DP2, a Carbohydrate Derivative, Enhances In Vitro Osteoblast Mineralisation.

Bone fracture healing is a complex biological process involving four phases coordinated over time: hematoma formation, granulation tissue formation, bony callus formation, and bone remodelling. Bone fractures represent a significant health problem, particularly among the elderly population and patients with comorbidities. Therapeutic strategies proposed to treat such fractures include the use of autografts, allografts, and tissue engineering strategies. It has been shown that bone morphogenetic protein 2 (BMP-2) has a therapeutic potential to enhance fracture healing. Despite the clinical efficacy of BMP-2 in osteoinduction and bone repair, adverse side effects and complications have been reported. Therefore, in this in vitro study, we propose the use of a disaccharide compound (DP2) to improve the mineralisation process. We first evaluated the effect of DP2 on primary human osteoblasts (HOb), and then investigated the mechanisms involved. Our findings showed that (i) DP2 improved osteoblast differentiation by inducing alkaline phosphatase activity, osteopontin, and osteocalcin expression; (ii) DP2 induced earlier in vitro mineralisation in HOb cells compared to BMP-2 mainly by earlier activation of Runx2; and (iii) DP2 is internalized in HOb cells and activates the protein kinase C signalling pathway. Consequently, DP2 is a potential therapeutical candidate molecule for bone fracture repair.

Dietary AhR Ligands Have No Anti-Fibrotic Properties in TGF-ß1-Stimulated Human Colonic Fibroblasts.

BACKGROUND: Intestinal fibrosis is a common complication in inflammatory bowel disease (IBD) patients without specific treatment. Aryl hydrocarbon receptor (AhR) activation is associated with better outcomes in intestinal inflammation. Development of novel therapies targeting fibrogenic pathways is required and we aimed to screen dietary AhR ligands for their anti-fibrotic properties in TGF-ß1-stimulated human colonic fibroblast cells. METHODS: The study was conducted using TGF-ß1-stimulated CCD-18Co, a human colonic fibroblast cell line in response to increased concentrations of dietary ligands of AhR such as FICZ, ITE, L-kynurenine and curcumin. Fibrosis markers such as α-SMA, COL1A1, COL3A1 and CTGF were assessed. AhR and ANRT RNA were evaluated. RESULTS: TGF-ß1 at 10 ng/mL significantly induced mRNA levels for ECM-associated proteins such as CTGF, COL1A1 and COL3A1 in CCD-18Co cells. FICZ from 10 to 1000 nM, L-kynurenine from 0.1 to 10 µM, ITE from 1 to 100 µM or curcumin from 5 to 20 µM had no significant effect on fibrosis markers in TGF-ß1-induced CCD-18Co. CONCLUSIONS: Our data highlight that none of the tested dietary AhR ligands had an effect on fibrosis markers in TGF-ß1-stimulated human colonic fibroblast cells in our experimental conditions. Further studies are now required to identify novel potential targets in intestinal fibrosis.

Extracellular Vesicles From LPS-Treated Macrophages Aggravate Smooth Muscle Cell Calcification by Propagating Inflammation and Oxidative Stress.

Background: Vascular calcification (VC) is a cardiovascular complication associated with a high mortality rate among patients with diseases such as atherosclerosis and chronic kidney disease. During VC, vascular smooth muscle cells (VSMCs) undergo an osteogenic switch and secrete a heterogeneous population of extracellular vesicles (EVs). Recent studies have shown involvement of EVs in the inflammation and oxidative stress observed in VC. We aimed to decipher the role and mechanism of action of macrophage-derived EVs in the propagation of inflammation and oxidative stress on VSMCs during VC. Methods: The macrophage murine cell line RAW 264.7 treated with lipopolysaccharide (LPS-EK) was used as a cellular model for inflammatory and oxidative stress. EVs secreted by these macrophages were collected by ultracentrifugation and characterized by transmission electron microscopy, cryo-electron microscopy, nanoparticle tracking analysis, and the analysis of acetylcholinesterase activity, as well as that of CD9 and CD81 protein expression by western blotting. These EVs were added to a murine VSMC cell line (MOVAS-1) under calcifying conditions (4 mM Pi-7 or 14 days) and calcification assessed by the o-cresolphthalein calcium assay. EV protein content was analyzed in a proteomic study and EV cytokine content assessed using an MSD multiplex immunoassay. Results: LPS-EK significantly decreased macrophage EV biogenesis. A 24-h treatment of VSMCs with these EVs induced both inflammatory and oxidative responses. LPS-EK-treated macrophage-derived EVs were enriched for pro-inflammatory cytokines and CAD, PAI-1, and Saa3 proteins, three molecules involved in inflammation, oxidative stress, and VC. Under calcifying conditions, these EVs significantly increase the calcification of VSMCs by increasing osteogenic markers and decreasing contractile marker expression. Conclusion: Our results show that EVs derived from LPS-EK-treated-macrophages are able to induce pro-inflammatory and pro-oxidative responses in surrounding cells, such as VSMCs, thus aggravating the VC process.

Dietary salt exacerbates intestinal fibrosis in chronic TNBS colitis via fibroblasts activation.

Intestinal fibrosis is a frequent complication in inflammatory bowel diseases (IBD). It is a challenge to identify environmental factors such as diet that may be driving this risk. Intestinal fibrosis result from accumulation of extracellular matrix (ECM) proteins secreted by myofibroblasts. Factors promoting intestinal fibrosis are unknown, but diet appears to be a critical component in its development. Consumption of salt above nutritional recommendations can exacerbate chronic inflammation. So far, high salt diet (HSD) have not been thoroughly investigated in the context of intestinal fibrosis associated to IBD. In the present study, we analyze the role of dietary salt in TNBS chronic colitis induced in rat, an intestinal fibrosis model, or in human colon fibroblast cells. Here, we have shown that high-salt diet exacerbates undernutrition and promoted ECM-associated proteins in fibroblasts. Taken together, our results suggested that dietary salt can activate intestinal fibroblasts, thereby contributing to exacerbation of intestinal fibrosis. Dietary salt may be considered as a putative environmental factor that drives intestinal fibrosis risk.

Effects of Chronic Kidney Disease and Uremic Toxins on Extracellular Vesicle Biology.

Vascular calcification (VC) is a cardiovascular complication associated with a high mortality rate, especially in patients with diabetes, atherosclerosis or chronic kidney disease (CKD). In CKD patients, VC is associated with the accumulation of uremic toxins, such as indoxyl sulphate or inorganic phosphate, which can have a major impact in vascular remodeling. During VC, vascular smooth muscle cells (VSMCs) undergo an osteogenic switch and secrete extracellular vesicles (EVs) that are heterogeneous in terms of their origin and composition. Under physiological conditions, EVs are involved in cell-cell communication and the maintenance of cellular homeostasis. They contain high levels of calcification inhibitors, such as fetuin-A and matrix Gla protein. Under pathological conditions (and particularly in the presence of uremic toxins), the secreted EVs acquire a pro-calcifying profile and thereby act as nucleating foci for the crystallization of hydroxyapatite and the propagation of calcification. Here, we review the most recent findings on the EVs' pathophysiological role in VC, the impact of uremic toxins on EV biogenesis and functions, the use of EVs as diagnostic biomarkers and the EVs' therapeutic potential in CKD.

Host Starvation and Female Sex Influence Enterobacterial ClpB Production: A Possible Link to the Etiology of Eating Disorders.

Altered signaling between gut bacteria and their host has recently been implicated in the pathophysiology of eating disorders, whereas the enterobacterial caseinolytic protease B (ClpB) may play a key role as an antigen mimetic of α-melanocyte-stimulating hormone, an anorexigenic neuropeptide. Here, we studied whether ClpB production by gut bacteria can be modified by chronic food restriction and female sex, two major risk factors for the development of eating disorders. We found that food restriction increased ClpB DNA in feces and ClpB protein in plasma in both male and female rats, whereas females displayed elevated basal ClpB protein levels in the lower gut and plasma as well as increased ClpB-reactive immunoglobulins (Ig)M and IgG. In contrast, direct application of estradiol in E. coli cultures decreased ClpB concentrations in bacteria, while testosterone had no effect. Thus, these data support a mechanistic link between host-dependent risk factors of eating disorders and the enterobacterial ClpB protein production.

GFOGER Peptide Modifies the Protein Content of Extracellular Vesicles and Inhibits Vascular Calcification.

OBJECTIVE: Vascular calcification (VC) is an active process during which vascular smooth muscle cells (VSMCs) undergo an osteogenic switch and release extracellular vesicles (EVs). In turn, the EVs serve as calcification foci via interaction with type 1 collagen (COL1). We recently showed that a specific, six-amino-acid repeat (GFOGER) in the sequence of COL1 was involved in the latter's interaction with integrins expressed on EVs. Our main objective was to test the GFOGER ability to inhibit VC. APPROACH: We synthesized the GFOGER peptide and tested its ability to inhibit the inorganic phosphate (Pi)-induced calcification of VSMCs and aortic rings. Using mass spectrometry, we studied GFOGER's effect on the protein composition of EVs released from Pi-treated VSMCs. RESULTS: Calcification of mouse VSMCs (MOVAS-1 cells), primary human VSMCs, and rat aortic rings was lower in the presence of GFOGER than with Pi alone (with relative decreases of 66, 58, and 91%, respectively; p < 0.001 for all) (no effect was observed with the scramble peptide GOERFG). A comparative proteomic analysis of EVs released from MOVAS-1 cells in the presence or absence of Pi highlighted significant differences in EVs' protein content. Interestingly, the expression of some of the EVs' proteins involved in the calcification process (such as osteogenic markers, TANK-binding kinase 1, and casein kinase II) was diminished in the presence of GFOGER peptide (data are available via ProteomeXchange with identifier PXD018169∗). The decrease of osteogenic marker expression observed in the presence of GFOGER was confirmed by q-RT-PCR analysis. CONCLUSION: GFOGER peptide reduces vascular calcification by modifying the protein content of the subsequently released EVs, in particular by decreasing osteogenicswitching in VSMCs.