ObjectiveClinical studies demonstrated a potential link between atherosclerosis and periodontitis. Porphyromonas gingivalis (Pg), one of the main periodontal pathogen, has been associated to atheromatous plaque worsening. However, synergism between infection and other endothelial stressors such as oxidized-LDL or TNF-α especially on endothelial cell (EC) death has not been investigated. This study aims to assess the role of Pg on EC death in an inflammatory context and to determine potential molecular pathways involved.MethodsHuman umbilical vein ECs (HUVECs) were infected with Pg (MOI 100) or stimulated by its lipopolysaccharide (Pg-LPS) (1μg/ml) for 24 to 48 hours. Cell viability was measured with AlamarBlue test, type of cell death induced was assessed using Annexin V/propidium iodide staining. mRNA expression regarding caspase-1, -3, -9, Bcl-2, Bax-1 and Apaf-1 has been evaluated with RT-qPCR. Caspases enzymatic activity and concentration of APAF-1 protein were evaluated to confirm mRNA results.ResultsPg infection and Pg-LPS stimulation induced EC death. A cumulative effect has been observed in Ox-LDL pre-treated ECs infected or stimulated. This effect was not observed in TNF-α pre-treated cells. Pg infection promotes EC necrosis, however, in infected Ox-LDL pre-treated ECs, apoptosis was promoted. This effect was not observed in TNF-α pre-treated cells highlighting specificity of molecular pathways activated. Regarding mRNA expression, Pg increased expression of pro-apoptotic genes including caspases-1,-3,-9, Bax-1 and decreased expression of anti-apoptotic Bcl-2. In Ox-LDL pre-treated ECs, Pg increased significantly the expression of Apaf-1. These results were confirmed at the protein level.ConclusionThis study contributes to demonstrate that Pg and its Pg-LPS could exacerbate Ox-LDL and TNF-α induced endothelial injury through increase of EC death. Interestingly, molecular pathways are differentially modulated by the infection in function of the pre-stimulation.

Alteration of the extracellular matrix (ECM) is one of the major events in the pathogenesis of brain lesions following ischemic stroke. Heparan sulfate mimetics (HSm) are synthetic pharmacologically active polysaccharides that promote ECM remodeling and tissue regeneration in various types of lesions. HSm bind to growth factors, protect them from enzymatic degradation and increase their bioavailability, which promotes tissue repair. As the ECM is altered during stroke and HSm have been shown to restore the ECM, we investigated the potential of HSm4131 (also named RGTA-4131®) to protect brain tissue and promote regeneration and plasticity after a stroke.Methods: Ischemic stroke was induced in rats using transient (1 h) intraluminal middle cerebral artery occlusion (MCAo). Animals were assigned to the treatment (HSm4131; 0.1, 0.5, 1.5, or 5 mg/kg) or vehicle control (saline) groups at different times (1, 2.5 or 6 h) after MCAo. Brain damage was assessed by MRI for the acute (2 days) and chronic (14 days) phases post-occlusion. Functional deficits were evaluated with a battery of sensorimotor behavioral tests. HSm4131-99mTc biodistribution in the ischemic brain was analyzed between 5 min and 3 h following middle cerebral artery reperfusion. Heparan sulfate distribution and cellular reactions, including angiogenesis and neurogenesis, were evaluated by immunohistochemistry, and growth factor gene expression (VEGF-A, Ang-2) was quantified by RT-PCR.Results: HSm4131, administered intravenously after stroke induction, located and remained in the ischemic hemisphere. HSm4131 conferred long-lasting neuroprotection, and significantly reduced functional deficits with no alteration of physiological parameters. It also restored the ECM, and increased brain plasticity processes, i.e., angiogenesis and neurogenesis, in the affected brain hemisphere.Conclusion: HSm represent a promising ECM-based therapeutic strategy to protect and repair the brain after a stroke and favor functional recovery.

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Sickle cell anemia (SCA) is a chronic illness that causes an increased risk of stroke and progressive brain and cognitive dysfunction. SCA-related cerebral vasculopathy includes vascular remodeling, abnormal arterial velocities and infarction. We studied the relationship between cytokines, velocities, and blood parameters in SCA-children enrolled in the "Drepagreffe" trial, a French prospective, Mendelian-randomized trial with 2 arms (transfusions/transplantation) defined by random-availability of a HLA-matched sibling. This trial enrolled SCA-children younger than 15, regularly transfused for abnormal-TCD history, with at least one non-SCA sibling, and parents agreeing to HLA-typing and transplantation. Between 12/2010 and 6/2013, 67 SCA-children (7 with stroke history) were enrolled. Thirty-two had a matched-sibling donor (MSD) and were transplanted, while 35 (no donor) were included in the transfusion arm. Hypoxia/angiogenesis and brain injury-related factor expression at 1-year was one of the trial secondary outcome. Elevated plasma BDNF and PDGF-AA have been shown to be significantly associated with high cerebral velocities (Hyacinth 2012). Chronic transfusion has been shown to reduce vascular endothelial activation and thrombogenicity in SCA-children with abnormal-TCD (Hyacinth 2014) but no study has been performed in transplanted SCA-children. Plasma samples were obtained at enrollment and 1-year post-enrollment and stored frozen. The expression of the following cytokines (VEGF, Ang-1, Ang-2, FGFb, HGF, PDGF-BB, BDNF) was assessed with a multiplex immunoassay (Bio-Techne). Ang-2, and BDNF levels were confirmed with specific enzyme-linked immunosorbent assays (ELISA, Bio-Techne). Blood parameters, velocities, ischemic lesions and stenoses were assessed at enrollment and 1-year post-enrollment. At 1-year, the percentage of patients with normalized-TCD (velocities<170cm/sec) was significantly higher in transplanted patients than in those maintained on chronic transfusion (27/32 (84%) vs 17/35 (49%), respectively; p=0.001). As shown (Table), leukocytes, neutrophils, platelets, reticulocytes, LDH, bilirubin, ferritin, HbS% were highly significantly lower in transplanted children than in those maintained on chronic transfusion, while hemoglobin and HbA% were highly significantly higher. Ang-2 and HGF were significantly lower in transplanted children than in those on chronic transfusion (p<0.001 and p=0.002, respectively). Velocities recorded in the artery with the highest values were significantly positively correlated with Ang-2 (r=0.385, p=0.015) and BDNF (r=0.444, p=0.005). Logistic regression analysis showed that TCD-normalization was significantly associated with the transplantation arm (OR=5.72 (95%CI:1.79-18.27); p=0.003). High hemoglobin (OR=1.49 per 1g/dL increase; 95%CI: 1.08-2.06; p=0.014) and HbA% (OR=1.05 per1% increase; 95%CI: 1.01-1.10; p=0.014) were significantly positively associated with TCD-normalization, but not independently. Higher levels of Ang-2 (OR=0.51 per 1 pg/mL increase, 95%CI:0.29-0.91; p=0.023) and BDNF (OR=0.69 per 1 pg/mL increase, 95%CI:0.50-0.94; p=0.02) were negatively and independently significantly associated with TCD-normalization. Multivariate analysis, also including the treatment arm, showed that BDNF remained an independent risk factor for a lack of TCD-normalization (OR=0.65, 95%CI:0.45-0.92: p=0.017). This study confirms the association between high levels of BDNF and high velocities, and suggests that transplantation increases the likelihood of TCD-normalization compared to transfusion, and is associated with reduced Ang-2 expression in plasma, which may reflect improved brain oxygenation. Disclosures Thuret: Addmedica: Research Funding; bluebird bio: Research Funding; Novartis: Research Funding. Pondarré:Addmedica: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Blue Bird Bio: Honoraria. Bernaudin:AddMedica: Honoraria; Pierre fabre: Research Funding; Cordons de Vie: Research Funding; BlueBirdBio: Consultancy.