Expanded Hemodialysis Therapy Ameliorates Uremia-Induced Systemic Microinflammation and Endothelial Dysfunction by Modulating VEGF, TNF-α and AP-1 Signaling

Systemic chronic microinflammation and altered cytokine signaling, with adjunct cardiovascular disease (CVD), endothelial maladaptation and dysfunction is common in dialysis patients suffering from end-stage renal disease and associated with increased morbidity and mortality. New hemodialysis filters might offer improvements. We here studied the impact of novel improved molecular cut-off hemodialysis filters on systemic microinflammation, uremia and endothelial dysfunction. Human endothelial cells (ECs) were incubated with uremic serum obtained from patients treated with two different hemodialysis regimens in the Permeability Enhancement to Reduce Chronic Inflammation (PERCI-II) crossover clinical trial, comparing High-Flux (HF) and Medium Cut-Off (MCO) membranes, and then assessed for their vascular endothelial growth factor (VEGF) production and angiogenesis. Compared to HF membranes, dialysis with MCO membranes lead to a reduction in proinflammatory mediators and reduced endothelial VEGF production and angiogenesis. Cytokine multiplex screening identified tumor necrosis factor (TNF) superfamily members as promising targets. The influence of TNF-alpha and its soluble receptors (sTNF-R1 and sTNF-R2) on endothelial VEGF promoter activation, protein release, and the involved signaling pathways was analyzed, revealing that this detrimental signaling was indeed induced by TNF-alpha and mediated by AP-1/c-FOS signaling. In conclusion, uremic toxins, in particular TNF-signaling, promote endothelial maladaptation, VEGF expression and aberrant angiogenesis, which can be positively modulated by dialysis with novel MCO membranes

Editorial: Mesenchymal stromal cell therapy for regenerative medicine

Mesenchymal stromal/stem cell (MSC) therapies are increasingly explored as novel regenerative and immunomodulatory approaches to treat or prevent diseases (Pittenger et al., 2019; Hmadcha et al., 2020; Moll et al., 2020b; Ringdén et al., 2022). These cells exhibit potent paracrine properties that can modulate host immune responses, lower inflammation, and orchestrate endogenous tissue repair, at both the local and the systemic level through multiple pathways (Singer and Caplan, 2011; Doorn et al., 2012). MSCs possess tropism toward damaged and inflamed tissues, where they can engraft short-term and exert their therapeutic effects by both direct and indirect mechanisms (Doorn et al., 2012; Galipeau and Sensebe, 2018; Soria et al., 2019). MSC products can be prepared from multiple sources (Moll et al., 2019, 2022), rapidly expanded and biobanked for clinical application. All these advantages make this cell type a versatile tool in regenerative medicine. The goal of our Research Topic is to highlight the latest advances in applications of MSCs for the treatment of a variety of diseases and their modes of action (MoA). A better understanding of the mechanisms underlying the therapeutic effect of MSCs can provide crucial insight into innovative strategies to enhance their effectiveness in clinical application (Singer and Caplan, 2011; Doorn et al., 2012; Galipeau and Sensebe, 2018; Moll et al., 2019, 2020b, 2022; Pittenger et al., 2019; Ringdén et al., 2022). The subjects covered within this Research Topic include: (a) Therapeutic application of MSCs for major clinical indications, (b) Cellular and molecular mechanisms underlying therapeutic effects of MSCs, and (c) Strategies for enhancement of the therapeutic effects of MSCs and their products. Here, we summarize the 37 manuscripts that were submitted to this Research Topic (Figure 1)

From acute injury to chronic disease: pathophysiological hypothesis of an epithelial/mesenchymal crosstalk alteration in CKD

Observational clinical studies link acute kidney injury to chronic kidney disease (CKD) progression. The pathophysiological mechanisms that underlie this process are currently unknown but recently published papers suggest that tubular epithelial cells and interstitial mesenchymal cells emerge as a single unit, and their integrity alteration as a whole might lead to renal fibrosis and CKD. The present article reviews the biological findings supporting the hypothesis of an altered epithelial/mesenchymal crosstalk in fibrosis development and progression toward CK

Effects of Atrazine exposure on human bone marrow-derived mesenchymal stromal cells assessed by combinatorial assay matrix

IntroductionMesenchymal Stromal/Stem cells (MSCs) are an essential component of the regenerative and immunoregulatory stem cell compartment of the human body and thus of major importance in human physiology. The MSCs elicit their beneficial properties through a multitude of complementary mechanisms, which makes it challenging to assess their phenotype and function in environmental toxicity screening. We here employed the novel combinatorial assays matrix approach/technology to profile the MSC response to the herbicide Atrazine, which is a common environmental xenobiotic, that is in widespread agricultural use in the US and other countries, but banned in the EU. Our here presented approach is representative for screening the impact of environmental xenobiotics and toxins on MSCs as an essential representative component of human physiology and well-being.MethodsWe here employed the combinatorial assay matrix approach, including a panel of well standardized assays, such as flow cytometry, multiplex secretome analysis, and metabolic assays, to define the phenotype and functionality of human-donor-derived primary MSCs exposed to the representative xenobiotic Atrazine. This assay matrix approach is now also endorsed for characterization of cell therapies by leading regulatory agencies, such as FDA and EMA.ResultsOur results show that the exposure to Atrazine modulates the metabolic activity, size, and granularity of MSCs in a dose and time dependent manner. Intriguingly, Atrazine exposure leads to a broad modulation of the MSCs secretome (both upregulation and downmodulation of certain factors) with the identification of Interleukin-8 as the topmost upregulated representative secretory molecule. Interestingly, Atrazine attenuates IFNγ-induced upregulation of MHC-class-II, but not MHC-class-I, and early phosphorylation signals on MSCs. Furthermore, Atrazine exposure attenuates IFNγ responsive secretome of MSCs. Mechanistic knockdown analysis identified that the Atrazine-induced effector molecule Interleukin-8 affects only certain but not all the related angiogenic secretome of MSCs.DiscussionThe here described Combinatorial Assay Matrix Technology identified that Atrazine affects both the innate/resting and cytokine-induced/stimulated assay matrix functionality of human MSCs, as identified through the modulation of selective, but not all effector molecules, thus vouching for the great usefulness of this approach to study the impact of xenobiotics on this important human cellular subset involved in the regenerative healing responses in humans

Multi-Parameter Analysis of Biobanked Human Bone Marrow Stromal Cells Shows Little Influence for Donor Age and Mild Comorbidities on Phenotypic and Functional Properties

Heterogeneous populations of human bone marrow-derived stromal cells (BMSC) are among the most frequently tested cellular therapeutics for treating degenerative and immune disorders, which occur predominantly in the aging population. Currently, it is unclear whether advanced donor age and commonly associated comorbidities affect the properties of ex vivo-expanded BMSCs. Thus, we stratified cells from adult and elderly donors from our biobank (n = 10 and n = 13, mean age 38 and 72 years, respectively) and compared their phenotypic and functional performance, using multiple assays typically employed as minimal criteria for defining multipotent mesenchymal stromal cells (MSCs). We found that BMSCs from both cohorts meet the standard criteria for MSC, exhibiting similar morphology, growth kinetics, gene expression profiles, and pro-angiogenic and immunosuppressive potential and the capacity to differentiate toward adipogenic, chondrogenic, and osteogenic lineages. We found no substantial differences between cells from the adult and elderly cohorts. As positive controls, we studied the impact of in vitro aging and inflammatory cytokine stimulation. Both conditions clearly affected the cellular properties, independent of donor age. We conclude that in vitro aging rather than in vivo donor aging influences BMSC characteristics

Freezing Medium Containing 5% DMSO Enhances the Cell Viability and Recovery Rate After Cryopreservation of Regulatory T Cell Products ex vivo and in vivo

Cell therapies have significant therapeutic potential in diverse fields including regenerative medicine, transplantation tolerance, and autoimmunity. Within these fields, regulatory T cells (Treg) have been deployed to ameliorate aberrant immune responses with great success. However, translation of the cryopreservation strategies employed for other cell therapy products, such as effector T cell therapies, to Treg therapies has been challenging. The lack of an optimized cryopreservation strategy for Treg products presents a substantial obstacle to their broader application, particularly as administration of fresh cells limits the window available for sterility and functional assessment. In this study, we aimed to develop an optimized cryopreservation strategy for our CD4+CD25+Foxp3+ Treg clinical product. We investigate the effect of synthetic or organic cryoprotectants including different concentrations of DMSO on Treg recovery, viability, phenotype, cytokine production, suppressive capacity, and in vivo survival following GMP-compliant manufacture. We additionally assess the effect of adding the extracellular cryoprotectant polyethylene glycol (PEG), or priming cellular expression of heat shock proteins as strategies to improve viability. We find that cryopreservation in serum-free freezing medium supplemented with 10% human serum albumin and 5% DMSO facilitates improved Treg recovery and functionality and supports a reduced DMSO concentration in Treg cryopreservation protocols. This strategy may be easily incorporated into clinical manufacture protocols for future studies

Expanded Hemodialysis ameliorates uremia-induced impairment of vasculoprotective KLF2 and concomitant proinflammatory priming of endothelial cells through an ERK/AP1/cFOS-dependent mechanism

AimsExpanded hemodialysis (HDx) therapy with improved molecular cut-off dialyzers exerts beneficial effects on lowering uremia-associated chronic systemic microinflammation, a driver of endothelial dysfunction and cardiovascular disease (CVD) in hemodialysis (HD) patients with end-stage renal disease (ESRD). However, studies on the underlying molecular mechanisms are still at an early stage. Here, we identify the (endothelial) transcription factor Krüppel-like factor 2 (KLF2) and its associated molecular signalling pathways as key targets and regulators of uremia-induced endothelial micro-inflammation in the HD/ESRD setting, which is crucial for vascular homeostasis and controlling detrimental vascular inflammation.Methods and resultsFirst, we found that human microvascular endothelial cells (HMECs) and other typical endothelial and kidney model cell lines (e.g. HUVECs, HREC, and HEK) exposed to uremic serum from patients treated with two different hemodialysis regimens in the Permeability Enhancement to Reduce Chronic Inflammation II (PERCI-II) crossover clinical trial - comparing High-Flux (HF) and Medium Cut-Off (MCO) membranes - exhibited strongly reduced expression of vasculoprotective KLF2 with HF dialyzers, while dialysis with MCO dialyzers led to the maintenance and restoration of physiological KLF2 levels in HMECs. Mechanistic follow-up revealed that the strong downmodulation of KLF2 in HMECs exposed to uremic serum was mediated by a dominant engagement of detrimental ERK instead of beneficial AKT signalling, with subsequent AP1-/c-FOS binding in the KLF2 promoter region, followed by the detrimental triggering of pleiotropic inflammatory mediators, while the introduction of a KLF2 overexpression plasmid could restore physiological KLF2 levels and downmodulate the detrimental vascular inflammation in a mechanistic rescue approach.ConclusionUremia downmodulates vasculoprotective KLF2 in endothelium, leading to detrimental vascular inflammation, while MCO dialysis with the novel improved HDx therapy approach can maintain physiological levels of vasculoprotective KLF2

RESTORE Survey on the Public Perception of Advanced Therapies and ATMPs in Europe — Why the European Union Should Invest More!

Advanced therapy medicinal products (ATMPs) are potential game changers in modern medical care with an anticipated major impact for patients and society. They are a new drug class often referred to as "living drugs," and are based on complex components such as vectors, cells and even tissues. The production of such ATMPs involves innovative biotechnological methods. In this survey, we have assessed the perception of European citizens regarding ATMPs and health care in Europe, in relation to other important topics, such as safety and security, data protection, climate friendly energy supply, migration, and others. A crucial question was to determine to what extent European citizens wish to support public funding of innovations in healthcare and reimbursement strategies for ATMPs. To answer this, we conducted an online survey in 13 European countries (representative of 85.3% of the entire EU population including the UK in 2020), surveying a total of 7,062 European citizens. The survey was representative with respect to adult age groups and gender in each country. Healthcare had the highest ranking among important societal topics. We found that 83% of the surveyed EU citizens were in support of more public funding of technologies in the field of ATMPs. Interestingly, 74% of respondents are in support of cross-border healthcare for patients with rare diseases to receive ATMP treatments and 61% support the reimbursement of very expensive ATMPs within the European health care system despite the current lack of long-term efficacy data. In conclusion, healthcare is a top ranking issue for European Citizens, who additionally support funding of new technologies to enable the wider application of ATMPs in Europe

Angiogenic Role of Mesothelium-Derived Chemokine CXCL1 During Unfavorable Peritoneal Tissue Remodeling in Patients Receiving Peritoneal Dialysis as Renal Replacement Therapy

Peritoneal dialysis (PD) is a valuable 'home treatment' option, even more so during the ongoing Coronavirus pandemic. However, the long-term use of PD is limited by unfavourable tissue remodelling in the peritoneal membrane, which is associated with inflammation-induced angiogenesis. This appears to be driven primarily through vascular endothelial growth factor (VEGF), while the involvement of other angiogenic signaling pathways is still poorly understood. Here, we have identified the crucial contribution of mesothelial cell-derived angiogenic CXC chemokine ligand 1 (CXCL1) to peritoneal angiogenesis in PD. CXCL1 expression and peritoneal microvessel density were analysed in biopsies obtained by the International Peritoneal Biobank (NCT01893710 at www.clinicaltrials.gov), comparing 13 children with end-stage kidney disease before initiating PD to 43 children on chronic PD. The angiogenic potential of mesothelial cell-derived CXCL1 was assessed in vitro by measuring endothelial tube formation of human microvascular endothelial cells (HMECs) treated with conditioned medium from human peritoneal mesothelial cells (HPMCs) stimulated to release CXCL1 by treatment with either recombinant IL-17 or PD effluent. We found that the capillary density in the human peritoneum correlated with local CXCL1 expression. Both CXCL1 expression and microvessel density were higher in PD patients than in the age-matched patients prior to initiation of PD. Exposure of HMECs to recombinant CXCL1 or conditioned medium from IL-17-stimulated HPMCs resulted in increased endothelial tube formation, while selective inhibition of mesothelial CXCL1 production by specific antibodies or through silencing of relevant transcription factors abolished the proangiogenic effect of HPMC-conditioned medium. In conclusion, peritoneal mesothelium-derived CXCL1 promotes endothelial tube formation in vitro and associates with peritoneal microvessel density in uremic patients undergoing PD, thus providing novel targets for therapeutic intervention to prolong PD therapy