Stem Cells in Kidney Ischemia: From Inflammation and Fibrosis to Renal Tissue Regeneration.

Ischemic nephropathy consists of progressive renal function loss due to renal hypoxia, inflammation, microvascular rarefaction, and fibrosis. We provide a literature review focused on kidney hypoperfusion-dependent inflammation and its influence on renal tissue's ability to self-regenerate. Moreover, an overview of the advances in regenerative therapy with mesenchymal stem cell (MSC) infusion is provided. Based on our search, we can point out the following conclusions: 1. endovascular reperfusion is the gold-standard therapy for RAS, but its success mostly depends on treatment timeliness and a preserved downstream vascular bed; 2. anti-RAAS drugs, SGLT2 inhibitors, and/or anti-endothelin agents are especially recommended for patients with renal ischemia who are not eligible for endovascular reperfusion for slowing renal damage progression; 3. TGF-ß, MCP-1, VEGF, and NGAL assays, along with BOLD MRI, should be extended in clinical practice and applied to a pre- and post-revascularization protocols; 4. MSC infusion appears effective in renal regeneration and could represent a revolutionary treatment for patients with fibrotic evolution of renal ischemia.

Ischemic Nephropaty: The Role of the Renal Artery Stenosis Revascularization on Renal Stem Cells.

We report the case of a 65-year-old man with acute GFR decline to 37 mL/min and uncontrolled high blood pressure. He was suspected for renovascular hypertension and underwent a renal color Doppler ultrasound scan that detected a bilateral atherosclerotic renal artery stenosis. A digital selective angiography by percutaneous transluminal angioplasty and stenting (PTRAs) was successfully performed. Blood pressure rapidly normalized, GFR increased within a few days, and proteinuria disappeared thereafter. These clinical goals were accompanied by a significant increase of circulating renal stem cells (RSC) and a slight increase of resistive index (RI) in both kidneys. This single observation suggests the need for extensive studies aimed at evaluating the predictive power of RI and RSC in detecting post-ischemic renal repair mechanisms.

Lipoxin A4 stimulates endothelial miR-126-5p expression and its transfer via microvesicles.

The proresolution lipid mediator lipoxin (LX)A4 bestows protective bioactions on endothelial cells. We examined the impact of LXA4 on transcellular endothelial signaling via microRNA (miR)-containing microvesicles. We report LXA4 inhibition of MV release by TNF-α-treated HUVECs, associated with the down-regulation of 18 miR in endothelial microvesicles (EMVs) and the up-regulation of miR-126-5p, both in HUVECs and in EMVs. LXA4 up-regulated miR-126-5p by ∼5-fold in HUVECs and promoted a release of microvesicles (LXA4-EMVs) that enhanced miR-126-5p by ∼7-fold in recipient HUVECs. In these cells, LXA4-EMVs abrogated the up-regulation of VCAM-1, induced in recipient HUVECs by EMVs released by untreated or TNF-α-treated HUVECs. LXA4-EMVs also reduced by ∼40% the expression of SPRED1, which we validated as an miR-126-5p target, whereas they stimulated monolayer repair in an in vitro wound assay. This effect was lost when the EMVs were depleted of miR-126-5p. These results provide evidence that changes in miR expression and microvesicle packaging and transfer represent a mechanism of action of LXA4, which may be relevant in vascular biology and inflammation.-Codagnone, M., Recchiuti, A., Lanuti, P., Pierdomenico, A. M., Cianci, E., Patruno, S., Mari, V. C., Simiele, F., Di Tomo, P., Pandolfi, A., Romano, M. Lipoxin A4 stimulates endothelial miR-126-5p expression and its transfer via microvesicles.

Mechanisms of endothelial cell dysfunction in cystic fibrosis.

Although cystic fibrosis (CF) patients exhibit signs of endothelial perturbation, the functions of the cystic fibrosis conductance regulator (CFTR) in vascular endothelial cells (EC) are poorly defined. We sought to uncover biological activities of endothelial CFTR, relevant for vascular homeostasis and inflammation. We examined cells from human umbilical cords (HUVEC) and pulmonary artery isolated from non-cystic fibrosis (PAEC) and CF human lungs (CF-PAEC), under static conditions or physiological shear. CFTR activity, clearly detected in HUVEC and PAEC, was markedly reduced in CF-PAEC. CFTR blockade increased endothelial permeability to macromolecules and reduced trans­endothelial electrical resistance (TEER). Consistent with this, CF-PAEC displayed lower TEER compared to PAEC. Under shear, CFTR blockade reduced VE-cadherin and p120 catenin membrane expression and triggered the formation of paxillin- and vinculin-enriched membrane blebs that evolved in shrinking of the cell body and disruption of cell-cell contacts. These changes were accompanied by enhanced release of microvesicles, which displayed reduced capability to stimulate proliferation in recipient EC. CFTR blockade also suppressed insulin-induced NO generation by EC, likely by inhibiting eNOS and AKT phosphorylation, whereas it enhanced IL-8 release. Remarkably, phosphodiesterase inhibitors in combination with a ß2 adrenergic receptor agonist corrected functional and morphological changes triggered by CFTR dysfunction in EC. Our results uncover regulatory functions of CFTR in EC, suggesting a physiological role of CFTR in the maintenance EC homeostasis and its involvement in pathogenetic aspects of CF. Moreover, our findings open avenues for novel pharmacology to control endothelial dysfunction and its consequences in CF.

Immunoresolving actions of oral resolvin D1 include selective regulation of the transcription machinery in resolution-phase mouse macrophages.

Resolvin D1 (RvD1; 7S,8R,17S-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid) is an endogenous immunoresolvent that regulates acute inflammation and orchestrates resolution. Here, we investigated anti-inflammatory and proresolving actions of RvD1 after oral administration. RvD1 rapidly accumulated in the mouse plasma after oral delivery and dose-dependently (1-100 ng/mouse) reduced leukocyte infiltration in zymosan A-induced acute peritonitis. Using mathematical resolution indices, RvD1 reduced Ψmax by ∼50%, shortened the resolution interval by 3 h, and significantly reduced total leukocyte (by ∼30-45%) and polymorphonuclear neutrophil (by ∼40-55%) accumulation when administered at the peak of peritonitis. RvD1 also improved course and outcome of severe peritonitis, shifting it toward resolution. In peritoneal macrophages (MΦs) from the resolution phase of peritonitis, RvD1 down-regulated (by 2- to 3-fold) select genes that control gene transcription, namely coactivator-associated arginine methyltransferase 1 (CARM1), and downstream genes, such as colony-stimulating factor 3, intercellular adhesion molecule 1, and monocyte inflammatory protein 2, which promote neutrophil infiltration and reduce MΦ phagocytosis. Congruently, CARM1 knockdown in human and murine MΦs induced a proresolving phenotype, recapitulating in vivo actions of RvD1. These results establish novel properties of RvD1 and demonstrate that RvD1 modifies the transcription control machinery in MΦs, as part of its mechanisms of action during the resolution of acute inflammation.-Recchiuti, A., Codagnone, M., Pierdomenico, A. M., Rossi, C., Mari, V. C., Cianci, E., Simiele, F., Gatta, V., Romano, M. Immunoresolving actions of oral resolvin D1 include selective regulation of the transcription machinery in resolution-phase mouse macrophages.

Transcriptional regulation of the human FPR2/ALX gene: evidence of a heritable genetic variant that impairs promoter activity.

Lipoxin (LX) A(4,) a main endogenous stop-signal of inflammation, activates the G-protein-coupled receptor FPR2/ALX, which triggers potent anti-inflammatory signaling in vivo. Thus, the regulation of FPR2/ALX expression may have pathophysiological and therapeutic relevance. Here, we mapped a nucleotide sequence with strong FPR2/ALX promoter activity. Chromatin immunoprecipitation revealed specificity protein 1 (Sp1) binding to the core promoter. Site-directed mutagenesis of the Sp1 cis-acting element and Sp1 overexpression established that this transcription factor is key for maximal promoter activity, which is instead suppressed by DNA methylation. LXA(4) enhanced FPR2/ALX promoter activity (+74%) and mRNA expression (+87.5%) in MDA-MB231 cells. A single nucleotide mutation (A/G) was detected in the core promoter of one subject with history of cardiovascular disease and of his two daughters. This mutation reduced by ∼35-90% the promoter activity in vitro. Moreover, neutrophils from individuals carrying the A/G variant displayed ∼10- and 3-fold reduction in FPR2/ALX mRNA and protein, respectively, compared with cells from their relatives or healthy volunteers expressing the wild-type allele. These results uncover FPR2/ALX transcriptional regulation and provide the first evidence of mutations that affect FPR2/ALX transcription, thus opening new opportunities for the understanding of the LXA(4)-FPR2/ALX axis in human disease.

Renal Stem Cells, Renal Resistive Index, and Neutrophil Gelatinase Associated Lipocalin Changes After Revascularization in Patients With Renovascular Hypertension and Ischemic Nephropathy.

BACKGROUND: Percutaneous transluminal renal angioplasty (PTRA) with or without stenting is the gold standard therapy in patients with atherosclerotic renal artery stenosis (aRAS). However, therapeutic success depends on the correct timing of revascularization and the reversibility of the renal damage. MATERIALS AND METHODS: We report a case series of patients treated with PTRA for renovascular hypertension and ischemic nephropathy. We measured bilateral renal resistive index (RRI), circulating renal stem cells (RSC), and Neutrophil Gelatinase Associated Lipocalin (NGAL) at baseline and after PTRA at different time points to understand their changes in post-revascularization. RESULTS: At baseline, the studied patients (n = 5) had different RSC levels. After PTRAs, all patients showed an improvement in blood pressure, while renal function varied differently within the studied subjects. RRI > 0.75 at baseline and the absence of NGAL decrease after PTRAs were associated with post-PTRA renal function worsening, despite an increase of RSC in all patients. CONCLUSION: Although limited to a few patients, our observation allowed the exploration of the behaviour of the studied parameters in different degrees of renal ischemia. This revealed different disease models suggesting the importance of further investigations in larger and homogeneous cohorts to confirm that a greater basal RSC percentage, low RRI values before PTRA, and a post-revascularization NGAL reduction could be related to better renal outcomes in aRAS patients.

Resolvin D1 Reduces Lung Infection and Inflammation Activating Resolution in Cystic Fibrosis.

Non-resolving lung inflammation and Pseudomonas aeruginosa infections are the underlying cause of morbidity and mortality in cystic fibrosis (CF). The endogenous lipid mediator resolvin (Rv) D1 is a potent regulator of resolution, and its roles, actions, and therapeutic potential in CF are of interest. Here, we investigated actions and efficacy of RvD1 in preclinical models of cystic fibrosis. Cftr knockout mice with chronic P. aeruginosa lung infection were treated with RvD1 to assess differences in lung bacterial load, inflammation, and tissue damage. Cells from volunteers with CF were treated with RvD1 during ex vivo infection with P. aeruginosa, and effects on phagocytosis and inflammatory signaling were determined. In CF mice, RvD1 reduced bacterial burden, neutrophil infiltration, and histological signs of lung pathology, improving clinical scores of diseases. Mechanistically, RvD1 increased macrophage-mediated bacterial and leukocyte clearance in vivo. The clinical significance of these findings is supported by actions in primary leukocytes and epithelial cells from volunteers with CF where RvD1 enhanced P. aeruginosa phagocytosis and reduced genes and proteins associated to NF-κB activation and leukocyte infiltration. Concentration of RvD1 in sputum from patients with CF was also inversely correlated to those of cytokines and chemokines involved in CF lung pathology. These findings demonstrate efficacy of RvD1 in enhancing resolution of lung inflammation and infections and provide proof of concept for its potential as a prototypic novel pro-resolutive therapeutic approach for CF.

Immobilization and delivery of biologically active Lipoxin A4 using electrospinning technology.

Lipoxin (LX)A4 is a lipoxygenase-formed arachidonic acid metabolite with potent anti-inflammatory, pro-resolution properties. Its therapeutic efficacy has been largely demonstrated in a variety of cellular, preclinical and clinical models. Among these, periodontal disease, where LXA4 promotes tissue repair, also by modulating functions of human periodontal ligament stem cells (hPDLSCs). As medicated biomembranes may be particularly useful in clinical settings, where local stimulation of tissue repair is needed, we used electrospinning to embed LXA4 in membranes made of poly(ethylene oxide) (PEO) and poly(d,l-lactide) (PDLLA). These membranes were fully characterized by scanning electron microscopy, differential scanning calorimetry and biocompatibility with hPDLSCs. Here, we report that LXA4 is retained in these membranes and that ∼15-20% of the total LXA4 amount added to the reaction can be eluted from the membranes using an aqueous buffered medium. The eluted LXA4 fully retained its capability to stimulate hPDLSC proliferation. A similar effect was obtained by adding directly the LXA4-containing membranes to cells. These results demonstrate for the first time that LXA4 can be incorporated into biomembranes, which may be useful to combat local inflammation and promote tissue repair in selected clinical settings.

Human Periodontal Stem Cells Release Specialized Proresolving Mediators and Carry Immunomodulatory and Prohealing Properties Regulated by Lipoxins.

UNLABELLED: Unresolved inflammation and tissue destruction are underlying mechanisms of periodontitis, which is linked to dysregulated polymorphonuclear neutrophil (PMN) functions. Lipoxin A4 (LXA4) is a specialized proresolving lipid mediator (SPM) that dampens excessive inflammation, promotes resolution, and protects from leukocyte-mediated tissue damage. Human periodontal ligament stem cells (hPDLSCs) represent key players during tissue regeneration and may contribute to resolution of inflammation; thus, they may represent a promising tool in regenerative dentistry. In the present study, we investigated the actions of hPDLSCs on PMN apoptosis and antimicrobial functions, and determined the impact of LXA4 on hPDLSCs. hPDLSCs significantly reduced apoptosis and stimulated microbicidal activity of human PMNs, via both cell-cell interactions and paracrine mechanisms. Lipid mediator metabololipidomics analysis demonstrated that hPDLSCs biosynthesize SPMs, including resolvin D1, D2, D5, and D6; protectin D1; maresins; and LXB4; as well as prostaglandins D2, E2, and F2α. LXA4 significantly enhanced proliferation, migration, and wound healing capacity of hPDLSCs through the activation of its cognate receptor ALX/FPR2, expressed on hPDLSCs. Together, these results demonstrate that hPDLSCs modulate PMN functions, and provide the first evidence that stem cells generate SPM and that the LXA4-ALX/FPR2 axis regulates regenerative functions of hPDLSCs by a novel receptor-mediated mechanism. SIGNIFICANCE: These findings uncovered unappreciated features of stem cells from the periodontal ligament, supporting the notion that these cells may act as master regulators of pathophysiological events through the release of mediators that promote the resolution of inflammation and bacterial killing. The study also demonstrated that it is possible to modulate important functions of periodontal stem cells using lipoxin A4, a potent endogenous stop signal of inflammation. Thus, this study revealed an unappreciated anti-inflammatory proregenerative circuit that may be exploited to combat periodontal pathologies using resident stem cells. Moreover, the data may represent a more general template to explain the immunomodulatory functions of stem cells.

Lipoxins and aspirin-triggered lipoxins in resolution of inflammation.

The resolution of the inflammatory response is highly regulated by the timely biosynthesis of a number of endogenous lipid mediators. Among these, lipoxins (LX) and their 15-epimers, aspirin triggered lipoxins (ATL) derived by the lipoxygenase (LO) route of arachidonic acid metabolism. In particular, they are formed and released by cells expressing 5-, 12- and 15-LO such as leukocytes, platelets, vascular endothelium and epithelium, alone or during transcellular interactions. ATL biosynthesis requires cyclooxygenase-2 acetylation by aspirin. LX and ATL exert potent bioactions on leukocytes, vascular and epithelial cells to stop inflammation and promote resolution. They have shown to be beneficial in a broad spectrum of preclinical models of disease as well as in some clinical trials. Counter-regulatory signaling by LXA4 and 15-epi-LXA4 follows the activation of a G protein-coupled receptor, termed ALX/FPR2, which is emerging as a key anti-inflammatory receptor.

Molecular and phenotypic characterization of human amniotic fluid-derived cells: a morphological and proteomic approach.

Mesenchymal Stem Cells derived from Amniotic Fluid (AFMSCs) are multipotent cells of great interest for regenerative medicine. Two predominant cell types, that is, Epithelial-like (E-like) and Fibroblast-like (F-like), have been previously detected in the amniotic fluid (AF). In this study, we examined the AF from 12 donors and observed the prevalence of the E-like phenotype in 5, whereas the F-like morphology was predominant in 7 samples. These phenotypes showed slight differences in membrane markers, with higher CD90 and lower Sox2 and SSEA-4 expression in F-like than in E-like cells; whereas CD326 was expressed only in the E-like phenotype. They did not show any significant differences in osteogenic, adipogenic or chondrogenic differentiation. Proteomic analysis revealed that samples with a predominant E-like phenotype (HC1) showed a different profile than those with a predominant F-like phenotype (HC2). Twenty-five and eighteen protein spots were differentially expressed in HC1 and HC2 classes, respectively. Of these, 17 from HC1 and 4 from HC2 were identified by mass spectrometry. Protein-interaction networks for both phenotypes showed strong interactions between specific AFMSC proteins and molecular chaperones, such as preproteasomes and mature proteasomes, both of which are important for cell cycle regulation and apoptosis. Collectively, our results provide evidence that, regardless of differences in protein profiling, the prevalence of E-like or F-like cells in AF does not affect the differentiation capacity of AFMSC preparations. This may be valuable information with a view to the therapeutic use of AFMSCs.

Calcium sensing receptor activation by calcimimetic R-568 in human amniotic fluid mesenchymal stem cells: correlation with osteogenic differentiation.

Human amniotic fluid mesenchymal stem cells (hAFMSCs) are promising for therapeutic applications in bone damage. Calcium sensing receptor (CaSR), a G protein-coupled receptor, plays a physiological role in the regulation of bone metabolism. Thus, the bone CaSR could be targeted by calcimimetic agonists, which may be potentially helpful in treating bone diseases. The aim of our study was to characterize CaSR expression in hAFMSCs and to assess the activity of calcimimetic R-568 during in vitro osteogenesis. Using western blotting, immunofluorescence, and flow cytometry, we consistently observed constitutive CaSR in osteo-differentiating hAFMSCs. Notably, both R-568 and calcium significantly enhanced hAFMSC osteogenic differentiation after exposure to osteogenic medium. To provide further evidence of the involvement of CaSR in osteogenesis, we correlated its expression with that of established osteogenic markers, that is, alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteopontin (OPN), and novel, not yet completely defined regulators of osteogenesis. Among these are ß-catenin and Slug, which are mediators of Wnt signaling, and nuclear factor of activated T cells c1 (NFATc1), which plays a critical role in calcium/calcineurin signaling. Taken together, our results demonstrate that CaSR is expressed in hAFMSCs, positively correlates with osteogenic markers, and is activated by R-568. Notably, downregulation of CaSR by RNA interference supports the conclusion that CaSR activation plays a central role in hAFMSC osteogenesis. Thus, this study provides significant information on the mechanisms of hAFMSC osteogenesis, which could provide additional molecular basis for the use of calcimimetics in bone regenerative medicine.

Wnt signaling behaves as a "master regulator" in the osteogenic and adipogenic commitment of human amniotic fluid mesenchymal stem cells.

Human amniotic fluid mesenchymal stem cells (huAFMSCs) are emerging as a promising therapeutic option in regenerative medicine. Here, we characterized huAFMSC phenotype and multipotentiality. When cultured in osteogenic medium, huAFMSC displayed a significant increase in: Alkaline Phosphatase (ALP) activity and mRNA expression, Alizarin Red S staining and Runx2 mRNA expression; whereas maintaining these cells in an adipogenic culture medium gave a time-dependent increase in PPARγ and FABP4 mRNA expression, glycerol-3-phosphate dehydrogenase (GPDH) activity and positivity to Oil Red Oil staining. These results confirm that huAFMSCs can differentiate toward osteogenic and adipogenic phenotypes. The canonical Wnt/ßcatenin signaling pathway appears to trigger huAFMSC osteoblastogenesis, since during early phases of osteogenic differentiation, the expression of Dishevelled-2 (Dvl-2), of the non-phosphorylated form of ß-catenin, and the phosphorylation of glycogen synthase kinase-3ß (GSK3ß) at serine 9 were upregulated. On the contrary, during adipogenic differentiation Dvl-2 expression decreased, whereas that of ß-catenin remained unchanged. This was associated with a late increase in GSK3ß phosphorylation. Consistent with this scenario, huAFMSCs exposure to Dickkopf-1, a selective inhibitor of the Wnt signaling, abolished Runx2 and ALP mRNA upregulation during huAFMSC osteogenic differentiation, whereas it enhanced FABP4 expression in adipocyte-differentiating cells. Taken together, these results unravel novel molecular determinants of huAFMSC commitment towards osteoblastogenesis, which may represent potential targets for directing the differentiation of these cells and improving their use in regenerative medicine.