Omar Benzakour

Expression of vascular endothelial growth factor receptors in smooth muscle cells

Vascular Endothelial Growth Factor (VEGF) has been typically considered to be an endothelial‐specific growth factor. However, it was recently demonstrated that VEGF can interact with non endothelial cells. In this study, we tested whether vascular smooth muscles cells (VSMCs) can express VEGF receptors, such as flk‐1, flt‐1, and neuropilin (NP)‐1, and respond to VEGF in vitro. In cultured VSMCs, flk‐1 and flt‐1 expression was inversely related to cell density. The expression of flk‐1 was down‐regulated with increasing passage numbers. However, NP‐1 levels were not affected by cell density or passage numbers. Flk‐1, Flt‐1, and NP‐1 protein levels were confirmed by Western Blotting. Although the functional mature form of Flk‐1 protein is expressed at low levels in VSMCs, phosphorylation of Flk‐1 following VEGF165 stimulation was still observed. SMCs migrated significantly in response to VEGF165 and VEGF‐E, whereas Placenta Growth Factor (PlGF) induced migration only at higher concentrations. Since VEGF‐E is a specific activator of flk‐1 while PlGF specifically activates only flt‐1, SMC migration induced by VEGF165 is likely to be mediated primarily through the flk‐1 receptor. VSMCs did not significantly proliferate in response to VEGF165, PlGF, and VEGF‐E. In conclusion, our studies demonstrate the presence of VEGF receptors on VSMCs that are functional. These studies also indicate that in vivo, VEGF may play a role in modulating the response of VSMCs.

Induction of Vascular SMC Proliferation by Urokinase Indicates a Novel Mechanism of Action in Vasoproliferative Disorders

The urokinase-type plasminogen activator (UPA) and its receptor are expressed in the vasculature and are involved in cell migration and remodeling of the extracellular matrix in the neointima. Vessels with atherosclerosis or neointimal hyperplasia, when compared with normal vessels, contain high UPA activity as well as increased levels of UPA receptor. In this study, we have identified the stimulation of vascular smooth muscle cell proliferation as a novel activity for UPA in the vessel wall. High-molecular-weight-UPA (12-200 nmol/L range) stimulated DNA synthesis and cell proliferation, which was half that induced by fetal calf serum or by platelet-derived growth factor-BB. UPA did not induce growth of endothelial cells, and tissue-type plasminogen activator showed no activity on either cell type. Induction of proliferation required the complete UPA molecule but was independent of the proteolytic activity of UPA, whereas neither the amino-terminal fragment nor the catalytic domain by itself was mitogenic. UPA also stimulated c-fos/c-myc mRNA expression and mitogen-activated protein kinase activity in smooth muscle cells. Blocking monoclonal antibodies against the UPA receptor and the enzymatic removal of receptors were ineffective in inhibiting the mitogenic effect of UPA, suggesting a UPA receptor-independent mechanism. Thus, we provide evidence for a novel function of UPA on vascular smooth muscle cell proliferation that, together with its previously documented involvement in regulating pericellular proteolysis-related events and cell migration, provides additional evidence for a role in the pathogenesis of atherosclerosis/restenosis.

Endogenous hepatocyte growth factor is a niche signal for subventricular zone neural stem cell amplification and self-renewal.

Neural stem cells persist in the adult mammalian brain, within the subventricular zone (SVZ). The endogenous mechanisms underpinning SVZ neural stem cell proliferation, self‐renewal, and differentiation are not fully elucidated. In the present report, we describe a growth‐stimulatory activity of liver explant‐conditioned media on SVZ cell cultures and identify hepatocyte growth factor (HGF) as a major player in this effect. HGF exhibited a mitogenic activity on SVZ cell cultures in a mitogen‐activated protein kinase (MAPK) (ERK1/2)‐dependent manner as U0126, a specific MAPK inhibitor, blocked it. Combining a functional neurosphere forming assay with immunostaining for c‐Met, along with markers of SVZ cells subtypes, demonstrated that HGF promotes the expansion of neural stem‐like cells that form neurospheres and self‐renew. Immunostaining, HGF enzyme‐linked immunosorbent assay and Madin‐Darby canine kidney cell scattering assay indicated that SVZ cell cultures produce and release HGF. SVZ cell‐conditioned media induced proliferation on SVZ cell cultures, which was blocked by HGF‐neutralizing antibodies, hence implying that endogenously produced HGF accounts for a major part in SVZ mitogenic activity. Brain sections immunostaining revealed that HGF is produced by nestin‐expressing cells and c‐Met is expressed within the SVZ by immature cells. HGF intracerebroventricular injection promoted SVZ cell proliferation and increased the ability of these cells exposed in vivo to HGF to form neurospheres in vitro, whereas intracerebroventricular injection of HGF‐neutralizing antibodies decreased SVZ cell proliferation. The present study unravels a major role, both in vitro and in vivo, for endogenous HGF in SVZ neural stem cell growth and self‐renewal. STEM CELLS 2009;27:408–419

An Endogenous Vitamin K-Dependent Mechanism Regulates Cell Proliferation in the Brain Subventricular Stem Cell Niche†‡§

Neural stem cells (NSC) persist in the adult mammalian brain, within the subventricular zone (SVZ). The endogenous mechanisms underpinning SVZ stem and progenitor cell proliferation are not fully elucidated. Vitamin K‐dependent proteins (VKDPs) are mainly secreted factors that were initially discovered as major regulators of blood coagulation. Warfarin ((S(−)‐3‐acetonylbenzyl)‐4‐hydroxycoumarin)), a widespread anticoagulant, is a vitamin K antagonist that inhibits the production of functional VKDP. We demonstrate that the suppression of functional VKDPs production, in vitro, by exposure of SVZ cell cultures to warfarin or, in vivo, by its intracerebroventricular injection to mice, leads to a substantial increase in SVZ cell proliferation. We identify the anticoagulant factors, protein S and its structural homolog Gas6, as the two only VKDPs produced by SVZ cells and describe the expression and activation pattern of their Tyro3, Axl, and Mer tyrosine kinase receptors. Both in vitro and in vivo loss of function studies consisting in either Gas6 gene invalidation or in endogenous protein S neutralization, provided evidence for an important novel regulatory role of these two VKDPs in the SVZ neurogenic niche. Specifically, we show that while a loss of Gas6 leads to a reduction in the numbers of stem‐like cells and in olfactory bulb neurogenesis, endogenous protein S inhibits SVZ cell proliferation. Our study opens up new perspectives for investigating further the role of vitamin K, VKDPs, and anticoagulants in NSC biology in health and disease. STEM CELLS 2012; 30:719–731

A chimerical phagocytosis model reveals the recruitment by Sertoli cells of autophagy for the degradation of ingested illegitimate substrates.

Phagocytosis and autophagy are typically dedicated to degradation of substrates of extrinsic and intrinsic origins respectively. Although overlaps between phagocytosis and autophagy were reported, the use of autophagy for ingested substrate degradation by nonprofessional phagocytes has not been described. Blood-separated tissues use their tissue-specific nonprofessional phagocytes for homeostatic phagocytosis. In the testis, Sertoli cells phagocytose spermatid residual bodies produced during germ cell differentiation. In the retina, pigmented epithelium phagocytoses shed photoreceptor tips produced during photoreceptor renewal. Spermatid residual bodies and shed photoreceptor tips are phosphatidylserine-exposing substrates. Activation of the tyrosine kinase receptor MERTK, which is implicated in phagocytosis of phosphatidylserine-exposing substrates, is a common feature of Sertoli and retinal pigmented epithelial cell phagocytosis. The major aim of our study was to investigate to what extent phagocytosis by Sertoli cells may be tissue specific. We analyzed in Sertoli cell cultures that were exposed to either spermatid residual bodies (legitimate substrates) or retina photoreceptor outer segments (illegitimate substrates) the course of the main phagocytosis stages. We show that whereas substrate binding and ingestion stages occur similarly for legitimate or illegitimate substrates, the degradation of illegitimate but not of legitimate substrates triggers autophagy as evidenced by the formation of double-membrane wrapping, MAP1LC3A-II/LC3-II clustering, SQSTM1/p62 degradation, and by marked changes in ATG5, ATG9 and BECN1/Beclin 1 protein expression profiles. The recruitment by nonprofessional phagocytes of autophagy for the degradation of ingested cell-derived substrates is a novel feature that may be of major importance for fundamentals of both apoptotic substrate clearance and tissue homeostasis.

The vitamin K–dependent anticoagulant factor, protein S, inhibits multiple VEGF-A–induced angiogenesis events in a Mer- and SHP2-dependent manner

Protein S is a vitamin K-dependent glycoprotein, which, besides its anticoagulant function, acts as an agonist for the tyrosine kinase receptors Tyro3, Axl, and Mer. The endothelium expresses Tyro3, Axl, and Mer and produces protein S. The interaction of protein S with endothelial cells and particularly its effects on angiogenesis have not yet been analyzed. Here we show that human protein S, at circulating concentrations, inhibited vascular endothelial growth factor (VEGF) receptor 2-dependent vascularization of Matrigel plugs in vivo and the capacity of endothelial cells to form capillary-like networks in vitro as well as VEGF-A-induced endothelial migration and proliferation. Furthermore, protein S inhibited VEGF-A-induced endothelial VEGFR2 phosphorylation and activation of mitogen-activated kinase-Erk1/2 and Akt. Protein S activated the tyrosine phosphatase SHP2, and the SHP2 inhibitor NSC 87877 reversed the observed inhibition of VEGF-A-induced endothelial proliferation. Using siRNA directed against Tyro3, Axl, and Mer, we demonstrate that protein S-mediated SHP2 activation and inhibition of VEGF-A-stimulated proliferation were mediated by Mer. Our report provides the first evidence for the existence of a protein S/Mer/SHP2 axis, which inhibits VEGFR2 signaling, regulates endothelial function, and points to a role for protein S as an endogenous angiogenesis inhibitor.

Dimeric Transferrin Inhibits Phagocytosis of Residual Bodies by Testicular Rat Sertoli Cells

Abstract Transferrin is well known as an iron transport glycoprotein. Dimeric or tetrameric transferrin forms have recently been reported to modulate phagocytosis by human leukocytes. It is mainly synthesized by the liver, and also by other sources, such as Sertoli cells of the testis. Sertoli cells show a strong phagocytic activity toward apoptotic germ cells and residual bodies. Here, we provide evidence that purified human dimeric transferrin from commercial sources decreased residual body phagocytosis, unlike monomeric transferrin. The presence of iron appeared essential for dimeric transferrin inhibitory activity. Importantly, dimeric transferrin could be visualized by immunoblotting in Sertoli cell lysates as well as in culture media, indicating that dimeric transferrin could be physiologically secreted by Sertoli cells. By siRNA-mediated knockdown, we show that endogenous transferrin significantly inhibited residual body ingestion by Sertoli cells. These results are the first to identify dimeric transferrin in Sertoli cells and to demonstrate its implication as a physiological modulator of residual body phagocytosis by Sertoli cells.

Evidence for a major role of endogenous fibroblast growth factor-2 in apoptotic cortex-induced subventricular zone cell proliferation

In the adult mammalian brain, neural stem cells persist in the subventricular zone (SVZ) of lateral ventricles. It is well established that cortical damage leads to SVZ cell proliferation and neuronal differentiation. We have previously demonstrated in rat that, when treated with the apoptosis‐inducing agent staurosporine, cortex explants release heat‐labile factors that promote SVZ cell culture proliferation. In the present report, we investigated in vitro mechanisms involved in cortex injury‐triggered neurogenesis in the rat. We demonstrated, using immunoblotting analysis and fibroblast growth factor (FGF)‐2 enzyme‐linked sandwich immunosorbent assay, that treatment of cortex explants with apoptosis‐inducing agents increases the release of FGF‐2. We next determined the effects of apoptotic cortex‐released factors in regulating SVZ cell proliferation and neuronal differentiation by using bromodeoxyuridine incorporation and microtubule‐associated protein 2 immunostaining assays, respectively. We found that conditioned media derived from staurosporine‐treated cortex explants enhanced SVZ cell culture proliferation and differentiation by over 50 and 80%, respectively. Finally, we showed that immunodepletion of FGF‐2 or pharmacological blockade of FGF‐2 receptor by SU5402 completely abolished staurosporine‐treated cortex mitogenic activity on SVZ cultures but did not alter its activity on neuronal cell differentiation. Altogether, the present report establishes that the release of endogenous FGF‐2 by apoptotic cortex explants plays a major role in the induction of SVZ cell proliferation but not neuronal differentiation, which probably depends on the release of other as yet unidentified cortical factors.

Endogenous factors derived from embryonic cortex regulate proliferation and neuronal differentiation of postnatal subventricular zone cell cultures

In rodents, the subventricular zone (SVZ) harbours neural stem cells that proliferate and produce neurons throughout life. Previous studies showed that factors released by the developing cortex promote neurogenesis in the embryonic ventricular zone. In the present report, we studied in the rat the possible involvement of endogenous factors derived from the embryonic cortex in the regulation of the development of postnatal SVZ cells. To this end, SVZ neurospheres were maintained with explants or conditioned media (CM) prepared from embryonic day (E) 13, E16 or early postnatal cortex. We demonstrate that early postnatal cortex‐derived factors have no significant effect on SVZ cell proliferation or differentiation. In contrast, E13 and E16 cortex release diffusible, heat‐labile factors that promote SVZ cell expansion through increased proliferation and reduced cell death. In addition, E16 cortex‐derived factors stimulate neuronal differentiation in both early postnatal and adult SVZ cultures. Fibroblast growth factor (FGF)‐2‐ but not epidermal growth factor (EGF)‐immunodepletion drastically reduces the mitogenic effect of E16 cortex CM, hence suggesting a major role of endogenous FGF‐2 released by E16 cortex in the stimulation of SVZ cell proliferation. The evidence we provide here for the regulation of SVZ cell proliferation and neuronal differentiation by endogenous factors released from embryonic cortex may be of major importance for brain repair research.

Vitamin K-dependent proteins: functions in blood coagulation and beyond.

Thromb Haemost 2008; 100: 527–529 Vitamin K or vitamin “Koagulation” (German spelling for coagulation) was discovered by the 1943 Nobel Prize winners Henrik Dam and Edward A. Doisy, as a fat soluble substance, the deficiency of which caused bleeding disorders.Vitamin K in its reduced form is required as a cofactor for the γ-glutamyl carboxylase enzyme that catalyses the γ-carboxylation of specific glutamic acid (Glu) residues of a subclass of proteins (1). This subclass of proteins was then termed vitamin K-dependent proteins (VKDP), or γ-carboxylated proteins or simply Gla-proteins. The enzymatic reaction generates γ-carboxyglutamate (Gla) and vitamin K 2,3,-epoxide which is then recycled back to the hydroquinone form by a reductase enzyme (1). Warfarin (3-[α-acetonyl-benzyl-4-hydroxycoumarin]) inhibits the activity of the vitamin K epoxide reductase blocking the vitamin K cycle.This property of warfarin has led to its widespread use in anticoagulant therapy (2). In 2000, warfarin was ranked among the top-selling drugs with a turnover estimated at 500 million dollars. The γ-carboxylation process appears to be required for the activities of all Gla-proteins studied to date. Carboxylase substrates synthesised in the presence of warfarin are undercarboxylated and have impaired biological activities (3). In the present theme issue, Shearer and Newman stress the importance of exploring vitamin K metabolism and catabolism pathways for a better understanding of pathologies linked to vitamin K deficiency (4). The occurrence of such pathologies is established in bone and arteries, two tissues that express many VKDP, whereby expression is hypothesised in others. The various molecular forms of vitamin K are different not only with regard to their co-factor activities but also with regard to their absorption, transport, cellular uptake, tissue distribution, turnover and catabolism.This paper provides a clear description of the nomenclature and chemical structure of K vitamins, namely phylloquinone (vitamin K1) and menaquinones (vitamins K2) as well as their dietary sources. Further research is certainly needed in this area to determine to what extent bacterially fermented food as well as intestinal flora contribute to the maintenance of vitamin K status. The effects of other putative non-cofactor functions of vitamin K, which potentially include the suppression of inflammation, prevention of brain oxidative damage and a role in sphingolipid synthesis as well as the possible existence of a receptor for vitamin K, are important issues that require long-term basic research to be clarified. The full consequences of dietary vitamin K deficiency on tissue distribution and metabolism of K vitamins as well as the consequences of oral anticoagulants on the metabolism of K vitamins remains to be assessed in the light of new findings on VKDP. Only few proteins are known to contain Gla residues. These include: (i) certain proteins of the blood coagulation system: prothrombin, factors VII, IX and X, protein C, protein S and protein Z; (ii) the protein encoded by the growth arrest specific gene GAS6, which presents strong structural homology with protein S; (iii) Gla proteins expressed in mineralised tissues: osteocalcin and matrix Gla-protein (MGP); (iv) Gla-containing snake venom proteins and conotoxins; (v) four transmembrane Gla-proteins PRGP1, PRGP2, TMG3 and TMG4 whose functions are as yet unknown; (vi) some members of the connexin family of proteins (5). The precise role of the γ-carboxylation modification for connexin protein function is not as yet known. Except for protein S and protein Z, VKDP of the blood-clotting cascade are serine protease zymogens and are activated by serine proteases. The role of protein Z in blood clotting is not fully clarified and neither cellular effects nor a specific pathology have been as yet associated with protein Z. The review by Vasse et al. (6) in the present issue covers this topic. Two further contributions in the present theme issue relate to the therapeutic functions of VKDP in haemostasis. In her concise review Ulla Hedner concentrates on the therapeutic use of factor VIIa in bleeding associated pathologies (7). The paper by Paul Monahan reviews the data obtained from factor IX knockout mice and other haemophilia B mouse models (8). Such mouse models have not only enabled a better understanding of the role of factor IX in haemostasis, thrombosis and wound healing but also pro-