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High cut-off dialysis mitigates pro-calcific effects of plasma on vascular progenitor cells.

ABSTRACT: Mortality of patients with end-stage renal disease tremendously exceeds that of the general population due to excess cardiovascular morbidity. Large middle-sized molecules (LMM) including pro-inflammatory cytokines are major drivers of uremic cardiovascular toxicity and cannot be removed sufficiently by conventional high-flux (HFL) hemodialysis. We tested the ability of plasma from 19 hemodialysis patients participating in a trial comparing HFL with high cut-off (HCO) membranes facilitating removal of LMM to induce calcification in mesenchymal stromal cells (MSC) functioning as vascular progenitors. HCO dialysis favorably changed plasma composition resulting in reduced pro-calcific activity. LMM were removed more effectively by HCO dialysis including FGF23, a typical LMM we found to promote osteoblastic differentiation of MSC. Protein-bound uremic retention solutes with known cardiovascular toxicity but not LMM inhibited proliferation of MSC without direct toxicity in screening experiments. We could not attribute the effect of HCO dialysis on MSC calcification to distinct mediators. However, we found evidence of sustained reduced inflammation that might parallel other anti-calcifying mechanisms such as altered generation of extracellular vesicles. Our findings imply protection of MSC from dysfunctional differentiation by novel dialysis techniques targeted at removal of LMM. HCO dialysis might preserve their physiologic role in vascular regeneration and improve outcomes in dialysis patients.

SUBMITTER: Schaub T

PROVIDER: S-EPMC7807056 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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High cut-off dialysis mitigates pro-calcific effects of plasma on vascular progenitor cells.

Schaub Theres T Janke Daniel D Zickler Daniel D Lange Claudia C Girndt Matthias M Schindler Ralf R Dragun Duska D Hegner Björn B

Scientific reports 20210113 1

Mortality of patients with end-stage renal disease tremendously exceeds that of the general population due to excess cardiovascular morbidity. Large middle-sized molecules (LMM) including pro-inflammatory cytokines are major drivers of uremic cardiovascular toxicity and cannot be removed sufficiently by conventional high-flux (HFL) hemodialysis. We tested the ability of plasma from 19 hemodialysis patients participating in a trial comparing HFL with high cut-off (HCO) membranes facilitating remo ...[more]

PMID: 33441772

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Project description:BackgroundCalcific tendonitis of the rotator cuff is due to carbonated apatite deposits in the shoulder tendons. During the evolution of the disease, an acute inflammatory episode may occur leading to the disappearance of the calcification. Although hydroxyapatite crystal-induced inflammation has been previously studied with synthetic crystals, no data are available with calcifications extracted from patients suffering from calcific tendinopathy. The objective of the study was to explore the inflammatory properties of human calcifications and the pathways involved.MethodsHuman calcifications and synthetic hydroxyapatite were used in vitro to stimulate human monocytes and macrophages, the human myeloid cell line THP-1, and human tenocytes. The release of IL-1β, IL-6, and IL-8 by cells was quantified by ELISA. The gene expression of pro- and anti-inflammatory cytokines was evaluated by quantitative PCR. NF-kB activation and NLRP3 involvement were assessed in THP-1 cells using a NF-kB inhibitor and a caspase-1 inhibitor. The inflammatory properties were then assessed in vivo using a mouse air pouch model.ResultsHuman calcifications were able to induce a significant release of IL-1β when incubated with monocytes, macrophages, and THP-1 only if they were first primed with LPS (monocytes and macrophages) or PMA (THP-1). Stimulation of THP-1 by human calcifications led to similar levels of IL-1β when compared to synthetic hydroxyapatite although these levels were significantly inferior in monocytes and macrophages. The patient's crystals enhanced mRNA expression of pro-IL-1β, as well as IL-18, NF-kB, and TGFβ when IL-6 and TNFα expression were not. IL-1β production was reduced by the inhibition of caspase-1 indicating the role of NLRP3 inflammasome. In vivo, injection of human calcifications or synthetic hydroxyapatite in the air pouch led to a significant increase in membrane thickness although significant overexpression of IL-1β was only observed for synthetic hydroxyapatite.ConclusionsAs synthetic hydroxyapatite, human calcifications were able to induce an inflammatory response resulting in the production of IL-1β after NF-kB activation and through NLRP3 inflammasome. In some experiments, IL-1β induction was lower with human calcifications compared to synthetic apatite. Differences in size, shape, and protein content may explain this observation.

Project description:BACKGROUND:The intimal endothelium is known to condition the underlying medial smooth muscle cell (SMC) layer of the vessel wall, and is highly responsive to receptor-activator of nuclear factor-?B ligand (RANKL) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), pro-calcific and anti-calcific agents, respectively. In this paper, we tested the hypothesis that RANKL-induced activation of endothelial NF-?B signalling is essential for pro-calcific activation of the underlying SMCs. METHODS:For these studies, human aortic endothelial and smooth muscle cell mono-cultures (HAECs, HASMCs) were treated with RANKL (0-25?ng/ml?±?5?ng/ml TRAIL) for 72?h. Non-contact transwell HAEC:HASMC co-cultures were also employed in which the luminal HAECs were treated with RANKL (± 5?ng/ml TRAIL), followed by analysis of pro-calcific markers in the underlying subluminal HASMCs. RESULTS:Treatment of either HAECs or HASMCs with RANKL activated the non-canonical NF-?B/p52 and canonical NF-?B/p65 pathways in both cell types. In RANKL?±?TRAIL-treated HAECs, recombinant TRAIL, previously demonstrated by our group to strongly attenuate the pro-calcific signalling effects of RANKL, was shown to specifically block the RANKL-mediated activation of non-canonical NF-?B/p52, clearly pointing to the mechanistic relevance of this specific pathway to RANKL function within endothelial cells. In a final series of HAEC:HASMC transwell co-culture experiments, RANKL treatment of HAECs that had been genetically silenced (via siRNA) for the NF-?B2 gene (the molecular forerunner to NF-?B/p52 generation) exhibited strongly attenuated pro-calcific activation of underlying HASMCs relative to scrambled siRNA controls. SUMMARY:These in vitro observations provide valuable mechanistic insights into how RANKL may potentially act upon endothelial cells through activation of the alternative NF-?B pathway to alter endothelial paracrine signalling and elicit pro-calcific responses within underlying vascular smooth muscle cells.

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High cut-off dialysis mitigates pro-calcific effects of plasma on vascular progenitor cells.

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High cut-off dialysis mitigates pro-calcific effects of plasma on vascular progenitor cells.

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