Hypercholesterolemia-induced increase in plasma oxidized LDL abrogated pro angiogenic response in kidney grafts.

BACKGROUND: Renal transplantation is increasingly associated with the presence of comorbidity factors such as dyslipidemia which could influence the graft outcome. We hypothesized that hypercholesterolemia could affect vascular repair processes and promote post-transplant renal vascular remodeling through the over-expression of the anti-angiogenic thrombospondin-1 interacting with vascular endothelial growth factor-A levels. METHODS: We tested this hypothesis in vitro, in vivo and in a human cohort using (1) endothelial cells; (2) kidney auto-transplanted pig subjected (n = 5) or not (n = 6) to a diet enriched in cholesterol and (3) a renal transplanted patient cohort (16 patients). RESULTS: Cells exposed to oxidized LDL showed reduced proliferation and an increased expression of thrombospondin-1. In pigs, 3 months after transplantation of kidney grafts, we observed a deregulation of the hypoxia inducible factor 1a-vascular endothelial growth factor-A axis induced in cholesterol-enriched diet animals concomitant with an overexpression of thrombospondin-1 and a decrease in cortical microvessel density promoting vascular remodeling. In patients, hypercholesterolemia was associated with decreased vascular endothelial growth factor-A plasma levels during early follow up after renal transplantation and increased chronic graft dysfunction. CONCLUSIONS: These results support a potential mechanism through which a high fat-diet impedes vascular repair in kidney graft and suggest the value of controlling cholesterolemia in recipient even at the early stage of renal transplantation.

Vectisol Formulation Enhances Solubility of Resveratrol and Brings Its Benefits to Kidney Transplantation in a Preclinical Porcine Model.

Current organ shortages have led centers to extend the acceptance criteria for organs, increasing the risk for adverse outcomes. Current preservation protocols have not been adapted so as to efficiently protect these organs. Herein, we target oxidative stress, the key mechanism of ischemia reperfusion injury. Vectisol® is a novel antioxidant strategy based on the encapsulation of resveratrol into a cyclodextrin, increasing its bioavailability. We tested this compound as an additive to the most popular static preservation solutions and machine perfusion (LifePort) in a preclinical pig model of kidney autotransplantation. In regard to static preservation, supplementation improved glomerular filtration and proximal tubular function early recovery. Extended follow-up confirmed the higher level of protection, slowing chronic loss of function (creatininemia and proteinuria) and the onset of histological lesions. Regarding machine perfusion, the use of Vectisol® decreased oxidative stress and apoptosis at the onset of reperfusion (30 min post declamping). Improved quality was confirmed with decreased early levels of circulating SOD (Superoxide Dismutase) and ASAT (asparagine amino transferase). Supplementation slowed the onset of chronic loss of function, as well as interstitial fibrosis and tubular atrophy. The simple addition of Vectisol® to the preservation solution significantly improved the performance of organ preservation, with long-term effects on the outcome. This strategy is thus a key player for future multi-drug therapy aimed at ischemia reperfusion in transplantation.

Strategies to optimize kidney recovery and preservation in transplantation: specific aspects in pediatric transplantation.

In renal transplantation, live donor kidney grafts are associated with optimum success rates due to the shorter period of ischemia during the surgical procedure. The current shortage of donor organs for adult patients has caused a shift towards deceased donors, often with co-morbidity factors, whose organs are more sensitive to ischemia-reperfusion injury, which is unavoidable during transplantation. Donor management is pivotal to kidney graft survival through the control of the ischemia-reperfusion sequence, which is known to stimulate numerous deleterious or regenerative pathways. Although the key role of endothelial cells has been established, the complexity of the injury, associated with stimulation of different cell signaling pathways, such as unfolded protein response and cell death, prevents the definition of a unique therapeutic target. Preclinical transplant models in large animals are necessary to establish relationships and kinetics and have already contributed to the improvement of organ preservation. Therapeutic strategies using mesenchymal stem cells to induce allograft tolerance are promising advances in the treatment of the pediatric recipient in terms of reducing/withdrawing immunosuppressive therapy. In this review we focus on the different donor management strategies in kidney graft conditioning and on graft preservation consequences by highlighting the role of endothelial cells. We also propose strategies for preventing ischemia-reperfusion, such as cell therapy.

The bacterial lysate OM-85 engages Toll-like receptors 2 and 4 triggering an immunomodulatory gene signature in human myeloid cells.

OM-85 is a bacterial lysate used in clinical practice to reduce duration and frequency of recurrent respiratory tract infections. Whereas knowledge of its regulatory effects in vivo has substantially advanced, the mechanisms of OM-85 sensing remain inadequately addressed. Here, we show that the immune response to OM-85 in the mouse is largely mediated by myeloid immune cells through Toll-like receptor (TLR) 4 in vitro and in vivo. Instead, in human immune cells, TLR2 and TLR4 orchestrate the response to OM-85, which binds to both receptors as shown by surface plasmon resonance assay. Ribonucleic acid-sequencing analyses of human monocyte-derived dendritic cells reveal that OM-85 triggers a pro-inflammatory signature and a unique gene set, which is not induced by canonical agonists of TLR2 or TLR4 and comprises tolerogenic genes. A largely overlapping TLR2/4-dependent gene signature was observed in individual subsets of primary human airway myeloid cells, highlighting the robust effects of OM-85. Collectively, our results suggest caution should be taken when relating murine studies on bacterial lysates to humans. Furthermore, our data shed light on how a standardized bacterial lysate shapes the response through TLR2 and TLR4, which are crucial for immune response, trained immunity, and tolerance.

CRISPR-AsCas12a Efficiently Corrects a GPR143 Intronic Mutation in Induced Pluripotent Stem Cells from an Ocular Albinism Patient.

Mutations in the GPR143 gene cause X-linked ocular albinism type 1 (OA1), a disease that severely impairs vision. We recently generated induced pluripotent stem cells (iPSCs) from skin fibroblasts of an OA1 patient carrying a point mutation in intron 7 of GPR143. This mutation activates a new splice site causing the incorporation of a pseudoexon. In this study, we present a high-performance CRISPR-Cas ribonucleoprotein strategy to permanently correct the GPR143 mutation in these patient-derived iPSCs. Interestingly, the two single-guide RNAs available for SpCas9 did not allow the cleavage of the target region. In contrast, the cleavage achieved with the CRISPR-AsCas12a system promoted homology-directed repair at a high rate. The CRISPR-AsCas12a-mediated correction did not alter iPSC pluripotency or genetic stability, nor did it result in off-target events. Moreover, we highlight that the disruption of the pathological splice site caused by CRISPR-AsCas12a-mediated insertions/deletions also rescued the normal splicing of GPR143 and its expression level.

Generation of a human Ocular Albinism type 1 iPSC line, SEIi001-A, with a mutation in GPR143.

Ocular albinism type 1 is a genetic eye disease caused by mutations in the GPR143 gene. Little is known about the molecular pathways involved in this disease and no therapeutic candidate has been identified as yet. Here we report the generation of an iPSC line from the skin fibroblasts of a patient with a mutation in the GPR143 gene using Sendai Virus vectors. This new iPSC line will allow a better understanding of the Ocular Albinism type 1 disease and to screen for potential therapeutic candidates.

Human embryonic stem cells extracellular vesicles and their effects on immortalized human retinal Müller cells.

Extracellular vesicles (EVs) released by virtually every cell of all organisms are involved in processes of intercellular communication through the delivery of their functional mRNAs, proteins and bioactive lipids. We previously demonstrated that mouse embryonic stem cell-released EVs (mESEVs) are able to transfer their content to different target retinal cells, inducing morphological and biochemical changes in them. The main objective of this paper is to characterize EVs derived from human embryonic stem cells (hESEVs) and investigate the effects that they have on cultured retinal glial, progenitor Müller cells, which are known to give rise to retinal neurons under specific conditions. This would allow us to establish if hESEVs have a pro-regenerative potential not yet described that could be used in the future for treatment of human retinal degenerative diseases. Initially, we showed that hESEVs are heterogeneous in size, contain mRNAs and proteins involved in the induction and maintenance of stem cell pluripotency and can be internalized by cultured Müller cells. After a single exposure to hESEVs these cells display changes in their gene expression profile, and with multiple exposures they de-differentiate and trans-differentiate into retinal neuronal precursors. hESEVs were then fractionated into microvesicles (MVs) and exosomes (EXOs), which were characterized by size, specific surface proteins and biochemical/molecular components. We demonstrate that despite the similar internalization of non-fractionated hESEVs, MVs and EXOs by Müller progenitor cells, in vitro, only the release of MVs' cargo into the cells' cytoplasm induces specific changes in their levels of pluripotency mRNAs and early retinal proteins. EXOs do not produce any detectable effect. Thus, we conclude that MVs and MVs-containing hESEVs are promising agents that possibly could promote the regeneration of diseased or damaged retinas in vivo through inducing glial Müller cells to become replacement neurons.

Renal auto-transplantation promotes cortical microvascular network remodeling in a preclinical porcine model.

The vascular network is a major target of ischemia-reperfusion, but has been poorly investigated in renal transplantation. The aim of this study was to characterize the remodeling of the renal vascular network that follows ischemia-reperfusion along with the most highly affected cortex section in a preclinical renal transplantation model. There were two experimental groups. The first was a grafted kidney group consisting of large white pigs for which the left kidney was harvested, cold flushed, preserved for 24 h in the University of Wisconsin's preservation solution, and then auto-transplanted (n = 5); the right kidney was removed to mimic the situation of human kidney transplantation. The second group (uni-nephrectomized kidney group) consisted of animals that underwent only right nephrectomy, but not left renal transplantation (n = 5). Three months after autotransplantation, the kidneys were studied by X-ray microcomputed tomography. Vessel morphology and density and tortuosity of the network were analyzed using a 3D image analysis method. Cortical blood flow was determined by laser doppler analysis and renal function and tissue injury assessed by plasma creatinine levels and histological analysis. Renal ischemia-reperfusion led to decreased vascular segment volume associated with fewer vessels of less than 30 µm, particularly in the inner cortex:0.79 ± 0.54% in grafted kidneys vs. 7.06 ± 1.44% in uni-nephrectomized kidneys, p < 0.05. Vessels showed higher connectivity throughout the cortex (the arborescence factor of the whole cortex was less in grafted than uni-nephrectomized kidneys 0.90 ± 0.04 vs. 1.07 ± 0.05, p < 0.05, with an increase in the number of bifurcations). Furthermore, cortical blood flow decreased early in kidney grafts and remained low three months after auto-transplantation. The decrease in microvasculature correlated with a deterioration of renal function, proteinuria, and tubular dysfunction, and was associated with the development of fibrous tissue. This work provides new evidence concerning the impact of ischemia-reperfusion injuries on the spectrum of renal vascular diseases and could potentially guide future therapy to preserve microvessels in transplantation ischemia-reperfusion injury.

Amniotic fluid-derived mesenchymal stem cells prevent fibrosis and preserve renal function in a preclinical porcine model of kidney transplantation.

It is well known that ischemia/reperfusion injuries strongly affect the success of human organ transplantation. Development of interstitial fibrosis and tubular atrophy is the main deleterious phenomenon involved. Stem cells are a promising therapeutic tool already validated in various ischemic diseases. Amniotic fluid-derived mesenchymal stem cells (af-MSCs), a subpopulation of multipotent cells identified in amniotic fluid, are known to secrete growth factors and anti-inflammatory cytokines. In addition, these cells are easy to collect, present higher proliferation and self-renewal rates compared with other adult stem cells (ASCs), and are suitable for banking. Consequently, af-MSCs represent a promising source of stem cells for regenerative therapies in humans. To determine the efficiency and the safety of af-MSC infusion in a preclinical porcine model of renal autotransplantation, we injected autologous af-MSCs in the renal artery 6 days after transplantation. The af-MSC injection improved glomerular and tubular functions, leading to full renal function recovery and abrogated fibrosis development at 3 months. The strong proof of concept generated by this translational porcine model is a first step toward evaluation of af-MSC-based therapies in human kidney transplantation.