Clinical, biological, and genetic features in an afibrinogenemia patient series in Algeria.

INTRODUCTION: The incidence of afibrinogenemia had not been previously reported in Algeria. Afibrinogenemia patients are prone to both haemorrhagic and thrombotic complications. Predictive markers of thrombosis in afibrinogenemia patients are not existent. AIMS AND METHODS: Clinical and biological data from 46 afibrinogenemia patients are reported. Biological investigations included routine tests, genetics analysis and thrombin generation. RESULTS: FGA mutations (four novel and four previously described) and FGB mutations (seven mutations; five novels) were homozygous in all but one family as a result of 28 consanguineous marriages out of 30 discrete families. Incidence of afibrinogenemia in Algeria is at least 3 per million births. Umbilical bleeding was reported in 39/46 cases and was the main discovery circumstance. We also report post trauma or post-surgery (3/46) bleeding and spontaneous deep vein thrombosis (DVT) in adulthood (1/46), as discovery circumstances. The median age (10.5-year-old) of the population reported here explains why there are few hemarthrosis and obstetrical or gynaecological complications in this series. Thrombotic events were reported in seven patients (four spontaneous). Endogenous Thrombin Potential was significantly increased in thrombosis-prone patients compared to afibrinogenemic patients with and without personal or familial history (1118 vs. 744 and 817 nM IIa × min, respectively). CONCLUSION: The incidence of afibrinogenemia in Algeria is the consequence of consanguineous marriage in families carrying private mutations. The thrombin generation test (TGT) could identify, among afibrinogenemic patients, those presenting a thrombotic risk.

Osteoprotegerin regulates cancer cell migration through SDF-1/CXCR4 axis and promotes tumour development by increasing neovascularization.

We previously reported that OPG is involved in ischemic tissue neovascularization through the secretion of SDF-1 by pretreated-OPG endothelial colony-forming cells (ECFCs). As the vascularization is one of the key factor influencing the tumour growth and cancer cell dissemination, we investigated whether OPG was able to modulate the invasion of human MNNG-HOS osteosarcoma and DU145 prostate cancer cell lines in vitro and in vivo. Cell motility was analysed in vitro by using Boyden chambers. Human GFP-labelled MMNG-HOS cells were inoculated in immunodeficient mice and the tumour nodules formed were then injected with OPG and/or FGF-2, AMD3100 or 0.9% NaCl (control group). Tumour growth was manually followed and angiogenesis was assessed by immunohistochemistry. In vitro, SDF-1 released by OPG-pretreated ECFCs markedly attracted both MNNG-HOS and DU145 cells and induced spontaneous migration of cancer cells. In vivo, tumour volumes were significantly increased in OPG-treated group compared to the control group and OPG potentiated the effect of FGF-2. Concomitantly, OPG alone or combined with FGF-2 increased the number of new vasculature compared to the control group. Interestingly AMD3100, an inhibitor of SDF-1, prevented the in vivo effects of OPG induced by SDF-1 This study provides experimental evidence that OPG promotes tumour development trough SDF-1/CXCR4 axis.

Low-Molecular-Weight Fucoidan Induces Endothelial Cell Migration via the PI3K/AKT Pathway and Modulates the Transcription of Genes Involved in Angiogenesis.

Low-molecular-weight fucoidan (LMWF) is a sulfated polysaccharide extracted from brown seaweed that presents antithrombotic and pro-angiogenic properties. However, its mechanism of action is not well-characterized. Here, we studied the effects of LMWF on cell signaling and whole genome expression in human umbilical vein endothelial cells and endothelial colony forming cells. We observed that LMWF and vascular endothelial growth factor had synergistic effects on cell signaling, and more interestingly that LMWF by itself, in the absence of other growth factors, was able to trigger the activation of the PI3K/AKT pathway, which plays a crucial role in angiogenesis and vasculogenesis. We also observed that the effects of LMWF on cell migration were PI3K/AKT-dependent and that LMWF modulated the expression of genes involved at different levels of the neovessel formation process, such as cell migration and cytoskeleton organization, cell mobilization and homing. This provides a better understanding of LMWF's mechanism of action and confirms that it could be an interesting therapeutic approach for vascular repair.

Tie2-dependent deletion of α6 integrin subunit in mice reduces tumor growth and angiogenesis.

The α6 integrin subunit (α6) has been implicated in cancer cell migration and in the progression of several malignancies, but its role in tumor angiogenesis is unclear. In mice, anti-α6 blocking antibodies reduce tumor angiogenesis, whereas Tie1-dependent α6 gene deletion enhances neovessel formation in melanoma and lung carcinoma. To clarify the discrepancy in these results we used the cre-lox system to generate a mouse line, α6fl/fl­Tie2Cre(+), with α6 gene deletion specifically in Tie2-lineage cells: endothelial cells, pericytes, subsets of hematopoietic stem cells, and Tie2-expressing monocytes/macrophages (TEMs), known for their proangiogenic properties. Loss of α6 expression in α6fl/fl­Tie2Cre(+) mice reduced tumor growth in a murine B16F10 melanoma model. Immunohistological analysis of the tumors showed that Tie2-dependent α6 gene deletion was associated with reduced tumor vascularization and with reduced infiltration of proangiogenic Tie2-expressing macrophages. These findings demonstrate that α6 integrin subunit plays a major role in tumor angiogenesis and TEM infiltration. Targeting α6 could be used as a strategy to reduce tumor growth.

Mechanistic study of the proangiogenic effect of osteoprotegerin.

Osteoprotegerin (OPG), a soluble tumour necrosis factor receptor superfamily member, inhibits RANKL-mediated osteoclastogenesis. We have previously reported that OPG enhances the proangiogenic properties of endothelial colony-forming cells (ECFCs) in vitro, and promotes vasculogenesis in vivo. Here we investigated how OPG promotes neovascularisation. Proteomic experiments showed that OPG pretreatment affected ECFCs protein expression in two ways, 23 spots being down-regulated and 6 upregulated. These spots corresponded to proteins involved in cell motility, adhesion, signal transduction and apoptosis. In keeping with these proteomic results, we found that OPG induced ECFCs adhesion to activated endothelium in shear stress conditions, promoting intermediate but not focal adhesion to fibronectin and collagen. Treatment with OPG induced a reorganization of the ECFCs cytoskeleton, with the emergence of cell protrusions characteristic of a migratory phenotype. These effects correlated with decreased FAK phosphorylation and enhanced integrin αVß3 expression. OPG drastically reduced caspase-3/7 activities and maintained ECFCs viability after 48 h of treatment. All these effects were significantly attenuated by ECFCs incubation with the CXCR4 antagonist AMD-3100, and by prior heparan sulphate proteoglycan disruption. The proangiogenic properties of OPG appeared to be mediated by the proteoglycan syndecan-1, although OPG 1-194 lacking its heparin-binding domain still had pro-vasculogenic effects in vitro and in vivo. These results suggest that OPG may interact with ECFCs by binding to HSPGs/syndecan-1, thereby induce an anti-adhesive effect and promoting ECFCs migration through a SDF-1/CXCR4 dependent pathway.

Tie2-dependent knockout of α6 integrin subunit in mice reduces post-ischaemic angiogenesis.

AIMS: Integrins α6ß1 and α6ß4 are receptors for laminins, the main components of the basement membrane underlying the endothelial cells. In vitro, α6 integrin subunit (α6) expression at the surface of endothelial cells and their progenitors (EPCs) is up-regulated by pro-angiogenic growth factors and is crucial for adhesion, migration, and pseudotube formation. We investigated the role for α6 in post-ischaemic vascular repair in vivo. METHODS AND RESULTS: We used the cre-lox system to generate a mouse line with specific α6 gene deletion in Tie2-lineage cells. In a model of hind-limb ischaemia, Tie2-dependent α6 deletion reduced neovessel formation and reperfusion of the ischaemic limb. Concerning the role for α6 in post-ischaemic vasculogenesis, we showed previously that α6 was required for EPC recruitment at the site of ischaemia. Here, we found that α6 deletion also reduced EPC mobilization from the bone marrow after ischaemia. Examination of the ischaemic muscles showed that Tie2-dependent α6 deletion decreased the recruitment of pro-angiogenic Tie2-expressing macrophages. In the Matrigel plug assay, fibroblast growth factor-2-induced vascularization was diminished in mice lacking endothelial α6. To specifically investigate the role for α6 in angiogenesis, aortic rings were embedded in Matrigel or collagen and cultured ex vivo. In Matrigel, neovessel outgrowth from rings lacking α6 was strongly diminished, whereas no genotype-dependent difference occurred for rings in collagen. CONCLUSION: α6 plays a major role in both post-ischaemic angiogenesis and vasculogenesis.

alpha6-integrin subunit plays a major role in the proangiogenic properties of endothelial progenitor cells.

OBJECTIVE: Alpha6 integrin subunit (alpha6) expression is increased by proangiogenic growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor. This increase correlates with enhanced in vitro tube formation by endothelial cells and their progenitors called endothelial colony-forming cells (ECFCs). We thus studied the role of alpha6 in vasculogenesis induced by human ECFCs, in a mouse model of hindlimb ischemia. METHODS AND RESULTS: We used small interfering RNA (siRNA) to inhibit alpha6 expression on the surface of ECFCs. For in vivo studies, human ECFCs were injected intravenously into a nude mouse model of unilateral hind limb ischemia. Transfection with siRNA alpha6 abrogated neovessel formation and reperfusion of the ischemic hind limb induced by ECFCs (P<0.01 and P<0.001, respectively). It also inhibited ECFC incorporation into the vasculature of the ischemic muscle (P<0.001). In vitro, siRNA alpha6 inhibited ECFC adhesion (P<0.01), pseudotube formation on Matrigel, migration, and AKT phosphorylation (P<0.0001), with no effect on cell proliferation or apoptosis. CONCLUSIONS: alpha6 Expression is required for ECFC migration, adhesion, recruitment at the site of ischemia, and the promotion of the postischemic vascular repair. Thus, we have demonstrated a major role of alpha6 in the proangiogenic properties of ECFCs.

Platelet microparticles: a potential predictive factor of survival in hormone-refractory prostate cancer patients treated with docetaxel-based chemotherapy.

BACKGROUND: Several studies suggest a causal relationship between platelet activation and cancer metastasis. Activated platelet microparticles (PMPs) release vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), which play a major role in angiogenesis. OBJECTIVE: We conducted a prospective, nonrandomised, single-centre study in hormone-refractory prostate cancer (HRPC) patients to determine the impact of PMPs on the outcome. DESIGN, SETTING, AND PARTICIPANTS: Eligible chemonaive and metastatic HRPC patients received docetaxel-based chemotherapy and a low dose of prednisone. INTERVENTION: PMPs in whole blood were quantified before the start of chemotherapy through flow cytometry using an anti-CD41a monoclonal antibody, and plasma VEGF and bFGF were determined with an enzyme-linked immunosorbent assay. MEASUREMENTS: The primary end point was to evaluate the impact of the PMPs on overall survival (OS). We also studied the statistical interaction between PMPs and platelets and their relationship with OS. The median PMP value was used to sort patients into two groups. RESULTS AND LIMITATIONS: Data of 43 consecutive HRPC patients treated in a single French centre were analysed. Significant correlations were observed between Eastern Cooperative Oncology Group performance status (ECOG PS), platelets, and PMP level. The median OS was significantly shorter for patients with >6867 PMPs per microl of whole blood than for those with lower values (16.7 vs 26.4 mo, p=0.013). A significant relationship was found between OS and PMPs, whereas a statistical interaction term between PMPs and platelets was significantly associated with OS (p=0.019). No association was found between OS and plasma VEGF and bFGF. In the multivariate analysis, only baseline prostate-specific antigen (PSA) and ECOG PS remained significantly predictive of risk of death. CONCLUSIONS: In HRPC patients, PMPs and their interaction with platelets were predictive of outcome. A biologic association between PMPs and the OS of HRPC patients, independent of chemotherapy regimen, should be demonstrated by confirmatory prospective studies.

Ex vivo priming of endothelial progenitor cells with SDF-1 before transplantation could increase their proangiogenic potential.

OBJECTIVE: As SDF-1 and its cognate receptor CXCR4 play a key role in the survival and mobilization of immature cells, we examined whether preconditioning of endothelial progenitor cells (EPCs) with SDF-1 could further promote their capacity to enhance angiogenesis. METHODS AND RESULTS: EPC exposure to 100 ng/mL SDF-1 for 30 min induced a proangiogenic phenotype, with cell migration and differentiation into vascular cords in Matrigel and increased their therapeutic potential in a nude mouse model of hindlimb ischemia. This pretreatment enhanced EPC adhesion to activated endothelium in physiological conditions of blood flow by stimulating integrin-mediated EPCs binding to endothelial cells. Pretreated EPCs showed significantly upregulated surface alpha4 and alphaM integrin subunit expression involved in the homing of immature cells to a neovasculature and enhanced FGF-2 and promatrix metalloproteinase (MMP)-2 secretion. All these effects were significantly attenuated by EPC incubation with AMD-3100, a CXCR4 antagonist, by prior HSPGs disruption and by HUVEC incubation with anti-intercellular adhesion molecule1 (ICAM-1) and anti-vascular cell adhesion molecule (VCAM) blocking antibodies. Pretreated EPCs adhered very rapidly (within minutes) and were resistant to shear stresses of up to 2500 s(-1). CONCLUSIONS: SDF-1 pretreatment during EPC expansion stimulates EPC adhesion to endothelial cells and thus augments the efficiency of cell therapy for ischemic vascular diseases.

Thromboxane synthase mutations in an increased bone density disorder (Ghosal syndrome).

Studying consanguineous families with Ghosal hematodiaphyseal dysplasia syndrome (GHDD), a disorder of increased bone density, we identified mutations in TBXAS1, which encodes thromboxane synthase (TXAS). TXAS, an enzyme of the arachidonic acid cascade, produces thromboxane A(2) (TXA(2)). Platelets from subjects with GHDD showed a specific deficit in arachidonic acid-produced aggregation. We also found that TXAS and TXA(2) modulated expression of TNFSF11 and TNFRSF11B (encoding RANKL and osteoprotegerin (OPG), respectively) in primary cultured osteoblasts.

A two adenine insertion polymorphism in the 3' untranslated region of factor VII gene is associated with peripheral arterial disease but not with venous thrombosis. Results of case-control studies.

Polymorphic variants of genes encoding blood coagulation proteins have been extensively studied as risk factors for venous or arterial thrombosis A variation in the 3' untranslated region (UTR) involved in the post-transcriptional regulation of factor VII (FVII) gene has been recently identified, a two adenine insertion/deletion at nucleotide 11293. In this study, we investigated its effect on the risk of thrombosis in the frame of two case-control studies, including patients suffering from peripheral arterial disease (PAD) or venous thromboembolic (VTE) disease. The 3'UTR FVII gene polymorphism was investigated i) in 181 patients who had symptomatic atherosclerotic disease of the lower limbs, ii) in 178 patients who had had at least one episode of objectively diagnosed deep venous thrombosis and iii) in controls matched for age and sex. Plasma FVII antigen (FVII: Ag) levels were lower in the presence of the 3'UTR 2A insertion (68.4 +/- 12.3%, 81.3 +/- 14.5% and 89.5 +/- 13.7% in 2A/2A, 2A/0 and 0/0 subjects respectively, p < 0.0001). No significant relationship was found with VTE disease. In the contrary we observed a lower risk of PAD for the 2A/2A compared to the 0/0 genotype after adjustment for traditional risk factors (hypercholesterolemia, smoking status, diabetes and hypertension), with an OR of 0.24 [95% CI 0.06-0.99]. In conclusion, the 2 adenine insertion in the 3'UTR of FVII gene, related to lower plasma FVII levels, is a genetic variation that may contribute to reduce the risk of PAD.

Low-molecular-weight fucoidan enhances the proangiogenic phenotype of endothelial progenitor cells.

Endothelial progenitor cell (EPC) transplantation is a potential means of inducing neovascularization in vivo. However, the number of circulating EPC is relatively small, it may thus be necessary to enhance their proangiogenic properties ex vivo prior to injection in vivo. Fucoidan has previously been shown to potentiate in vitro tube formation by mature endothelial cells in the presence of basic fibroblast growth factor (FGF-2). We therefore examined whether fucoidan, alone or combined with FGF-2, could increase EPC proangiogenic potency in vitro. EPC exposure to 10 microg/ml fucoidan induced a proangiogenic phenotype, including cell proliferation (p < 0.01) and migration (p < 0.01); moreover, differentiation into vascular cords occurred in the presence of FGF-2 (p < 0.01). This latter effect correlated with upregulation of the cell-surface #alpha6 integrin subunit of the laminin receptor (p < 0.05). Compared to untreated HUVEC, untreated EPC #alpha6 expression and adhesion to laminin were enhanced two-fold. Fucoidan treatment further enhanced HUVEC but not EPC adhesion to laminin. These results show that fucoidan enhances the proangiogenic properties of EPC and suggest that ex vivo fucoidan preconditioning of EPC might lead to increased neovascularization when injected into ischemic tissues.

Effect of an oversulfated exopolysaccharide on angiogenesis induced by fibroblast growth factor-2 or vascular endothelial growth factor in vitro.

The aim of this study was to determine the angiogenic properties of an oversulfated exopolysaccharide (OS-EPS) derived from a polysaccharide secreted by the mesophilic bacterium Alteromonas infernus. We compared the effect of this OS-EPS with that of a non-oversulfated exopolysaccharide (EPS) on human umbilical vein endothelial cell (HUVEC) proliferation, migration and differentiation induced by basic fibroblast growth factor (FGF-2) or vascular endothelial growth factor (VEGF). OS-EPS enhanced HUVEC proliferation by 58% when used alone, and by respectively 30% and 70% in the presence of FGF-2 and VEGF. OS-EPS also increased the density of tubular structures on Matrigel in the presence of FGF-2 or VEGF. Vascular tube formation was related to alpha(6) integrin subunit expression, which was enhanced by 50% in the presence of the growth factors. Indeed, a monoclonal anti-alpha(6) blocking antibody abolished this vascular tube formation. EPS had no effect in any of the experimental conditions, underlying the importance of sulfation in the angiogenic effects of exopolysaccharide. By potentiating the angiogenic activity of FGF-2 and/or VEGF, OS-EPS, which possesses low anticoagulant activity and thus a low hemorrhagic risk, could potentially be used to accelerate vascular wound healing or to promote the growth of collateral blood vessels in ischemic tissues.

Low molecular weight fucoidan and heparin enhance the basic fibroblast growth factor-induced tube formation of endothelial cells through heparan sulfate-dependent alpha6 overexpression.

Basic fibroblast growth factor (FGF-2) activates its high-affinity receptors (FGFRs) but also acts through interaction with heparan sulfate proteoglycans (HSPG). Exogenous polysaccharides also modulate the angiogenic activity of FGF-2. We investigated the effect and mechanism of action of a low molecular weight fucoidan derivative (LMWF) on tube formation by human endothelial cells. LMWF has a better arterial antithrombotic potential in animals than low molecular weight heparin (LMWH). After stimulation of human umbilical vein endothelial cells (HUVEC) by FGF-2 and LMWF (or LMWH), we observed 1) using flow cytometry, an increase in the amount of the alpha6 integrin subunit; 2) using quantitative reverse transcription-polymerase chain reaction, an increase in alpha6 mRNA (higher with LMWF than with LMWH); and 3) using a Matrigel model, an increase in vascular tube formation (also higher with LMWF than with LMWH). A direct link between alpha6 overexpression and vascular tube formation was confirmed by use of an anti-alpha6 antibody: in its presence, there was no capillary network formation on Matrigel. Unexpectedly, an anti-FGFR blocking antibody had no effect on alpha6 over-expression, whereas stripping off the heparan sulfate with heparitinases abolished overexpression. Overall, our data suggest that FGF-2 stimulates alpha6 over-expression in HUVEC, through HSPG but independently from FGFR, and that LMWF (or LMWH) modulates this interaction. Expression of heparan sulfate proteoglycan increases after ischemic injury. Given its antithrombotic properties and its ability to potentiate tube formation of endothelial cells, LMWF may have to be considered for revascularization of ischemic areas.

A fucosylated chondroitin sulfate from echinoderm modulates in vitro fibroblast growth factor 2-dependent angiogenesis.

Fucosylated chondroitin sulfate (FucCS), a glycosaminoglycan obtained from sea cucumber, has the same structure as mammalian chondroitin sulfate, but some of the glucuronic acid residues display sulfated fucose branches. This new polysaccharide has a more favorable effect than heparin on vascular cell growth. It inhibits smooth muscle cell proliferation as heparin, and it has a potent enhancing effect on endothelial cell proliferation and migration in the presence of heparin-binding growth factors. We now extend our studies to the effect of this glycosaminoglycan on endothelial cells to an in vitro angiogenesis model on Matrigel. FucCS, in the presence of fibroblast growth factor-2 (FGF-2), strongly increases the capacity of endothelial cells to form vascular tubes on Matrigel with a well-organized capillary-like network and typical closed structures. Comparison between the activity of native and chemically modified chondroitin sulfate from sea cucumber reveals that the sulfated fucose branches are the structural motif for the proangiogenic activity. Heparin does not induce angiogenesis in this experimental model. We also have evidence for the proposition that endothelial cell proliferation is not the sole event involved in the in vitro FGF-2-induced angiogenesis. It implies a variety of other modifications of the endothelial cells and of their interaction with the extracellular matrix, such as integrin expression and actin cytoskeleton reorganization. Finally, the proangiogenic effect of FucCS, concomitant with its capacity to prevent venous and arterial thrombosis, in animal models makes this new glycosaminoglycan a promising molecule with possible beneficial effects in pathological conditions affecting blood vessels such as the neovascularization of ischemic areas.

Effect of fucoidan on fibroblast growth factor-2-induced angiogenesis in vitro.

Fucoidans are sulfated polysaccharides extracted from brown marine algae. A purified fucoidan fraction exhibits the same venous antithrombotic activity as heparin in rabbits, but with a lower anticoagulant effect. Because of its heparin-like structure, we postulated that fucoidan might modulate heparin-binding angiogenic growth factor activity. We thus studied its effect, at antithrombotic concentrations, on fibroblast growth factor (FGF)-2-induced proliferation and differentiation of human umbilical vein endothelial cells. The fucoidan effect on endothelial cell differentiation was evaluated by studying the expression of surface proteins (i.e. integrin, adhesion molecule) known to be modulated by FGF-2 and involved in angiogenesis, and by quantifying closed areas delimited by vascular tubes formed on reconstituted basement membrane. Fucoidan had no modulatory effect on the mitogenic activity of FGF-2, but significantly increased tubular structure density induced by FGF-2. Fucoidan alone increased alpha(6) integrin subunit expression with only partially organized tubular structure. In the presence of FGF-2, fucoidan enhanced alpha(6), beta(1) and PECAM-1 and inhibited alpha(v)beta(3) integrin expression. Heparin had no effect in these systems. The most striking effect of fucoidan was observed on alpha(6) expression and tube formation was abolished by monoclonal anti-alpha(6) antibodies. Fucoidan plus FGF-2 effect on alpha(6) expression was markedly decreased by monoclonal anti-FGF-2 antibodies, indicating that fucoidan acts mainly via FGF-2. These results show that, at antithrombotic concentrations, contrary to heparin, fucoidan can enhance vascular tube formation induced by FGF-2 with a modulation of the expression of surface proteins (mainly alpha(6)) involved in angiogenesis.