Porphyromonas gingivalis Participates in Pathogenesis of Human Abdominal Aortic Aneurysm by Neutrophil Activation. Proof of Concept in Rats

International audienceBACKGROUND: Abdominal Aortic Aneurysms (AAAs) represent a particular form of atherothrombosis where neutrophil proteolytic activity plays a major role. We postulated that neutrophil recruitment and activation participating in AAA growth may originate in part from repeated episodes of periodontal bacteremia. METHODS AND FINDINGS: Our results show that neutrophil activation in human AAA was associated with Neutrophil Extracellular Trap (NET) formation in the IntraLuminal Thrombus, leading to the release of cell-free DNA. Human AAA samples were shown to contain bacterial DNA with high frequency (11/16), and in particular that of Porphyromonas gingivalis (Pg), the most prevalent pathogen involved in chronic periodontitis, a common form of periodontal disease. Both DNA reflecting the presence of NETs and antibodies to Pg were found to be increased in plasma of patients with AAA. Using a rat model of AAA, we demonstrated that repeated injection of Pg fostered aneurysm development, associated with pathological characteristics similar to those observed in humans, such as the persistence of a neutrophil-rich luminal thrombus, not observed in saline-injected rats in which a healing process was observed. CONCLUSIONS: Thus, the control of periodontal disease may represent a therapeutic target to limit human AAA progression

Synthesis and activation of an iron oxide immobilized drug-mimicking reporter under conventional and pulsed X-ray irradiation conditions

International audienceAn efficient nano-sized delivery system is presented here allowing the immobilized, picolinium-tethered organic ligand to be released by X-ray irradiation. A marked difference was observed in the fragmentation efficiency by using conventional Cs-137 vs. pulsed sources

Leukocyte mimetic polysaccharide microparticles tracked in vivo on activated endothelium and in abdominal aortic aneurysm

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USPIO–PEG nanoparticles functionalized with a highly specific collagen-binding peptide: a step towards MRI diagnosis of fibrosis

International audienceFibrosis is characterized by a pathologic deposition of collagen I, leading to impaired function of organs. Tissue biopsy is the gold standard method for the diagnosis of fibrosis but this is an invasive procedure, subject to sampling errors. Several non-invasive techniques such as magnetic resonance imaging (MRI) using non-specific probes have been developed but they are not fully satisfying as they allow diagnosis at a late stage. In this study, collagelin, a collagen-binding peptide has been covalently linked using click chemistry to pegylated Ultra Small Super Paramagnetic Iron Oxide Nanoparticles (USPIO–PO–PEG–collagelin NPs) with the aim of diagnosing fibrosis at an early stage by MRI. USPIO–PO–PEG–collagelin NPs showed a high affinity for collagen I, two times higher than that of free collagelin whereas not peptide labeled USPIO NPs (USPIO–PO–PEG-yne) did not present any affinity. NPs were not toxic for macrophages and fibroblasts. Diffusion through collagen hydrogels concentrated at 3 and 10 mg mL−1 revealed a large accumulation of USPIO–PO–PEG–collagelin NPs within the collagen network after 72 hours, ca. 3 times larger than that of unlabeled USPIO, thereby evidencing the specific targeting of collagen I. Moreover, the quantity of USPIO–PO–PEG–collagelin NPs accumulated within hydrogels was proportional to the collagen concentration. Subsequently, the NPs diffusion through collagen hydrogels was monitored by MRI. The MRI T2 time relaxation decreased much more significantly with depth for USPIO–PO–PEG–collagelin NPs compared to unlabeled ones. Taken together, these results show that USPIO–PEG–collagelin NPs are promising as effective MRI nanotracers for molecular imaging of fibrosis at an early stage

Bimodal Fucoidan-Coated Zinc Oxide/Iron Oxide-Based Nanoparticles for the Imaging of Atherothrombosis

A polyol method was used to obtain ultrasmall ZnO nanoparticles (NPs) doped with iron ions and coated with a low molecular weight fucoidan in order to perform in vivo MR and ex vivo fluorescence imaging of athrothrombosis. During the synthesis, the early elimination of water by azeotropic distillation with toluene allowed us to produce NPs which size, determined by XRD and TEM, decreased from 7 nm to 4 nm with the increase of iron/zinc ratios from 0.05 to 0.50 respectively. For the highest iron content (NP-0.50) NPs were evidenced as a mixture of nanocrystals made of wurtzite and cubic phase with a molar ratio of 2.57:1, although it was not possible to distinguish one from the other by TEM. NP-0.50 were superparamagnetic and exhibited a large emission spectrum at 470 nm when excited at 370 nm. After surface functionalization of NP-0.50 with fucoidan (fuco-0.50), the hydrodynamic size in the physiological medium was 162.0 ± 0.4 nm, with a corresponding negative zeta potential of −48.7 ± 0.4 mV, respectively. The coating was evidenced by FT-IR spectra and thermogravimetric analysis. Aqueous suspensions of fuco-0.50 revealed high transverse proton relaxivities (T2) with an r2 value of 173.5 mM−1 s−1 (300 K, 7.0 T) and remained stable for more than 3 months in water or in phosphate buffer saline without evolution of the hydrodynamic size and size distribution. No cytotoxic effect was observed on human endothelial cells up to 48 h with these NPs at a dose of 0.1 mg/mL. After injection into a rat model of atherothrombosis, MR imaging allowed the localization of diseased areas and the subsequent fluorescence imaging of thrombus on tissue slices

Detection of bacteria in human AAA samples.

<p>(A) Endotoxin levels from gram-negative bacteria were quantified in the conditioned medium of the Intra-luminal thrombus (ILT) and the arterial wall of AAA (n = 16) and control aortas (n = 10) using the Limulus Amebocyte Lysate chromogenic assay kit. *p<0.05; **p<0.01 (Mann-Whitney Analysis). DNA was extracted from the ILT and associated arterial wall before amplification by PCR using a ubiquitous set of primers targeting bacterial 16S rRNA (B) or <i>Pg</i> 16S rRNA (C) gene. Amplification products were separated by electrophoresis in a 1% agarose gel.</p

<i>P. gingivalis</i> (<i>Pg</i>) promotes NET formation.

<p>Freshly isolated human neutrophils were plated on Lab-tek® chamber slides and then stimulated or not by f-MLP (100 nM), a bacterial peptide used as a positive control, or by <i>Pg</i> (1.10⁷ CFU) for 2 hours at 37°C. (A) Immunofluorescence detection of histone H1 (red) and citrullinated histone H4 (green) was performed without permeabilization. (B) Cell-free DNA (cf-DNA) concentration was determined in the culture medium of neutrophils stimulated or not either with f-MLP (100 nM) or increasing concentrations of Pg. *p<0.05; **p<0.01 <i>vs</i> ctl (Mann-Whitney analysis). The trapping of Pg (red) by externalized nucleosomes (histone H1, green) was visualized by epifluorescence (C) and confocal microscopy (D). The bottom panel represents a virtual section constructed according to the Z axis, confirming the intracellular presence of <i>Pg</i> subsequent to phagocytosis by neutrophils.</p

<i>P. gingivalis</i> (<i>Pg</i>) infection promoted neutrophil recruitment, NET formation and inhibited healing.

<p>(A) Hematoxylin/Eosin staining showing the presence of a thrombus and neutrophil accumulation at its luminal pole (right, inset) in <i>Pg</i>-infected rats. The presence of mesenchymatous cells is observed in saline-injected rats (left, inset). (B) Masson's trichrome staining. Fibrosis associated with healing is observed in green in saline-injected rats whereas red staining highlights the presence of a thrombus in <i>Pg</i>-infected rats. (C) Immunostaining for histone H1 (red), nuclei appear in blue (DAPI). Merged images show the presence of extracellular H1 associated with disorganized DNA (inset), but also intact neutrophils characterized by their multilobed nuclei (bottom, right).</p

Increased MMP9 activity and MPO released by AAA samples of <i>P. gingivalis</i> (<i>Pg</i>) infected rats.

<p>(A) gelatin zymography analysis of saline- and <i>Pg</i>-injected rats (respectively rats R1,2,3 and R4,5,6). MW: molecular weight, Ref: reference containing pro- and active MMP-9. Graphs represent spatial density quantification of pro- and active MMP9 lysis areas (Image J software). (B) MPO concentration was determined by ELISA in conditioned medium and in plasma. *p<0.05, **p<0.01 (Mann-Whitney Analysis).</p

Cell-free DNA (Cf-DNA) and MPO-DNA complexes are increased in rats infected with <i>P. gingivalis</i> (<i>Pg</i>).

<p>(A) Concentration of cf-DNA released by the AAA segment (thrombus + wall) of saline- or <i>Pg</i>-infected rats or by the thoracic aorta (T. Ao) from <i>Pg</i>-infected rats. Cf-DNA was also quantified in plasma. (B) Quantification of MPO-DNA complexes in conditioned medium or plasma of saline-or <i>Pg</i>-injected rats. **p<0.01; ***p<0.0001 (Mann-Whitney analysis). The correlation between AAA diameter and Cf-DNA concentration was determined by the Least Squares method.</p