin wound healing is a natural and intricate process that takes place after injury, involving different sequential phases such as hemostasis, inflammatory phase, proliferative phase, and remodeling that are associated with complex biochemical events. The interruption or failure of wound healing leads to chronic nonhealing wounds or fibrosis-associated diseases constituting a major health problem where, unfortunately, medicines are not very effective. The objective of this study was to evaluate the capacity of Cicaderma ointment (Boiron, Lyon, France) to accelerate ulcer closure without fibrosis and investigate wound healing dynamic processes. We used a necrotic ulcer model in mice induced by intradermal doxorubicin injection, and after 11 days, when the ulcer area was maximal, we applied Vaseline petroleum jelly or Cicaderma every 2 days. Topical application of Cicaderma allowed a rapid recovery of mature epidermal structure, a more compact and organized dermis and collagen bundles compared with the Vaseline group. Furthermore, the expression of numerous cytokines/molecules in the ulcer was increased 11 days after doxorubicin injection compared with healthy skin. Cicaderma rapidly reduced the level of proinflammatory cytokines, mainly tumor necrosis factor (TNF)-α and others of the TNF pathway, which can be correlated to a decrease of polymorphonuclear recruitment. It is noteworthy that the modulation of inflammation through TNF-α, macrophage inflammatory protein-1α, interleukin (IL)-12, IL-4, and macrophage-colony-stimulating factor was maintained 9 days after the first ointment application, facilitating the wound closure without affecting angiogenesis. These cytokines seem to be potential targets for therapeutic approaches in chronic wounds. Our results confirm the use of Cicaderma for accelerating skin wound healing and open new avenues for sequential treatments to improve healing.

Angiogenesis Is Differentially Modulated by Platelet-Derived Products.

Platelet-derived preparations are being used in clinic for their role in tissue repair and regenerative processes. The release of platelet-derived products such as autologous growth factors, cytokines and chemokines can trigger therapeutic angiogenesis. In this in vitro study, we evaluated and compared the ability of three platelet-derived preparations: platelet-rich-plasma (PRP), PRP-hyaluronic acid (PRP-HA) and platelet lysates (PL) at various concentrations (5-40%) to modulate human umbilical vein endothelial cells (HUVEC) biological effects on metabolism, viability, senescence, angiogenic factors secretion and angiogenic capacities in 2D (endothelial tube formation assay or EFTA) and in 3D (fibrin bead assay or FBA). HUVEC exocytosis was stimulated with PRP and PRP-HA. Cell viability was strongly increased by PRP and PRP-HA but mildly by PL. The three preparations inhibit HUVEC tube formation on Matrigel, while PRP enhanced the complexity of the network. In the fibrin bead assay (FBA), PRP and PRP-HA stimulated all steps of the angiogenic process resulting in massive sprouting of a branched microvessel network, while PL showed a weaker angiogenic response. Secretome profiling revealed modulation of 26 human angiogenic proteins upon treatment with the platelet derived preparations. These in vitro experiments suggest that PRP and PRP-HA are effective biological therapeutic tools when sustained therapeutic angiogenesis is needed.

Pharmacologic Normalization of Pancreatic Cancer-Associated Fibroblast Secretome Impairs Prometastatic Cross-Talk With Macrophages.

BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs) from pancreatic adenocarcinoma (PDA) present high protein synthesis rates. CAFs express the G-protein-coupled somatostatin receptor sst1. The sst1 agonist SOM230 blocks CAF protumoral features in vitro and in immunocompromised mice. We have explored here the therapeutic potential of SOM230, and underlying mechanisms, in immunocompetent models of murine PDA mimicking the heavy fibrotic and immunosuppressive stroma observed in patient tumors. METHODS: Large-scale mass spectrometry analyses were performed on media conditioned from 9 patient PDA-derived CAF primary cultures. Spontaneous transgenic and experimental (orthotopic co-graft of tumor cells plus CAFs) PDA-bearing mice were longitudinally ultrasound-monitored for tumor and metastatic progression. Histopathology and flow cytometry analyses were performed on primary tumors and metastases. Stromal signatures were functionally validated through bioinformatics using several published, and 1 original, PDA database. RESULTS: Proteomics on the CAF secretome showed that SOM230 controls stromal activities including inflammatory responses. Among the identified secreted proteins, we validated that colony-stimulating factor 1 (CSF-1) (a macrophage growth factor) was reduced by SOM230 in the tumor and plasma of PDA-harboring mice, alongside intratumor stromal normalization (reduced CAF and macrophage activities), and dramatic metastasis reduction. In transgenic mice, these SOM230 benefits alleviate the chemotherapy-induced (gemcitabine) immunosuppressive stroma reshaping. Mechanistically, SOM230 acts in vivo on CAFs through sst1 to disrupt prometastatic CAF production of CSF-1 and cross-talk with macrophages. We found that in patients, stromal CSF-1 was associated with aggressive PDA forms. CONCLUSIONS: We propose SOM230 as an antimetastatic therapy in PDA for its capacity to remodel the fibrotic and immunosuppressive myeloid stroma. This pharmacotherapy should benefit PDA patients treated with chemotherapies.

Angiogenesis Analyzer for ImageJ - A comparative morphometric analysis of "Endothelial Tube Formation Assay" and "Fibrin Bead Assay".

Angiogenesis assays based on in vitro capillary-like growth of endothelial cells (EC) are widely used, either to evaluate the effect of anti- and pro-angiogenesis drugs of interest, or to test and compare the functional capacities of various types of EC and progenitor cells. Among the different methods applied to study angiogenesis, the most commonly used is the "Endothelial Tube Formation Assay" (ETFA). In suitable culture conditions, EC form two-dimensional (2D) branched structures that can lead to a meshed pseudo-capillary network. An alternative approach to ETFA is the "Fibrin Bead Assay" (FBA), based on the use of Cytodex 3 microspheres, which promote the growth of 3D capillary-like patterns from coated EC, suitable for high throughput in vitro angiogenesis studies. The analytical evaluation of these two widely used assays still remains challenging in terms of observation method and image analysis. We previously developed the "Angiogenesis Analyzer" for ImageJ (AA), a tool allowing analysis of ETFA-derived images, according to characteristics of the pseudo-capillary networks. In this work, we developed and implemented a new algorithm for AA able to recognize microspheres and to analyze the attached capillary-like structures from the FBA model. Such a method is presented for the first time in fully automated mode and using non-destructive image acquisition. We detailed these two algorithms and used the new AA version to compare both methods (i.e. ETFA and FBA) in their efficiency, accuracy and statistical relevance to model angiogenesis patterns of Human Umbilical Vein EC (HUVEC). Although the two methods do not assess the same biological step, our data suggest that they display specific and complementary information on the angiogenesis processes analysis.

A collagen Vα1-derived fragment inhibits FGF-2 induced-angiogenesis by modulating endothelial cells plasticity through its heparin-binding site.

Type V collagen (ColV) is a component of the endothelial basement membrane zone. During angiogenesis, extracellular matrix remodelling results in the release of active protein fragments that display pro- or anti-angiogenic properties. The latter often exert their activity through their heparin-binding site. We previously characterized a ColVα1-derived fragment called HEPV that contains a high affinity-binding site for heparin and heparan sulphate chains. Here we show that HEPV binds to FGF2 through its heparin-binding site. Using in vitro and in vivo angiogenesis assays, we show that HEPV but not the HEPV mutant at the heparin-binding site, inhibits FGF2-dependant angiogenesis. On the opposite, HEPV does not bind to VEGFA and has no effect on VEGFA-mediated angiogenesis. In 3D collagen gels, the addition of HEPV abrogates endothelial cell invasion and sprouting induced by FGF2. Interestingly, in vivo experiments reveal that HEPV anti-angiogenic activity is associated with the appearance of endothelial to mesenchymal transition (EndMT) markers. Together, these findings indicate that the ColVα1-derived fragment HEPV functions as an anti-angiogenic factor that represses FGF2-mediated angiogenesis through the regulation of endothelial cell plasticity. Previous observations showing that ColV overexpression negatively regulates pathological angiogenesis were left unexplained. Our data provide insights into the possible molecular mechanisms.

Vitamin-D3 (α-1, 25(OH) 2D3) Protects Retinal Pigment Epithelium From Hyperoxic Insults.

Purpose: Oxidative stress affects the retinal pigment epithelium (RPE) leading to development of vascular eye diseases. Cholecalciferol (VIT-D) is a known modulator of oxidative stress and angiogenesis. This in vitro study was carried out to evaluate the protective role of VIT-D on RPE cells incubated under hyperoxic conditions. Methods: Cadaver primary RPE (PRPE) cells were cultured in hyperoxia (40% O2) with or without VIT-D (α-1, 25(OH) 2D3). The functional and physiological effects of PRPE cells with VIT-D treatment were analyzed using molecular and biochemical tools. Results: Vascular signaling modulators, such as vascular endothelial growth factor (VEGF) and Notch, were reduced in hyperoxic conditions but significantly upregulated in the presence of VIT-D. Additionally, PRPE conditioned medium with VIT-D induced the tubulogenesis in primary human umbilical vein endothelial cells (HUVEC) cells. VIT-D supplementation restored phagocytosis and transmembrane potential in PRPE cells cultured under hyperoxia. Conclusions: VIT-D protects RPE cells and promotes angiogenesis under hyperoxic insult. These findings may give impetus to the potential of VIT-D as a therapeutic agent in hyperoxia induced retinal vascular diseases.

3-O-sulfated heparan sulfate interactors target synaptic adhesion molecules from neonatal mouse brain and inhibit neural activity and synaptogenesis in vitro.

Heparan sulfate (HS) chains, covalently linked to heparan sulfate proteoglycans (HSPG), promote synaptic development and functions by connecting various synaptic adhesion proteins (AP). HS binding to AP could vary according to modifications of HS chains by different sulfotransferases. 3-O-sulfotransferases (Hs3sts) produce rare 3-O-sulfated HSs (3S-HSs), of poorly known functions in the nervous system. Here, we showed that a peptide known to block herpes simplex virus by interfering with 3S-HSs in vitro and in vivo (i.e. G2 peptide), specifically inhibited neural activity, reduced evoked glutamate release, and impaired synaptic assembly in hippocampal cell cultures. A role for 3S-HSs in promoting synaptic assembly and neural activity is consistent with the synaptic interactome of G2 peptide, and with the detection of Hs3sts and their products in synapses of cultured neurons and in synaptosomes prepared from developing brains. Our study suggests that 3S-HSs acting as receptors for herpesviruses might be important regulators of neuronal and synaptic development in vertebrates.

Glycosaminoglycans from Alzheimer's disease hippocampus have altered capacities to bind and regulate growth factors activities and to bind tau.

Glycosaminoglycans (GAGs), including heparan sulfates and chondroitin sulfates, are major components of the extracellular matrix. Upon interacting with heparin binding growth factors (HBGF), GAGs participate to the maintaintenance of tissue homeostasis and contribute to self-healing. Although several processes regulated by HBGF are altered in Alzheimer's disease, it is unknown whether the brain GAG capacities to bind and regulate the function of HBGF or of other heparin binding proteins, as tau, are modified in this disease. Here, we show that total sulfated GAGs from hippocampus of Alzheimer's disease have altered capacities to bind and potentiate the activities of growth factors including FGF-2, VEGF, and BDNF while their capacity to bind to tau is remarkable increased. Alterations of GAG structures and capacities to interact with and regulate the activity of heparin binding proteins might contribute to impaired tissue homeostasis in the Alzheimer's disease brain.

Initial clonogenic potential of human endothelial progenitor cells is predictive of their further properties and establishes a functional hierarchy related to immaturity.

Endothelial progenitor cells (EPCs) generate in vitro Endothelial Colony Forming Cells (ECFCs) combining features of endothelial and stem/progenitor cells. Their angiogenic properties confer them a therapeutic potential for treating ischemic lesions. They may be isolated from umbilical cord blood (CB-ECFCs) or peripheral adult blood (AB-ECFCs). It is generally accepted that CB-ECFCs are more clonogenic, proliferative and angiogenic than AB-ECFCs. Nevertheless, only a few studies have focused on the functional heterogeneity of CB-ECFCs from different individuals. Moreover, AB-ECFC loss of function is yet to be precisely described. We have focused on these two issues that are critical for clinical perspectives. The detailed clonogenic profile of CB-ECFCs and AB-ECFCs was obtained and revealed a high inter individual heterogeneity and the absence of correlation with age. Most CB-ECFCs yielded initial colonies and had functional properties similar to those of AB-ECFCs. Conversely, a high clonogenicity was associated with an enhanced proliferative and angiogenic potential and stemness gene overexpression, confirming that immaturity, lost by AB-ECFCs, was a prerequisite to functionality. We thus demonstrated the importance of selecting CB-ECFCs according to specific criteria, and we propose using the initial clonogenicity as a relevant marker of their potential efficacy on vascular repair.

Increased signaling by the autism-related Engrailed-2 protein enhances dendritic branching and spine density, alters synaptic structural matching, and exaggerates protein synthesis.

Engrailed 1 (En1) and 2 (En2) code for closely related homeoproteins acting as transcription factors and as signaling molecules that contribute to midbrain and hindbrain patterning, to development and maintenance of monoaminergic pathways, and to retinotectal wiring. En2 has been suggested to be an autism susceptibility gene and individuals with autism display an overexpression of this homeogene but the mechanisms remain unclear. We addressed in the present study the effect of exogenously added En2 on the morphology of hippocampal cells that normally express only low levels of Engrailed proteins. By means of RT-qPCR, we confirmed that En1 and En2 were expressed at low levels in hippocampus and hippocampal neurons, and observed a pronounced decrease in En2 expression at birth and during the first postnatal week, a period characterized by intense synaptogenesis. To address a putative effect of Engrailed in dendritogenesis or synaptogenesis, we added recombinant En1 or En2 proteins to hippocampal cell cultures. Both En1 and En2 treatment increased the complexity of the dendritic tree of glutamatergic neurons, but only En2 increased that of GABAergic cells. En1 increased the density of dendritic spines both in vitro and in vivo. En2 had similar but less pronounced effect on spine density. The number of mature synapses remained unchanged upon En1 treatment but was reduced by En2 treatment, as well as the area of post-synaptic densities. Finally, both En1 and En2 elevated mTORC1 activity and protein synthesis in hippocampal cells, suggesting that some effects of Engrailed proteins may require mRNA translation. Our results indicate that Engrailed proteins can play, even at low concentrations, an active role in the morphogenesis of hippocampal cells. Further, they emphasize the over-regulation of GABA cell morphology and the vulnerability of excitatory synapses in a pathological context of En2 overexpression.