Dominique Helley

Marine Polysaccharides: A Source of Bioactive Molecules for Cell Therapy and Tissue Engineering

The therapeutic potential of natural bioactive compounds such as polysaccharides, especially glycosaminoglycans, is now well documented, and this activity combined with natural biodiversity will allow the development of a new generation of therapeutics. Advances in our understanding of the biosynthesis, structure and function of complex glycans from mammalian origin have shown the crucial role of this class of molecules to modulate disease processes and the importance of a deeper knowledge of structure-activity relationships. Marine environment offers a tremendous biodiversity and original polysaccharides have been discovered presenting a great chemical diversity that is largely species specific. The study of the biological properties of the polysaccharides from marine eukaryotes and marine prokaryotes revealed that the polysaccharides from the marine environment could provide a valid alternative to traditional polysaccharides such as glycosaminoglycans. Marine polysaccharides present a real potential for natural product drug discovery and for the delivery of new marine derived products for therapeutic applications.

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.

Increased VEGFR2 expression during human late endothelial progenitor cells expansion enhances in vitro angiogenesis with up-regulation of integrin α6

In vitro expansion of late endothelial progenitor cells (EPCs) might yield a cell therapy product useful for myocardial and leg ischaemia, but the influence of EPC expansion on the angiogenic properties of these cells is unknown. In the present study, we investigated the effect of in vitro EPC expansion on vascular endothelial growth factor (VEGF) receptor expression. EPCs were obtained from CD34+ cord blood cells and expanded for up to 5 weeks. Real‐time quantitative reverse‐transcription polymerase chain reaction (RT‐PCR) showed that VEGFR2 expression, contrary to VEGFR1 and VEGFR3 expression, was significantly higher on expanded EPCs than on freshly isolated CD34+ cells or on human umbilical vein endothelial cells (HUVECs). Quantitative flow cytometry confirmed that VEGFR2 density on EPCs increased during the expansion process and was significantly higher than on HUVECs. The impact of VEGFR2 increase was studied on the three theoretical steps of angiogenesis, i.e., EPC proliferation, migration and differentiation. VEGFR2 up‐regulation had no effect on VEGF‐induced cell proliferation, but significantly enhanced EPC migration and pseudotubes formation dependent on integrin α6 subunit overexpression. In vitro expansion of late EPCs increases the expression of VEGFR2, the main VEGF receptor, with possible implications for EPC‐based angiogenic therapy.

Evaluation of hemostasis and endothelial function in patients with paroxysmal nocturnal hemoglobinuria receiving eculizumab

Background Paroxysmal nocturnal hemoglobinuria (PNH) is associated with an increased risk of thrombosis through unknown mechanisms. Design and Methods We studied 23 patients with PNH, before and after five and 11 weeks of treatment with eculizumab. We examined markers of thrombin generation and reactional fibrinolysis (prothrombin fragment 1+2 (F1+2), D-dimers, and plasmin antiplasmin complexes (P-AP), and endothelial dysfunction tissue plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1), soluble thrombomodulin (sTM), intercellular adhesion molecule 1 (sICAM-1), vascular cell adhesion molecule (sVCAM-1), endothelial microparticles (EMPs), and tissue factor pathway inhibitor (TFPI). Results At baseline, vWF, sVCAM-1, the EMP count, and F1+2 and D-dimer levels were significantly elevated in the patients, including those with no history of clinical thrombosis. Treatment with eculizumab was associated with significant decreases in plasma markers of coagulation activation (F1+2, P=0.012, and D-dimers, P=0.01), and reactional fibrinolysis (P-AP, P=0.0002). Eculizumab treatment also significantly reduced plasma markers of endothelial cell activation (t-PA, P=0.0005, sVCAM-1, P<0.0001, and vWF, P=0.0047) and total (P=0.0008) and free (P=0.0013) TFPI plasma levels. Conclusions Our results suggest a new understanding of the contribution of endothelial cell activation to the pathogenesis of thrombosis in PNH. The terminal complement inhibitor, eculizumab, induced a significant and sustained decrease in the activation of both the plasma hemostatic system and the vascular endothelium, likely contributing to the protective effect of eculizumab on thrombosis in this setting.

Successful use of eculizumab in a patient with post‐transplant thrombotic microangiopathy

Transplantation-associated thrombotic microangiopathy (TA-TMA) represents a challenge after allogeneic haematopoietic stem cell transplantation (HSCT) because of diagnostic uncertainties, lack of established treatment, and an overall poor prognosis (Laskin et al, 2011). We report the case of a 61-year-old man who was diagnosed with multiple myeloma in 2009. He was initially treated with a combination of bortezomib, doxorubicin and dexamethasone, followed by highdose melphalan with autologous HSCT. Following disease relapse in 2011, he received three courses of bortezomib, thalidomide dexamethasone, and was then included in a sequential autologous-allogeneic tandem approach that comprised high-dose melphalan and auto-HSCT followed by two Gray total body irradiation-conditioned allo-HSCT (Karlin et al, 2011). Two months post-HSCT, the patient was diagnosed with graft-versus-host disease (GvHD) of the skin (stage 3) and gut (stage 1), which responded to methylprednisone 2 mg/kg. Three months later, GvHD of the gut reappeared during steroid tapering. Although the GvHD responded to the increased doses of steroids, the patient developed a severe TA-TMA (Fig 1). As already reported in such a situation, acute renal failure was absent (Cho et al, 2010). A concomitant diagnosis of cytomegalovirus (CMV) & Epstein Barr virus (EBV) infections was made. Ciclosporin was stopped, treatment with Foscanet was initiated and the patient received one injection of mabthera. Seven days later, he developed a progressive involvement of the central nervous system, with confusion and peripheral facial paralysis, while the biological characteristics of TA-TMA remained stable (Fig 1). Ciclosporin was no longer detectable in the blood. CMV & EBV infections responded to treatment at that time. Magnetic resonance imaging and a lumbar puncture were normal. Complement proteins (C3, C4, Factor H, Factor I) were in the normal ranges. Testing for anti-complement Factor H antibodies was negative. No decrease in ADAMTS 13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) activity was identified. Given the devastating prognosis, we administered eculizumab to this patient accordingly to atypical haemolytic and uraemic syndrome dosage (900 mg weekly for four weeks, followed by 1200 mg every two weeks). The patient’s neurological status improved dramatically within 48 h after the first infusion of eculizumab. Clinical improvement was associated with rapid normalization of disease activity markers: platelet counts increased, and lactate dehydrogenase levels decreased quickly (Fig 1). Complete C5 blockage, defined by a 50% haemolytic complement (CH50) activity below 10% was observed 24 h after the first infusion of eculizumab and during the entire study period. Concomitantly, the number of circulating endothelial cells (CEC), a known prognostic marker in thrombotic microangiopathy (Erdbruegger et al, 2006), decreased drastically. The levels of CEC were about 1200/ml before eculizumab infusion compared with 512/ml at 36 h and 5/ml at 3 days post-injection. The patient was discharged two weeks later with normal neurological status. Three months later, eculizumab treatment was stopped because there were no signs of TMA. After a 3-month follow-up period, the patient had completely recovered and continues to do very well. Given the progressive course of the disease after the mabthera injection, it seemed unlikely that the recovery was due to this treatment. The rapid clinical response to eculizumab supports the concept that TA-TMA might involve aberrant and autonomous complement activation despite the control of the potential causes (calcineurin inhibitors, GvHD & herpes virus infections). The dramatic resolution of symptoms after eculizumab administration suggests that TA-TMA is an area deserving further careful investigation of therapeutic complement blockade.

α6-Integrin Subunit Plays a Major Role in the Proangiogenic Properties of Endothelial Progenitor Cells

Objective—Alpha6 integrin subunit (&agr;6) 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 &agr;6 in vasculogenesis induced by human ECFCs, in a mouse model of hindlimb ischemia. Methods and Results—We used small interfering RNA (siRNA) to inhibit &agr;6 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 &agr;6 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 &agr;6 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. Conclusion—&agr;6 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 &agr;6 in the proangiogenic properties of ECFCs.

Endothelial dysfunction in systemic lupus patients with low disease activity: evaluation by quantification and characterization of circulating endothelial microparticles, role of anti-endothelial cell antibodies

OBJECTIVE We attempted to evaluate endothelial dysfunction and the role of AECAs in systemic lupus (SL) with low disease activity. We quantified endothelial microparticles (EMps) and attempted to find the best flow cytometry method for that purpose. METHODS CD105, CD144 and CD146 were tested, individually or in combination, on EMp-enriched plasma. Twenty-three healthy blood donors and 27 SL patients were evaluated. SL patients with a SLEDAI <10 (median 2.6) were evaluated in our outpatient clinic. For each patient, EMps (CD105-CD146(+), CD45(-)) and AECAs were quantified and characterized. RESULTS The monochrome composite marker CD105-CD146 appeared to be the most efficient in detecting EMps. SL patients had more circulating EMps than healthy donors: respective median values were 2575 and 130 EMps/microl (P < 0.001). SL patients had more CD54(+) and CD54(-) EMps than healthy donors (496 vs 34 EMps CD54(+)/microl, P < 0.0001; 1875 vs 89 EMps CD54(-)/microl, P < 0.0001). The ratio CD54(+) EMps/total EMps was lower for lupus patients than for healthy individuals (20.3 vs 33.7%, P = 0.03). Twenty-four patients (89%) were positive for AECAs. EMp counts were not significantly higher for patients with AECAs. CONCLUSION Monochrome composite marker is efficient in detecting the whole population of EMps using flow cytometry. SL patients with low disease activity have a marked endothelial dysfunction. EMps released from the endothelium originate from activated and non-activated cells. AECAs do not seem to be the main cause of endothelial dysfunction in this population.

Synergy of Radiotherapy and a Cancer Vaccine for the Treatment of HPV-Associated Head and Neck Cancer

There is growing interest in the association of radiotherapy and immunotherapy for the treatment of solid tumors. Here, we report an extremely effective combination of local irradiation (IR) and Shiga Toxin B (STxB)–based human papillomavirus (HPV) vaccination for the treatment of HPV-associated head and neck squamous cell carcinoma (HNSCC). The efficacy of the irradiation and vaccine association was tested using a model of HNSCC obtained by grafting TC-1/luciferase cells at a submucosal site of the inner lip of immunocompetent mice. Irradiation and the STxB-E7 vaccine acted synergistically with both single and fractionated irradiation schemes, resulting in complete tumor clearance in the majority of the treated mice. A dose threshold of 7.5 Gy was required to elicit the dramatic antitumor response. The combined treatment induced high levels of tumor-infiltrating, antigen-specific CD8+ T cells, which were required to trigger the antitumor activity. Treatment with STxB-E7 and irradiation induced CD8+ T-cell memory, which was sufficient to exert complete antitumor responses in both local recurrences and distant metastases. We also report for the first time that a combination therapy based on local irradiation and vaccination induces an increased pericyte coverage (as shown by αSMA and NG2 staining) and ICAM-1 expression on vessels. This was associated with enhanced intratumor vascular permeability that correlated with the antitumor response, suggesting that the combination therapy could also act through an increased accessibility for immune cells. The combination strategy proposed here offers a promising approach that could potentially be transferred into early-phase clinical trials. Mol Cancer Ther; 14(6); 1336–45. ©2015 AACR.

Effect of Low Molecular Weight Fucoidan and Low Molecular Weight Heparin in a Rabbit Model of Arterial Thrombosis

Background: Therapeutic use of unfractionated heparin and low molecular weight heparins (LMWHs) is limited by hemorrhagic adverse effects. We compared the antithrombotic effect of LMW fucoidan (LMWF) and LMWH in an experimental model. Methods: Thrombosis was induced in femoral arteries of male New Zealand White rabbits by in situ induction of endothelial apoptosis with staurosporine (10–5M for 30 min). Starting the day before apoptosis induction, the animals received subcutaneous LMWF (15 mg/kg), LMWH (enoxaparin 2.5 mg/kg) or saline solution (control group) twice a day for 4 days. Results: The degrees of apoptosis and endothelial denudation were similar in the 3 groups. The thrombotic score was significantly lower in the LMWF group than in the LMWH and control groups (p = 0.01). Tissue factor expression was significantly lower in the LMWF group than in the control and LMWH groups (p = 0.01). The plasma concentration of tissue factor pathway inhibitor was significantly increased after LMWF injection (137 ± 28 vs. 102 ± 17; p = 0.01), whereas no change was observed after LMWH treatment. LMWF did not prolong the bleeding time or decrease platelet aggregation. Conclusions: LMWF appeared to be more effective than LMWH for preventing arterial thrombosis in this experimental model. LMWF also had a lower hemorrhagic risk than LMWH.

Factors V Leiden and II 20210A in patients with symptomatic pulmonary embolism and deep vein thrombosis

PURPOSE Factor V Leiden and factor II 20210A are inherited disorders of the clotting system that occur frequently in patients with deep vein thrombosis. We conducted this study to determine whether these factors are also common in patients with pulmonary embolism. SUBJECTS AND METHODS We determined the prevalence of factor V Leiden and factor II 20210A in 773 consecutive patients with objectively documented symptomatic deep vein thrombosis or symptomatic pulmonary embolism, or with a combination of these disorders. RESULTS Isolated symptomatic deep vein thrombosis occurred in 345 patients; isolated symptomatic pulmonary embolism occurred in 236; and both anomalies occurred in 192. Factor V Leiden was present in 21 (9%) of the patients with isolated symptomatic pulmonary embolism, in 30 (16%) with both manifestations, and in 63 (18%) with isolated symptomatic deep vein thrombosis (P = 0.007). Factor V Leiden was more common among patients with deep vein thrombosis (odds ratio [OR] = 2.1; 95% confidence interval [CI]: 1.2 to 3.7; P = 0.006) or both pulmonary embolism and deep vein thrombosis (OR = 1.8; 95% CI: 1.0 to 3.3; P = 0.07) than among patients with isolated pulmonary embolism. Factor V Leiden was less common in massive pulmonary embolism (5% [7 of 127]) than in submassive pulmonary embolism (13% [21 of 155], P = 0.03). We found no significant difference in the prevalence of factor II 20210A among the three groups. CONCLUSION Factors V Leiden and II 20210A vary in prevalence among patients with pulmonary embolism and deep vein thrombosis, suggesting that the risk of pulmonary embolization may vary among patients who have different causes of venous thromboses.