The Effect of the Varietal Type, Ripening Stage, and Growing Conditions on the Content and Profile of Sugars and Capsaicinoids in Capsicum Peppers.

Peppers (Capsicum sp.) are used both as vegetables and/or spice and their fruits are used in a plethora of recipes, contributing to their flavor and aroma. Among flavor-related traits, pungency (capsaicinoids) and lately volatiles have been considered the most important factors. However, the knowledge of sugars is low, probably due to the fact peppers were historically considered tasteless. Here, using HPLC, we studied the content and profile of major sugars and capsaicinoids in a comprehensive collection of varietal types (genotype, G), grown under different growing systems (environment, E) in two years (Y) and considered the two main ripening stages (R). We found a major contribution to the ripening stage and the genotype in total and individual sugars and capsaicinoids. The year was also significant in most cases, as well as the G × E and G × Y interactions, while the growing system was low or nil. Ripening increased considerably in sugars (from 19.6 to 36.1 g kg-1 on average) and capsaicinoids (from 97 to 142 mg kg-1 on average), with remarkable differences among varieties. Moreover, sugars in fully ripe fruits ranged between 7.5 and 38.5 g kg-1 in glucose and between 5.2 and 34.3 g kg-1 in fructose, and several accessions reached total sugars between 40 and 70 g kg-1, similar to tomatoes. The results reveal the importance of the genotype and the ripening for these traits, particularly sugars, which should be considered key for the improvement of taste and flavor in peppers.

The Combination of Monochromatic LEDs and Elicitation with Stressors Enhances the Accumulation of Glucosinolates in Mustard Sprouts with Species-Dependency.

This work studies the enhancement of glucosinolates (GSLs) in mustard sprouts as health promoters. Sprouts of Sinapis alba, Brassica nigra, and B. carinata were grown under broad-spectrum, monochromatic blue or red light-emitting diode (LED) lamps, irrigated with 0-100 mM sodium chloride (NaCl), and sprayed with 0-250 µM methyl jasmonate (MeJA) as elicitor. The use of LEDs did not result in increased sprout biomass in any case. The effect of the applied treatments on the GSLs depended on the species and were restricted to Brassica spp. The red LEDs produced an overall increase in GSLs over 500% in B. carinata (from 12 to 81 mg 100 g-1 F.W.), compared to the white broad-spectrum lights, although the highest increase in content was obtained in treated sprouts with 250 µM MeJA (104 an 105 mg 101 g-1 F.W., under the red and blue LEDs, respectively). The combination of blue LEDs, 100 mM NaCl, and 250 µM MeJA enhanced the levels of GLSs in B. nigra to the maximum (81 mg 100 g-1 F.W.). Overall, these results indicate that by modifying the growing conditions for a given sprout, enhancement in the accumulation of GSLs as health promoters is possible. The use of these treatments is a sustainable alternative to genetic modification when looking for bioactive-enriched foods, delivering natural plant foods rich in bioactive ingredients (e.g., glucosinolates). Nevertheless, the response to the treatments varies among species, indicating that treatments will require adjustment across sprouts. Further research continues with producing cruciferous sprouts to obtain GSL-enriched formulas for further studying the effects of their bioavailability and bioactivity on health-promotion.

Potential In Vitro Inhibition of Selected Plant Extracts against SARS-CoV-2 Chymotripsin-Like Protease (3CLPro) Activity.

Antiviral treatments inhibiting Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication may represent a strategy complementary to vaccination to fight the ongoing Coronavirus disease 19 (COVID-19) pandemic. Molecules or extracts inhibiting the SARS-CoV-2 chymotripsin-like protease (3CLPro) could contribute to reducing or suppressing SARS-CoV-2 replication. Using a targeted approach, we identified 17 plant products that are included in current and traditional cuisines as promising inhibitors of SARS-CoV-2 3CLPro activity. Methanolic extracts were evaluated in vitro for inhibition of SARS-CoV-2 3CLPro activity using a quenched fluorescence resonance energy transfer (FRET) assay. Extracts from turmeric (Curcuma longa) rhizomes, mustard (Brassica nigra) seeds, and wall rocket (Diplotaxis erucoides subsp. erucoides) at 500 µg mL-1 displayed significant inhibition of the 3CLPro activity, resulting in residual protease activities of 0.0%, 9.4%, and 14.9%, respectively. Using different extract concentrations, an IC50 value of 15.74 µg mL-1 was calculated for turmeric extract. Commercial curcumin inhibited the 3CLPro activity, but did not fully account for the inhibitory effect of turmeric rhizomes extracts, suggesting that other components of the turmeric extract must also play a main role in inhibiting the 3CLPro activity. Sinigrin, a major glucosinolate present in mustard seeds and wall rocket, did not have relevant 3CLPro inhibitory activity; however, its hydrolysis product allyl isothiocyanate had an IC50 value of 41.43 µg mL-1. The current study identifies plant extracts and molecules that can be of interest in the search for treatments against COVID-19, acting as a basis for future chemical, in vivo, and clinical trials.

Consumers acceptance and volatile profile of wall rocket (Diplotaxis erucoides).

Wall rocket (Diplotaxis erucoides) is a wild edible herb traditionally consumed in the Mediterranean regions with a characteristic, pungent flavour. However, little is known about its acceptance as a potential new crop. In the present study, an hedonic test with 98 volunteers was performed in order to evaluate the potential of wall rocket as a new crop. Three products were tested corresponding to microgreens, seedlings and baby-leaves. The volatile constituents were also studied due to their probable influence on acceptance, and compared to Dijon's mustard and wasabi. The degree of acceptance was mainly related to taste and pungency. Microgreens were well accepted, whereas seedlings and baby-leaves were mainly appreciated by individuals that enjoy pungent tastes. The purchase intent was also highly related to the acceptance of taste and pungency. The volatiles profile revealed that wall rocket was rich in allyl isothiocyanate, like mustard and wasabi. This compound may be greatly responsible of the relationship between the acceptance of mustard, wasabi and wall rocket. Microgreens displayed the highest levels of isothiocyanates, although the quantity of product tested by panellists did not probably allow the appreciation of such compounds. In baby-leaves, a significant decrease in isothiocyanates GC area and relative abundances was observed. These results suggest that wall rocket microgreens would be accepted by a significant proportion of the general public since pungency is lowly perceived in the product, despite its high levels of isothiocyanates. By contrast, baby-leaves may become a crop for a cohort of consumers that enjoy pungent flavours.

HS-SPME analysis of the volatiles profile of water celery (Apium nodiflorum), a wild vegetable with increasing culinary interest.

Water celery (Apium nodiflorum) is a wild plant traditionally harvested in some Mediterranean areas for being consumed raw. Despite its appreciated organoleptic properties, the aromatic profile of the fresh vegetable remains to be studied. In the present study, volatile compounds from five wild populations were extracted by the headspace-solid phase microextraction technique, analysed by gas cromatography-mass spectrometry, and compared to related crops. The wild species had a high number of aromatic compounds. It was rich in monoterpenes (49.2%), sesquiterpenes (39.4%) and phenylpropanoids (9.6%), with quantitative differences among populations, in absolute terms and relative abundance. On average, germacrene D was the main compound (16.6%), followed by allo-ocimene (11.9%) and limonene (11.1%). Only in one population, the levels of limonene were greater than those of germacrene D. Among phenylpropanoids, dillapiol displayed the highest levels, and co-occurred with myristicin in all populations except one. These differences may have a genetic component, which would indicate the possibility of establishing selection programmes for the development of water celery as a crop adapted to different market preferences. On the other hand, comparison with related crops revealed some similarities among individual volatiles present in the different crops, which would be responsible of the common aroma notes. However, water celery displayed a unique profile, which was in addition quantitatively richer than others. Thus, this differentiation may promote the use of water celery as a new crop.

Vorinostat synergizes with antioxidant therapy to target myeloproliferative neoplasms.

BCR-ABL-negative myeloproliferative neoplasms (MPNs) are driven by JAK-STAT pathway activation, but epigenetic alterations also play an important pathophysiological role. These can be pharmacologically manipulated with histone deacetylase inhibitors (HDACIs), which have proven to be clinically effective in the treatment of MPNs but exhibit dose-limiting toxicity. The treatment of primary MPN cells with vorinostat modulates the expression of genes associated with apoptosis, cell cycle, inflammation, and signaling. The induction of this transcriptional program results in decreased cellular viability, paralleled by a decrease in levels of reactive oxygen species (ROS). In vitro manipulation of ROS levels revealed that the reduction of ROS levels promoted apoptosis. When vorinostat was combined with antioxidant agents, the apoptosis of MPN cells increased in a synergistic manner. The results described here suggest a novel and promising therapeutic strategy combining HDACIs with ROS-reducing agents to treat MPNs.

Wild edible fool's watercress, a potential crop with high nutraceutical properties.

BACKGROUND: Fool's watercress (Apium nodiflorum) is an edible vegetable with potential as a new crop. However, little information is available regarding the antioxidant properties of the plant and the individual phenolics accounting for this capacity are unknown. METHODS: The antioxidant properties of twenty-five wild populations were analysed and individual phenolics present in the species reported and compared with celery and parsley. The antioxidant activity was measured as the 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) free radical scavenging capacity, and the total phenolics content (TPC) via the Folin-Ciocalteu procedure. The individual phenolics constituents were determined via high performance liquid chromatography (HPLC) as aglycones. RESULTS: The average DPPH and TPC of fool's watercress were 28.1 mg Trolox g-1 DW and 22.3 mg of chlorogenic acid equivalents g-1 DW, respectively, much higher than those of celery and parsley. Significant differences for both DPPH and TPC, which may be explained by either genotype or environmental factors, were detected among groups established according to geographical origin. Quercetin was identified as the major phenolic present in the leaves of the species, unlike parsley and celery, in which high amounts of apigenin and luteolin were determined. Quercetin represented 61.6% of the phenolics targeted in fool's watercress, followed by caffeic acid derivatives as main hydroxycinnamic acids. DISCUSSION: The study reports the high antioxidant properties of fool's watercress based on a large number of populations. Results suggest that quercetin accounts for an important share of the antioxidant capacity of this potential new crop. The study also provides a basis for future breeding programs, suggesting that selection by geographical locations may result in differences in the antioxidant properties.

Therapeutic activity of superoxide dismutase-containing enzymosomes on rat liver ischaemia-reperfusion injury followed by magnetic resonance microscopy.

Liver ischaemia-reperfusion injury (IRI) may occur during hepatic surgery and is unavoidable in liver transplantation. Superoxide dismutase enzymosomes (SOD-enzymosomes), liposomes where SOD is at the liposomal surface expressing enzymatic activity in intact form without the need of liposomal disruption, were developed with the aim of having a better insight into its antioxidant therapeutic outcome in IRI. We also aimed at validating magnetic resonance microscopy (MRM) at 7T as a tool to follow IRI. SOD-enzymosomes were characterized and tested in a rat ischaemia-reperfusion model and the therapeutic outcome was compared with conventional long circulating SOD liposomes and free SOD using biochemical liver injury biomarkers, histology and MRM. MRM results correlated with those obtained using classical biochemical biomarkers of liver injury and liver histology. Moreover, MRM images suggested that the therapeutic efficacy of both SOD liposomal formulations used was related to prevention of peripheral biliary ductular damage and disrupted vascular architecture. Therefore, MRM at 7T is a useful technique to follow IRI. SOD-enzymosomes were more effective than conventional liposomes in reducing liver ischaemia-reperfusion injury and this may be due to a short therapeutic window.

Hydrogen peroxide regulates angiogenesis-related factors in tumor cells.

Tumor angiogenesis is required for tumor development and growth, and is regulated by several factors including ROS. H2O2 is a ROS with an important role in cell signaling, but how H2O2 regulates tumor angiogenesis is still poorly understood. We have xenografted tumor cells with altered levels of H2O2 by catalase overexpression into zebrafish embryos to study redox-induced tumor neovascularization. We found that vascular recruitment and invasion were impaired if catalase was overexpressed. In addition, the overexpression of catalase altered the transcriptional levels of several angiogenesis-related factors in tumor cells, including TIMP-3 and THBS1. These two anti-angiogenic factors were found to be H2O2-regulated by two different mechanisms: TIMP-3 expression in a cell-autonomous manner; and, THBS1 expression that was non-cell-autonomous. Our work shows that intracellular H2O2 regulates the expression of angiogenic factors and the formation of a vessel network. Understanding the molecular mechanisms that govern this multifunctional effect of H2O2 on tumor angiogenesis could be important for the development of more efficient anti-angiogenic therapies.

Opi1p translocation to the nucleus is regulated by hydrogen peroxide in Saccharomyces cerevisiae.

During exposure of yeast cells to low levels of hydrogen peroxide (H2 O2 ), the expression of several genes is regulated for cells to adapt to the surrounding oxidative environment. Such adaptation involves modification of plasma membrane lipid composition, reorganization of ergosterol-rich microdomains and altered gene expression of proteins involved in lipid and vesicle traffic, to decrease permeability to exogenous H2 O2 . Opi1p is a transcriptional repressor that is inactive when present at the nuclear membrane/endoplasmic reticulum, but represseses transcription of inositol upstream activating sequence (UASINO )-containing genes, many of which are involved in the synthesis of phospholipids and fatty acids, when it is translocated to the nucleus. We investigated whether H2 O2 in concentrations inducing adaptation regulates Opi1p function. We found that, in the presence of H2 O2 , GFP-Opi1p fusion protein translocates to the nucleus and, concomitantly, the expression of UASINO -containing genes is affected. We also investigated whether cysteine residues of Opi1p were implicated in the H2 O2 -mediated translocation of this protein to the nucleus and identified cysteine residue 159 as essential for this process. Our work shows that Opi1p is redox-regulated and establishes a new mechanism of gene regulation involving Opi1p, which is important for adaptation to H2 O2 in yeast cells. Copyright © 2017 John Wiley & Sons, Ltd.

Studies on the mechanism of action of antitumor bis(aminophenolate) ruthenium(III) complexes.

Two recently published Ru(III) complexes bearing (N2O2) tetradentate bis(aminophenolate) ligands, formulated as [Ru(III)(salan)(PPh3)Cl] (salan is the tetradentate ligand 6,6'-(1S,2S)-cyclohexane-1,2-diylbis(azanediyl)bis(methylene)bis(3-methoxyphenol) in complex 1, or 2,2'-(1S,2S)-cyclohexane-1,2-diylbis(azanediyl)bis(methylene)bis(4-methoxyphenol) in complex 2; PPh3 is triphenylphosphane) and found very active against ovarian and breast adenocarcinoma human cells were studied to outline their antitumor mode of action. The human cisplatin-sensitive ovarian adenocarcinoma line A2780 was used herein as the cell model. At a 24h challenge (similarly as found before for 72h) both complexes are active, their cytotoxicity being comparable to that of cisplatin in the same conditions. As a possible target in the cell for their action, the interaction of 1 and 2 with DNA was assessed through displacement of well-established DNA fluorescent probes (ethidium bromide, EB, and 4',6-diamidino-2-phenylindole, DAPI) through steady-state and time-resolved fluorescence spectroscopy. The whole emission spectra were analyzed globally for the binary DNA-probe and ternary DNA-probe-Ru(III) complex systems. Both Ru(III) complexes can displace EB and bind to DNA with similar and moderate strong affinity with conditional stability constants of logK'=(5.05±0.01) for 1 and logK'=(4.79±0.01) for 2. The analysis of time-domain fluorescence intensity decays confirmed both qualitatively and quantitatively the model used to describe the binding and competition processes. Cell studies indicated that apoptosis is the major mechanism of cell death for both complexes, with 2 (the more active complex) promoting that process more efficiently than 1. Transmission electron micrographs revealed clear alterations on intracellular organization consistent with the induction of programmed cell death processes.

Data on intracellular localization of RPSA upon alteration of its redox state.

Ribosomal Protein SA (RPSA), a component of the 40S ribosomal subunit, was identified as a H2O2 target in HeLa cells [1]. In order to analyze the intracellular localization of RPSA in different redox states we overexpressed wild-type RPSA (RPSAwt) or RPSA containing two cysteine to serine residue substitutions at positions 148 and 163 (RPSAmut) in HeLa cells. The transfected cells were exposed to H2O2 or N-acetylcysteine (NAC) and RPSA subcellular localization was assessed by immunofluorescence in permeabilized cells. In addition, co-immunofluorescence for RPSA and Ribosomal Protein S6 (RPS6) was performed in cells overexpressing RPSAwt or RPSAmut. Finally, the ribosomal expression of endogenous RPSA in the presence or absence of H2O2 was analyzed by Western blot. The data presented in this work is related to the research article entitled "Hydrogen peroxide regulates cell adhesion through the redox sensor RPSA" [1].

Hydrogen peroxide regulates cell adhesion through the redox sensor RPSA.

To become metastatic, a tumor cell must acquire new adhesion properties that allow migration into the surrounding connective tissue, transmigration across endothelial cells to reach the blood stream and, at the site of metastasis, adhesion to endothelial cells and transmigration to colonize a new tissue. Hydrogen peroxide (H2O2) is a redox signaling molecule produced in tumor cell microenvironment with high relevance for tumor development. However, the molecular mechanisms regulated by H2O2 in tumor cells are still poorly known. The identification of H2O2-target proteins in tumor cells and the understanding of their role in tumor cell adhesion are essential for the development of novel redox-based therapies for cancer. In this paper, we identified Ribosomal Protein SA (RPSA) as a target of H2O2 and showed that RPSA in the oxidized state accumulates in clusters that contain specific adhesion molecules. Furthermore, we showed that RPSA oxidation improves cell adhesion efficiency to laminin in vitro and promotes cell extravasation in vivo. Our results unravel a new mechanism for H2O2-dependent modulation of cell adhesion properties and identify RPSA as the H2O2 sensor in this process. This work indicates that high levels of RPSA expression might confer a selective advantage to tumor cells in an oxidative environment.

The extracellular matrix modulates H2O2 degradation and redox signaling in endothelial cells.

The molecular processes that are crucial for cell function, such as proliferation, migration and survival, are regulated by hydrogen peroxide (H2O2). Although environmental cues, such as growth factors, regulate redox signaling, it was still unknown whether the ECM, a component of the cell microenvironment, had a function in this process. Here, we showed that the extracellular matrix (ECM) differently regulated H2O2 consumption by endothelial cells and that this effect was not general for all types of cells. The analysis of biophysical properties of the endothelial cell membrane suggested that this modification in H2O2 consumption rates was not due to altered membrane permeability. Instead, we found that the ECM regulated GPx activity, a known H2O2 scavenger. Finally, we showed that the extent of PTEN oxidation was dependent on the ECM, indicating that the ECM was able to modulate H2O2-dependent protein oxidation. Thus, our results unraveled a new mechanism by which the ECM regulates endothelial cell function by altering redox balance. These results pinpoint the ECM as an important component of redox-signaling.

Hydrogen peroxide sensing, signaling and regulation of transcription factors.

The regulatory mechanisms by which hydrogen peroxide (H2O2) modulates the activity of transcription factors in bacteria (OxyR and PerR), lower eukaryotes (Yap1, Maf1, Hsf1 and Msn2/4) and mammalian cells (AP-1, NRF2, CREB, HSF1, HIF-1, TP53, NF-κB, NOTCH, SP1 and SCREB-1) are reviewed. The complexity of regulatory networks increases throughout the phylogenetic tree, reaching a high level of complexity in mammalians. Multiple H2O2 sensors and pathways are triggered converging in the regulation of transcription factors at several levels: (1) synthesis of the transcription factor by upregulating transcription or increasing both mRNA stability and translation; (ii) stability of the transcription factor by decreasing its association with the ubiquitin E3 ligase complex or by inhibiting this complex; (iii) cytoplasm-nuclear traffic by exposing/masking nuclear localization signals, or by releasing the transcription factor from partners or from membrane anchors; and (iv) DNA binding and nuclear transactivation by modulating transcription factor affinity towards DNA, co-activators or repressors, and by targeting specific regions of chromatin to activate individual genes. We also discuss how H2O2 biological specificity results from diverse thiol protein sensors, with different reactivity of their sulfhydryl groups towards H2O2, being activated by different concentrations and times of exposure to H2O2. The specific regulation of local H2O2 concentrations is also crucial and results from H2O2 localized production and removal controlled by signals. Finally, we formulate equations to extract from typical experiments quantitative data concerning H2O2 reactivity with sensor molecules. Rate constants of 140 M(-1) s(-1) and ≥1.3 × 10(3) M(-1) s(-1) were estimated, respectively, for the reaction of H2O2 with KEAP1 and with an unknown target that mediates NRF2 protein synthesis. In conclusion, the multitude of H2O2 targets and mechanisms provides an opportunity for highly specific effects on gene regulation that depend on the cell type and on signals received from the cellular microenvironment.

Cellular polarity in aging: role of redox regulation and nutrition.

Cellular polarity concerns the spatial asymmetric organization of cellular components and structures. Such organization is important not only for biological behavior at the individual cell level, but also for the 3D organization of tissues and organs in living organisms. Processes like cell migration and motility, asymmetric inheritance, and spatial organization of daughter cells in tissues are all dependent of cell polarity. Many of these processes are compromised during aging and cellular senescence. For example, permeability epithelium barriers are leakier during aging; elderly people have impaired vascular function and increased frequency of cancer, and asymmetrical inheritance is compromised in senescent cells, including stem cells. Here, we review the cellular regulation of polarity, as well as the signaling mechanisms and respective redox regulation of the pathways involved in defining cellular polarity. Emphasis will be put on the role of cytoskeleton and the AMP-activated protein kinase pathway. We also discuss how nutrients can affect polarity-dependent processes, both by direct exposure of the gastrointestinal epithelium to nutrients and by indirect effects elicited by the metabolism of nutrients, such as activation of antioxidant response and phase-II detoxification enzymes through the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2). In summary, cellular polarity emerges as a key process whose redox deregulation is hypothesized to have a central role in aging and cellular senescence.

Bone marrow-derived endothelial progenitors expressing Delta-like 4 (Dll4) regulate tumor angiogenesis.

Neo-blood vessel growth (angiogenesis), which may involve the activation of pre-existing endothelial cells (EC) and/or the recruitment of bone marrow-derived vascular precursor cells (BM-VPC), is essential for tumor growth. Molecularly, besides the well established roles for Vascular endothelial growth factor (VEGF), recent findings show the Notch signalling pathway, in particular the ligand Delta-like 4 (Dll4), is also essential for adequate tumor angiogenesis; Dll4 inhibition results in impaired, non-functional, angiogenesis and reduced tumor growth. However, the role of BM-VPC in the setting of Notch pathway modulation was not addressed and is the subject of the present report. Here we show that SDF-1 and VEGF, which are produced by tumors, increase Dll4 expression on recruited BM-VPC. Mechanistically, BM-VPC activated, in a Dll4-dependent manner, a transcriptional program on mature EC suggestive of EC activation and stabilization. BM-VPC induced ICAM-2 and Fibronectin expression on EC, an effect that was blocked by a Dll4-specific neutralizing antibody. In vivo, transplantation of BM-VPC with decreased Dll4 into tumor-bearing mice resulted in the formation of microvessels with decreased pericyte coverage and reduced fibronectin expression. Consequently, transplantation of BM-VPC with decreased Dll4 resulted in impaired tumor angiogenesis, increased tumor hypoxia and apoptosis, and decreased tumor growth. Taken together, our data suggests that Dll4 expression by BM-VPC affects their communication with tumor vessel endothelial cells, thereby modulating tumor angiogenesis by affecting vascular stability.

Notch pathway modulation on bone marrow-derived vascular precursor cells regulates their angiogenic and wound healing potential.

Bone marrow (BM) derived vascular precursor cells (BM-PC, endothelial progenitors) are involved in normal and malignant angiogenesis, in ischemia and in wound healing. However, the mechanisms by which BM-PC stimulate the pre-existing endothelial cells at sites of vascular remodelling/recovery, and their contribution towards the formation of new blood vessels are still undisclosed. In the present report, we exploited the possibility that members of the Notch signalling pathway, expressed by BM-PC during endothelial differentiation, might regulate their pro-angiogenic or pro-wound healing properties. We demonstrate that Notch pathway modulates the adhesion of BM-PC to extracellular matrix (ECM) in vitro via regulation of integrin alpha3beta1; and that Notch pathway inhibition on BM-PC impairs their capacity to stimulate endothelial cell tube formation on matrigel and to promote endothelial monolayer recovery following wounding in vitro. Moreover, we show that activation of Notch pathway on BM-PC improved wound healing in vivo through angiogenesis induction. Conversely, inoculation of BM-PC pre-treated with a gamma secretase inhibitor (GSI) into wounded mice failed to induce angiogenesis at the wound site and did not promote wound healing, presumably due to a lower frequency of BM-PC at the wound area. Our data suggests that Notch pathway regulates BM-PC adhesion to ECM at sites of vascular repair and that it also regulates the capacity of BM-PC to stimulate angiogenesis and to promote wound healing. Drug targeting of the Notch pathway on BM-PC may thus represent a novel strategy to modulate neo-angiogenesis and vessel repair.

Endothelial progenitors in vascular repair and angiogenesis: how many are needed and what to do?

Defects in the regulation of neo blood vessel growth (angiogenesis) or in vessel repair are major complications in many diseases, such as cancer, diabetes, atherosclerosis and myocardial infarction. In these diseases it was shown that the number of circulating endothelial progenitor cells (EPC) was altered. This has been associated with the angiogenic status and patient prognosis. However, the regulation of angiogenesis depends not only on the number of circulating EPC but also on their functions. EPC are bone marrow derived cells that are recruited into the peripheral blood in situations of vascular repair/angiogenesis or vascular stress. EPC are believed to exert their function using mainly two strategies: activating locally the endothelial cells and/or differentiating into mature endothelial cells that integrate the damaged vessels. To do this, EPC must home to "angiogenic active" sites, adhere to the activated/damaged endothelial cells or to the extracellular matrix and participate in the endothelial activation/repair process. In vitro and in vivo experiments using animal models revealed the importance of various signalling pathways in these processes and, in patients, new therapeutic strategies are being developed based on the specific functions of EPC. Although the role of EPC in vessel repair in disease is not totally understood, it becomes clear that the activation state of these cells is critical for the vessel repair process. Our previous work generated a detailed gene expression profile of EPC during the endothelial differentiation process in vitro. With this information, it has been possible to identify numerous molecular targets crucial for EPC differentiation and function and to test their involvement in EPC function during wound healing or tumor angiogenesis. The importance of EPC identification, activation state and function in vascular repair and in angiogenesis in disease will be discussed in this review.

Neural crest progenitors and stem cells.

In the vertebrate embryo, multiple cell types originate from a common structure, the neural crest (NC), which forms at the dorsal tips of the neural epithelium. The NC gives rise to migratory cells that colonise a wide range of embryonic tissues and later differentiate into neurones and glial cells of the peripheral nervous system (PNS), pigment cells (melanocytes) in the skin and endocrine cells in the adrenal and thyroid glands. In the head and the neck, the NC also yields mesenchymal cells that form craniofacial cartilages, bones, dermis, adipose tissue, and vascular smooth muscle cells. The NC is therefore a model system to study cell diversification during embryogenesis and phenotype maintenance in the adult. By analysing the developmental potentials of quail NC cells in clonal cultures, we have shown that the migratory NC is a collection of heterogeneous progenitors, including various types of intermediate precursors and highly multipotent cells, some of which being endowed of self-renewal capacity. We also have identified common progenitors for mesenchymal derivatives and neural/melanocytic cells in the cephalic NC. These results are consistent with a hierarchical model of lineage segregation wherein environmental cytokines control the fate of progenitors and stem cells. One of these cytokines, the endothelin3 peptide, promotes the survival, proliferation, and self-renewal capacity of common progenitors for glial cells and melanocytes. At post-migratory stages, when they have already differentiated, NC-derived cells exhibit phenotypic plasticity. Epidermal pigment cells and Schwann cells from peripheral nerves in single-cell culture are able to reverse into multipotent NC-like progenitors endowed with self-renewal. Therefore, stem cell properties are expressed by a variety of NC progenitors and can be re-acquired by differentiated cells of NC origin, suggesting potential function for repair.