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1.
Cell Stem Cell ; 30(5): 549-570, 2023 05 04.
Article in English | MEDLINE | ID: mdl-37146580

ABSTRACT

The growing clinical success of hematopoietic stem/progenitor cell (HSPC) gene therapy (GT) relies on the development of viral vectors as portable "Trojan horses" for safe and efficient gene transfer. The recent advent of novel technologies enabling site-specific gene editing is broadening the scope and means of GT, paving the way to more precise genetic engineering and expanding the spectrum of diseases amenable to HSPC-GT. Here, we provide an overview of state-of-the-art and prospective developments of the HSPC-GT field, highlighting how advances in biological characterization and manipulation of HSPCs will enable the design of the next generation of these transforming therapeutics.


Subject(s)
CRISPR-Cas Systems , Hematopoietic Stem Cells , Prospective Studies , Gene Editing , Genetic Therapy , Biology
2.
Sci Transl Med ; 15(698): eabq3679, 2023 05 31.
Article in English | MEDLINE | ID: mdl-37256933

ABSTRACT

Clinical evidence highlights a relationship between the blood and the bone, but the underlying mechanism linking these two tissues is not fully elucidated. Here, we used ß-thalassemia as a model of congenital anemia with bone and bone marrow (BM) niche defects. We demonstrate that fibroblast growth factor 23 (FGF23) is increased in patients and mice with ß-thalassemia because erythropoietin induces FGF23 overproduction in bone and BM erythroid cells via ERK1/2 and STAT5 pathways. We show that in vivo inhibition of FGF23 signaling by carboxyl-terminal FGF23 peptide is a safe and efficacious therapeutic strategy to rescue bone mineralization and deposition in mice with ß-thalassemia, normalizing the expression of niche factors and restoring hematopoietic stem cell (HSC) function. FGF23 may thus represent a molecular link connecting anemia, bone, and the HSC niche. This study provides a translational approach to targeting bone defects and rescuing HSC niche interactions, with potential clinical relevance for improving HSC transplantation and gene therapy for hematopoietic disorders.


Subject(s)
Hematopoietic Stem Cell Transplantation , beta-Thalassemia , Animals , Mice , beta-Thalassemia/therapy , Bone Marrow , Bone Marrow Cells/metabolism , Hematopoietic Stem Cells/metabolism , Stem Cell Niche , Humans
3.
Pharmaceuticals (Basel) ; 15(5)2022 May 11.
Article in English | MEDLINE | ID: mdl-35631417

ABSTRACT

In the last decade, research on pathophysiology and therapeutic solutions for ß-thalassemia (BThal) and sickle cell disease (SCD) has been mostly focused on the primary erythroid defect, thus neglecting the study of hematopoietic stem cells (HSCs) and bone marrow (BM) microenvironment. The quality and engraftment of HSCs depend on the BM microenvironment, influencing the outcome of HSC transplantation (HSCT) both in allogeneic and in autologous gene therapy settings. In BThal and SCD, the consequences of severe anemia alter erythropoiesis and cause chronic stress in different organs, including the BM. Here, we discuss the recent findings that highlighted multiple alterations of the BM niche in BThal and SCD. We point out the importance of improving our understanding of HSC biology, the status of the BM niche, and their functional crosstalk in these disorders towards the novel concept of combined therapies by not only targeting the genetic defect, but also key players of the HSC-niche interaction in order to improve the clinical outcomes of transplantation.

4.
Genes (Basel) ; 12(9)2021 08 24.
Article in English | MEDLINE | ID: mdl-34573276

ABSTRACT

Mitochondria are dynamic organelles undergoing continuous fusion and fission with Drp1, encoded by the DNM1L gene, required for mitochondrial fragmentation. DNM1L dominant pathogenic variants lead to progressive neurological disorders with early exitus. Herein we report on the case of a boy affected by epileptic encephalopathy carrying two heterozygous variants (in cis) of the DNM1L gene: a pathogenic variant (PV) c.1085G>A (p.Gly362Asp) accompanied with a variant of unknown significance (VUS) c.1535T>C (p.Ile512Thr). Amplicon sequencing of the mother's DNA revealed the presence of the PV and VUS in 5% of cells, with the remaining cells presenting only VUS. Functional investigations performed on the patient and his mother's cells unveiled altered mitochondrial respiratory chain activities, network architecture and Ca2+ homeostasis as compared with healthy unrelated subjects' samples. Modelling Drp1 harbouring the two variants, separately or in combination, resulted in structural changes as compared with Wt protein. Considering the clinical history of the mother, PV transmission by a maternal germline mosaicism mechanism is proposed. Altered Drp1 function leads to changes in the mitochondrial structure and bioenergetics as well as in Ca2+ homeostasis. The novel VUS might be a modifier that synergistically worsens the phenotype when associated with the PV.


Subject(s)
Dynamins/genetics , Germ-Line Mutation , Maternal Inheritance , Mitochondrial Diseases/genetics , Mosaicism , Spasms, Infantile/genetics , Adult , Alleles , Calcium/metabolism , Cells, Cultured , Child , Dynamins/chemistry , Dynamins/metabolism , Female , Heterozygote , Humans , Infant , Male , Mitochondrial Diseases/metabolism , Mitochondrial Diseases/pathology , Mitochondrial Dynamics , Mutation, Missense , Protein Conformation , Spasms, Infantile/metabolism , Spasms, Infantile/pathology
5.
Blood ; 136(5): 610-622, 2020 07 30.
Article in English | MEDLINE | ID: mdl-32344432

ABSTRACT

Hematopoietic stem cells (HSCs) are regulated by signals from the bone marrow (BM) niche that tune hematopoiesis at steady state and in hematologic disorders. To understand HSC-niche interactions in altered nonmalignant homeostasis, we selected ß-thalassemia, a hemoglobin disorder, as a paradigm. In this severe congenital anemia, alterations secondary to the primary hemoglobin defect have a potential impact on HSC-niche cross talk. We report that HSCs in thalassemic mice (th3) have an impaired function, caused by the interaction with an altered BM niche. The HSC self-renewal defect is rescued after cell transplantation into a normal microenvironment, thus proving the active role of the BM stroma. Consistent with the common finding of osteoporosis in patients, we found reduced bone deposition with decreased levels of parathyroid hormone (PTH), which is a key regulator of bone metabolism but also of HSC activity. In vivo activation of PTH signaling through the reestablished Jagged1 and osteopontin levels correlated with the rescue of the functional pool of th3 HSCs by correcting HSC-niche cross talk. Reduced HSC quiescence was confirmed in thalassemic patients, along with altered features of the BM stromal niche. Our findings reveal a defect in HSCs in ß-thalassemia induced by an altered BM microenvironment and provide novel and relevant insight for improving transplantation and gene therapy approaches.


Subject(s)
Bone Marrow/pathology , Hematopoietic Stem Cells/pathology , Stem Cell Niche , beta-Thalassemia/pathology , Animals , Female , Hematopoiesis/physiology , Humans , Male , Mice , Mice, Inbred C57BL
6.
Mol Genet Genomic Med ; 8(1): e1056, 2020 01.
Article in English | MEDLINE | ID: mdl-31851782

ABSTRACT

BACKGROUND: Chromosomal microarray analysis (CMA) is nowadays widely used in the diagnostic path of patients with clinical phenotypes. However, there is no ascertained evidence to date on how to assemble single/combined clinical categories of developmental phenotypic findings to improve the array-based detection rate. METHODS: The Italian Society of Human Genetics coordinated a retrospective study which included CMA results of 5,110 Italian patients referred to 17 genetics laboratories for variable combined clinical phenotypes. RESULTS: Non-polymorphic copy number variants (CNVs) were identified in 1512 patients (30%) and 615 (32%) present in 552 patients (11%) were classified as pathogenic. CNVs were analysed according to type, size, inheritance pattern, distribution among chromosomes, and association to known syndromes. In addition, the evaluation of the detection rate of clinical subgroups of patients allowed to associate dysmorphisms and/or congenital malformations combined with any other single clinical sign to an increased detection rate, whereas non-syndromic neurodevelopmental signs and non-syndromic congenital malformations to a decreased detection rate. CONCLUSIONS: Our retrospective study resulted in confirming the high detection rate of CMA and indicated new clinical markers useful to optimize their inclusion in the diagnostic and rehabilitative path of patients with developmental phenotypes.


Subject(s)
Chromosome Aberrations , Developmental Disabilities/genetics , Genetic Testing/standards , Oligonucleotide Array Sequence Analysis/standards , Practice Guidelines as Topic , DNA Copy Number Variations , Developmental Disabilities/classification , Developmental Disabilities/diagnosis , Genetic Testing/methods , Genetics, Medical/organization & administration , Humans , Italy , Oligonucleotide Array Sequence Analysis/methods , Phenotype , Sensitivity and Specificity , Societies, Medical/standards
7.
J Clin Invest ; 129(4): 1566-1580, 2019 02 25.
Article in English | MEDLINE | ID: mdl-30830876

ABSTRACT

BACKGROUND: The human bone marrow (BM) niche contains a population of mesenchymal stromal cells (MSCs) that provide physical support and regulate hematopoietic stem cell (HSC) homeostasis. ß-Thalassemia (BT) is a hereditary disorder characterized by altered hemoglobin beta-chain synthesis amenable to allogeneic HSC transplantation and HSC gene therapy. Iron overload (IO) is a common complication in BT patients affecting several organs. However, data on the BM stromal compartment are scarce. METHODS: MSCs were isolated and characterized from BM aspirates of healthy donors (HDs) and BT patients. The state of IO was assessed and correlated with the presence of primitive MSCs in vitro and in vivo. Hematopoietic supportive capacity of MSCs was evaluated by transwell migration assay and 2D coculture of MSCs with human CD34+ HSCs. In vivo, the ability of MSCs to facilitate HSC engraftment was tested in a xenogenic transplant model, whereas the capacity to sustain human hematopoiesis was evaluated in humanized ossicle models. RESULTS: We report that, despite iron chelation, BT BM contains high levels of iron and ferritin, indicative of iron accumulation in the BM niche. We found a pauperization of the most primitive MSC pool caused by increased ROS production in vitro which impaired MSC stemness properties. We confirmed a reduced frequency of primitive MSCs in vivo in BT patients. We also discovered a weakened antioxidative response and diminished expression of BM niche-associated genes in BT-MSCs. This caused a functional impairment in MSC hematopoietic supportive capacity in vitro and in cotransplantation models. In addition, BT-MSCs failed to form a proper BM niche in humanized ossicle models. CONCLUSION: Our results suggest an impairment in the mesenchymal compartment of BT BM niche and highlight the need for novel strategies to target the niche to reduce IO and oxidative stress before transplantation. FUNDING: This work was supported by the SR-TIGET Core grant from Fondazione Telethon and by Ricerca Corrente.


Subject(s)
Bone Marrow Cells/metabolism , Hematopoiesis , Hematopoietic Stem Cells/metabolism , Oxidative Stress , beta-Thalassemia/metabolism , Animals , Bone Marrow Cells/pathology , Coculture Techniques , Hematopoietic Stem Cells/pathology , Humans , Mice , Stromal Cells/metabolism , Stromal Cells/pathology , beta-Thalassemia/pathology
8.
Nat Med ; 25(2): 234-241, 2019 02.
Article in English | MEDLINE | ID: mdl-30664781

ABSTRACT

ß-thalassemia is caused by ß-globin gene mutations resulting in reduced (ß+) or absent (ß0) hemoglobin production. Patient life expectancy has recently increased, but the need for chronic transfusions in transfusion-dependent thalassemia (TDT) and iron chelation impairs quality of life1. Allogeneic hematopoietic stem cell (HSC) transplantation represents the curative treatment, with thalassemia-free survival exceeding 80%. However, it is available to a minority of patients and is associated with morbidity, rejection and graft-versus-host disease2. Gene therapy with autologous HSCs modified to express ß-globin represents a potential therapeutic option. We treated three adults and six children with ß0 or severe ß+ mutations in a phase 1/2 trial ( NCT02453477 ) with an intrabone administration of HSCs transduced with the lentiviral vector GLOBE. Rapid hematopoietic recovery with polyclonal multilineage engraftment of vector-marked cells was achieved, with a median of 37.5% (range 12.6-76.4%) in hematopoietic progenitors and a vector copy number per cell (VCN) of 0.58 (range 0.10-1.97) in erythroid precursors at 1 year, in absence of clonal dominance. Transfusion requirement was reduced in the adults. Three out of four evaluable pediatric participants discontinued transfusions after gene therapy and were transfusion independent at the last follow-up. Younger age and persistence of higher VCN in the repopulating hematopoietic cells are associated with better outcome.


Subject(s)
Blood Transfusion , Bone and Bones/pathology , Genetic Therapy , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , beta-Thalassemia/genetics , beta-Thalassemia/therapy , Adolescent , Adult , Child , Child, Preschool , Female , Humans , Male , Treatment Outcome
9.
Mol Ther Methods Clin Dev ; 11: 9-28, 2018 Dec 14.
Article in English | MEDLINE | ID: mdl-30320151

ABSTRACT

Gene therapy clinical trials require rigorous non-clinical studies in the most relevant models to assess the benefit-to-risk ratio. To support the clinical development of gene therapy for ß-thalassemia, we performed in vitro and in vivo studies for prediction of safety. First we developed newly GLOBE-derived vectors that were tested for their transcriptional activity and potential interference with the expression of surrounding genes. Because these vectors did not show significant advantages, GLOBE lentiviral vector (LV) was elected for further safety characterization. To support the use of hematopoietic stem cells (HSCs) transduced by GLOBE LV for the treatment of ß-thalassemia, we conducted toxicology, tumorigenicity, and biodistribution studies in compliance with the OECD Principles of Good Laboratory Practice. We demonstrated a lack of toxicity and tumorigenic potential associated with GLOBE LV-transduced cells. Vector integration site (IS) studies demonstrated that both murine and human transduced HSCs retain self-renewal capacity and generate new blood cell progeny in the absence of clonal dominance. Moreover, IS analysis showed an absence of enrichment in cancer-related genes, and the genes targeted by GLOBE LV in human HSCs are well known sites of integration, as seen in other lentiviral gene therapy trials, and have not been associated with clonal expansion. Taken together, these integrated studies provide safety data supporting the clinical application of GLOBE-mediated gene therapy for ß-thalassemia.

10.
Haematologica ; 102(4): e120-e124, 2017 04.
Article in English | MEDLINE | ID: mdl-28034992
11.
Transl Res ; 169: 31-9.e1-4, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26581433

ABSTRACT

Detection of hemoglobin (Hb) variants represents an important issue for diagnosis and adequate treatment of hemoglobinopathies. The Capillarys 2 Flex Piercing analyzer (Capillarys) by Sebia is routinely used in our clinical laboratories to detect Hb variants in peripheral blood (PB). This automated method separates Hb fractions by capillary electrophoresis, giving a spectrophotometric measure of their relative proportion. The scientific research in the field of hemoglobinopathies needs robust procedures to evaluate the efficacy of experimental therapies, as gene therapy. We investigated for the first time the feasibility to use Capillarys on cellular lysates from in vitro erythroid cultures. Because total Hb concentration in erythroid lysates is up to 20-fold lower than in hemolysates from PB, we analyzed diluted blood samples, thanks to the manual mode included in the Capillarys setting. We compared analytical precision, accuracy, sensitivity, and specificity of this procedure to the automatic method, routinely used in diagnostics. For instance, adult Hb intra- and interassay precision were estimated as coefficient of variation 0.2% and 0.3%, respectively. The manual mode is less robust for detection of fractions <3% and the lower level of sensitivity is 2 g/L of total Hb. Specificity of manual and automatic settings was equivalent. We confirmed the performance of the method by analyzing erythroid lysates from thalassemic patients' cultures. Our study demonstrated that the Capillarys 2 Flex Piercing manual method is comparable to the automatic one. The analysis is very robust at low Hb concentrations, as in erythroid cultures from patients affected by hemoglobinopathies, representing a useful tool also in translational research.


Subject(s)
Electrophoresis, Capillary/instrumentation , Erythrocytes/chemistry , Hemoglobins/analysis , Translational Research, Biomedical , Cells, Cultured , Erythrocytes/cytology , Hemoglobins/isolation & purification , Humans , Limit of Detection , Reproducibility of Results
12.
Stem Cells ; 32(5): 1267-77, 2014 May.
Article in English | MEDLINE | ID: mdl-24446190

ABSTRACT

Oxidative metabolism and redox signaling prove to play a decisional role in controlling adult hematopoietic stem/progenitor cells (HSPCs) biology. However, HSPCs reside in a hypoxic bone marrow microenvironment raising the question of how oxygen metabolism might be ensued. In this study, we provide for the first time novel functional and molecular evidences that human HSPCs express myoglobin (Mb) at level comparable with that of a muscle-derived cell line. Optical spectroscopy and oxymetry enabled to estimate an O2-sensitive heme-containing protein content of approximately 180 ng globin per 10(6) HSPC and a P50 of approximately 3 µM O2. Noticeably, expression of Mb mainly occurs through a HIF-1-induced alternative transcript (Mb-V/Mb-N = 35 ± 15, p < .01). A search for other Mb-related globins unveiled significant expression of neuroglobin (Ngb) but not of cytoglobin. Confocal microscopy immune detection of Mb in HSPCs strikingly revealed nuclear localization in cell subsets expressing high level of CD34 (nuclear/cytoplasmic Mb ratios 1.40 ± 0.02 vs. 0.85 ± 0.05, p < .01) whereas Ngb was homogeneously distributed in all the HSPC population. Dual-color fluorescence flow cytometry indicated that while the Mb content was homogeneously distributed in all the HSPC subsets that of Ngb was twofold higher in more immature HSPC. Moreover, we show that HSPCs exhibit a hypoxic nitrite reductase activity releasing NO consistent with described noncanonical functions of globins. Our finding extends the notion that Mb and Ngb can be expressed in nonmuscle and non-neural contexts, respectively, and is suggestive of a differential role of Mb in HSPC in controlling oxidative metabolism at different stages of commitment.


Subject(s)
Gene Expression , Globins/genetics , Hematopoietic Stem Cells/metabolism , Myoglobin/genetics , Nerve Tissue Proteins/genetics , Adaptation, Physiological , Antigens, CD34/metabolism , Globins/metabolism , Hematopoietic Stem Cells/cytology , Humans , Hypoxia/physiopathology , Immunoblotting , Microscopy, Confocal , Myoglobin/metabolism , Nerve Tissue Proteins/metabolism , Neuroglobin , Nitric Oxide/metabolism , Nitrite Reductases/metabolism , Oxidative Stress/physiology , Oxygen/metabolism , Reverse Transcriptase Polymerase Chain Reaction
13.
Free Radic Biol Med ; 52(5): 916-27, 2012 Mar 01.
Article in English | MEDLINE | ID: mdl-22240155

ABSTRACT

Gap junctions play a central role in coordinating intercellular signal-transduction pathways to control tissue homeostasis. Deregulation of gap junctional intercellular communication is a common phenotype of cancer cells and supports its involvement in the carcinogenesis process. Many carcinogens, like environmental heavy-metal chemical pollutants, are known to activate various signal transduction mechanisms and modulate GJIC. They act as tumor promoters on preexisting "initiated" cells, rather than as genotoxic initiators, albeit their mode of action is often unknown. In this study we investigated the effect of Hg(II) (HgCl(2)) on GJIC in cultured human keratinocytes. It is shown that subcytotoxic concentrations of HgCl(2) as low as 10 nM cause inhibition of the GJIC, assessed by dye transfer assay, despite enhanced expression of connexins. In addition, HgCl(2)-treated keratinocytes exhibited a decrease of free thiols and accumulation of mitochondria-derived reactive oxygen species, albeit no effect on the respiratory chain activity was observed. Treatment of HgCl(2)-exposed keratinocytes with the PKC inhibitor calphostin C and with all-trans retinoic acid resulted in rescue of the mitochondrial ROS overproduction and full recovery of the GJIC. Similar results were obtained with the PKA activator db-cAMP. Overall, the presented results support a cross-talk between the altered intracellular redox tone and PKA- and PKC-mediated signaling in HgCl(2)-challenged keratinocytes. These events, although not cytotoxic, lead to inhibition of GJIC and possibly to carcinogenic priming.


Subject(s)
Cell Communication/drug effects , Cytotoxins/pharmacology , Gap Junctions/metabolism , Mercuric Chloride/pharmacology , Cells, Cultured , Cyclic AMP-Dependent Protein Kinases/metabolism , Fluorescent Dyes/metabolism , Gap Junctions/drug effects , Glutathione/metabolism , Glutathione Disulfide/metabolism , Humans , Isoquinolines/metabolism , Keratinocytes/drug effects , Keratinocytes/metabolism , Mitochondria/drug effects , Mitochondria/metabolism , Oxidation-Reduction , Oxygen Consumption/drug effects , Phosphorylation , Protein Kinase C/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction , Tretinoin/pharmacology
14.
Stem Cells Dev ; 21(4): 634-46, 2012 Mar 01.
Article in English | MEDLINE | ID: mdl-21561312

ABSTRACT

Bone marrow-derived hematopoietic stem/progenitor cells (HSPCs) encompass a wide array of cell subsets with different capacities of engraftment and injured tissue-regenerating potential. The characterization/isolation of the stem cell subpopulations represents a major challenge to improve the efficacy of transplantation protocols used in regenerative medicine. Cystic fibrosis (CF) is one of the diseases whose hope of cure relies on the successful application of cell-based gene therapy. This study was aimed at characterizing murine HSPCs on the basis of their bioenergetic competence and CF transmembrane conductance regulator (CFTR) expression. Positively immunoselected Sca-1(+) HSPCs encompassed 2 populations distinguished by their different size, Sca-1 expression and mitochondrial content. The smaller were the cells, the higher was Sca-1 expression and the lower was the intracellular density of functional mitochondria. Reverse transcription-polymerase chain reaction and western blotting revealed that HSPCs expressed CFTR mRNA and protein, which was also functional, as assessed by spectrofluorimetric and patch-clamp techniques. Inhibition of mitochondrial oxidative phosphorylation by oligomycin resulted in a 70% decrease of both the intracelluar adenosine triphosphate content and CFTR-mediated channel activity. Finally, HSPCs with lower Sca-1 expression and higher mitochondrial content displayed higher CFTR levels. Our findings identify 2 subpopulations in HSPCs and unveil a so-far unappreciated relationship between bioenergetic metabolism and CFTR in HSPC biology.


Subject(s)
Antigens, Ly/biosynthesis , Cystic Fibrosis , Energy Metabolism , Gene Expression Regulation , Hematopoietic Stem Cells , Membrane Proteins/biosynthesis , Mitochondria , Animals , Cystic Fibrosis/metabolism , Cystic Fibrosis/pathology , Cystic Fibrosis Transmembrane Conductance Regulator/biosynthesis , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/pathology , Male , Mice , Mitochondria/metabolism , Mitochondria/pathology
15.
Hepatology ; 55(5): 1333-43, 2012 May.
Article in English | MEDLINE | ID: mdl-22135208

ABSTRACT

UNLABELLED: Alisporivir (Debio-025) is an analogue of cyclosporine A and represents the prototype of a new class of non-immunosuppressive cyclophilin inhibitors. In vitro and in vivo studies have shown that alisporivir inhibits hepatitis C virus (HCV) replication, and ongoing clinical trials are exploring its therapeutic potential in patients with chronic hepatitis C. Recent data suggest that the antiviral effect is mediated by inhibition of cyclophilin A, which is an essential host factor in the HCV life cycle. However, alisporivir also inhibits mitochondrial permeability transition by binding to cyclophilin D. Because HCV is known to affect mitochondrial function, we explored the effect of alisporivir on HCV protein-mediated mitochondrial dysfunction. Through the use of inducible cell lines, which allow to investigate the effects of HCV polyprotein expression independent from viral RNA replication and which recapitulate the major alterations of mitochondrial bioenergetics observed in infectious cell systems, we show that alisporivir prevents HCV protein-mediated decrease of cell respiration, collapse of mitochondrial membrane potential, overproduction of reactive oxygen species and mitochondrial calcium overload. Strikingly, some of the HCV-mediated mitochondrial dysfunctions could even be rescued by alisporivir. CONCLUSION: These observations provide new insights into the pathogenesis of HCV-related liver disease and reveal an additional mechanism of action of alisporivir that is likely beneficial in the treatment of chronic hepatitis C.


Subject(s)
Cyclosporine/pharmacology , Hepacivirus/drug effects , Mitochondria, Liver/drug effects , Virus Replication/drug effects , Antiviral Agents/pharmacology , Apoptosis/drug effects , Calcium/metabolism , Cell Respiration/drug effects , Cells, Cultured/drug effects , Cyclophilins/antagonists & inhibitors , Hepacivirus/physiology , Humans , Immunohistochemistry , Membrane Potentials , Mitochondria, Liver/metabolism , Reactive Oxygen Species/metabolism , Sensitivity and Specificity
16.
J Cell Mol Med ; 15(2): 375-95, 2011 Feb.
Article in English | MEDLINE | ID: mdl-19863698

ABSTRACT

Dyslipidemia is a well-established condition proved to accelerate the progression of chronic kidney disease leading to tubulo-interstitial injury. However, the molecular aspects of the dyslipidemia-induced renal damage have not been fully clarified and in particular the role played by low-density lipoproteins (LDLs). This study aimed to examine the effects of native non-oxidized LDL on cellular oxidative metabolism in cultured human proximal tubular cells. By means of confocal microscopy imaging combined to respirometric and enzymatic assays it is shown that purified native LDL caused a marked increase of cellular reactive oxygen species (ROS) production, which was mediated by activation of NADPH oxidase(s) and by mitochondrial dysfunction by means of a ROS-induced ROS release mechanism. The LDL-dependent mitochondrial alterations comprised inhibition of the respiratory chain activity, enhanced ROS production, uncoupling of the oxidative phosphorylation efficiency, collapse of the mtΔΨ, increased Ca(2+) uptake and loss of cytochrome c. All the above LDL-induced effects were completely abrogated by chelating extracellular Ca(2+) as well as by inhibition of the Ca(2+) -activated cytoplasmic phospholipase A2, NADPH oxidase and mitochondrial permeability transition. We propose a mechanicistic model whereby the LDL-induced intracellular redox unbalance is triggered by a Ca(2+) inward flux-dependent commencement of cPLA2 followed by activation of a lipid- and ROS-based cross-talking signalling pathway. This involves first oxidants production via the plasmamembrane NADPH oxidase and then propagates downstream to mitochondria eliciting redox- and Ca(2+) -dependent dysfunctions leading to cell-harming conditions. These findings may help to clarify the mechanism of dyslipidemia-induced renal damage and suggest new potential targets for specific therapeutic strategies to prevent oxidative stress implicated in kidney diseases.


Subject(s)
Kidney Tubules, Proximal/metabolism , Lipoproteins, LDL/metabolism , Oxidative Stress , Reactive Oxygen Species/metabolism , Calcium/metabolism , Calcium Signaling , Cell Line , Cytochromes c/metabolism , Dyslipidemias/metabolism , Humans , Mitochondria/pathology , NADPH Oxidases/antagonists & inhibitors , NADPH Oxidases/biosynthesis , NADPH Oxidases/metabolism , Phospholipase A2 Inhibitors , Phospholipases A2/metabolism , Signal Transduction
17.
J Virol ; 84(1): 647-60, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19846525

ABSTRACT

Hepatitis C virus (HCV) infection induces a state of oxidative stress by affecting mitochondrial-respiratory-chain activity. By using cell lines inducibly expressing different HCV constructs, we showed previously that viral-protein expression leads to severe impairment of mitochondrial oxidative phosphorylation and to major reliance on nonoxidative glucose metabolism. However, the bioenergetic competence of the induced cells was not compromised, indicating an efficient prosurvival adaptive response. Here, we show that HCV protein expression activates hypoxia-inducible factor 1 (HIF-1) by normoxic stabilization of its alpha subunit. In consequence, expression of HIF-controlled genes, including those coding for glycolytic enzymes, was significantly upregulated. Similar expression of HIF-controlled genes was observed in cell lines inducibly expressing subgenomic HCV constructs encoding either structural or nonstructural viral proteins. Stabilization and transcriptional activation of HIF-1alpha was confirmed in Huh-7.5 cells harboring cell culture-derived infectious HCV and in liver biopsy specimens from patients with chronic hepatitis C. The HCV-related HIF-1alpha stabilization was insensitive to antioxidant treatment. Mimicking an impairment of mitochondrial oxidative phosphorylation by treatment of inducible cell lines with oligomycin resulted in stabilization of HIF-1alpha. Similar results were obtained by treatment with pyruvate, indicating that accumulation of intermediate metabolites is sufficient to stabilize HIF-1alpha. These observations provide new insights into the pathogenesis of chronic hepatitis C and, possibly, the HCV-related development of hepatocellular carcinoma.


Subject(s)
Adaptation, Physiological , Glycolysis , Hepacivirus/pathogenicity , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mitochondria/virology , Antioxidants/pharmacology , Cell Line, Tumor , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/analysis , Liver/metabolism , Mitochondria/metabolism , Oxidative Phosphorylation , Protein Stability , Viral Proteins
18.
Stem Cells ; 26(11): 2843-54, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18787213

ABSTRACT

Retroviral vectors are used in human gene therapy trials to stably introduce therapeutic genes in the genome of patients' cells. Their applicability, however, is frustrated by the limited viability of transformed cells and/or by risks linked to selection of oncogene-mutated clones. The reasons for these drawbacks are not yet completely understood. In this study, we show that LXSN-NeoR gene/interleukin-7-engineered mesenchymal stromal cells exhibited a marked enhancement of reactive oxygen species production compared with untransfected cells. This effect resulted to be independent on the product of the gene carried by the retroviral vehicle as it was reproducible in cells transfected with the empty vector alone. Stable transfection of mesenchymal stromal cells with the different retroviral vectors pBabe-puro and PINCO-puro and the lentiviral vector pSico PGK-puro caused similar redox imbalance, unveiling a phenomenon of more general impact. The enhanced production of reactive oxygen species over the basal level was attributable to mitochondrial dysfunction and brought back to altered activity of the NADH-CoQ oxidoreductase (complex I) of the respiratory chain. The oxidative stress in transfected mesenchymal stem cells was completely reversed by treatment with a cAMP analog, thus pointing to alteration in the protein kinase A-dependent signaling pathway of the host cell. Transfection of mesenchymal stromal cells with a PINCO-parental vector harboring the green fluorescent protein gene as selection marker in place of the puromycin-resistance gene resulted in no alteration of the redox phenotype. These novel findings provide insights and caveats to the applicability of cell- or gene-based therapies and indicate possible intervention to improve them. Disclosure of potential conflicts of interest is found at the end of this article.


Subject(s)
Bone Marrow Cells/cytology , Cyclic AMP/metabolism , Cyclic AMP/physiology , Mitochondria/physiology , Reactive Oxygen Species/metabolism , Cell Line , Cell Respiration/physiology , Cell Transformation, Viral , Cyclic AMP-Dependent Protein Kinases/physiology , Electron Transport Complex I/antagonists & inhibitors , Electron Transport Complex I/physiology , Humans , Interleukin-7/metabolism , Lentivirus/genetics , Mesoderm/cytology , Moloney murine leukemia virus/genetics , Oxidation-Reduction , Stromal Cells/cytology , Transduction, Genetic
19.
Neurochem Res ; 33(12): 2565-74, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18473170

ABSTRACT

In the present study mitochondrial respiratory function of fibroblasts from a patient affected by early-onset parkinsonism carrying the homozygous W437X nonsense mutation in the PINK1 gene has been thoroughly characterized. When compared with normal fibroblasts, the patient's fibroblast mitochondria exhibited a lower respiratory activity and a decreased respiratory control ratio with cellular ATP supply relying mainly on enhanced glycolytic production. The quantity, specific activity and subunit pattern of the oxidative phosphorylation complexes were normal. However, a significant decrease of the cellular cytochrome c content was observed and this correlated with a reduced cytochrome c oxidase in situ-activity. Measurement of ROS revealed in mitochondria of the patient's fibroblasts enhanced O(2)(*-) and H(2)O(2) production abrogated by inhibition of complex I. No change in the glutathione-based redox buffering was, however, observed.


Subject(s)
Mitochondria/physiology , Mutation , Parkinson Disease/genetics , Protein Kinases/genetics , Adenosine Triphosphate/metabolism , Adult , Cells, Cultured , Cytochromes c/metabolism , Glutathione/metabolism , Humans , Immunohistochemistry , Microscopy, Confocal , Mitochondria/enzymology , Mitochondria/metabolism , Oxidative Phosphorylation , Parkinson Disease/metabolism , Parkinson Disease/physiopathology , Reactive Oxygen Species/metabolism , Subcellular Fractions/metabolism
20.
FEBS Lett ; 581(16): 3111-9, 2007 Jun 26.
Article in English | MEDLINE | ID: mdl-17568584

ABSTRACT

The hypoxia-inducible factor (HIF) transcriptional system enables cell adaptation to limited O(2) availability, transducing this signal into patho-physiological responses such as angiogenesis, erythropoiesis, vasomotor control, and altered energy metabolism, as well as cell survival decisions. However, other factors beyond hypoxia are known to activate this pleiotropic transcription factor. The aim of this study was to characterize HIF in human hematopoietic stem cells (HSCs) and evidence is provided that granulocyte colony stimulating factor-mobilized CD34+- and CD133+-HSCs express a stabilized cytoplasmic form of HIF-1alpha under normoxic conditions. It is shown that HIF-1alpha stabilization correlates with down-regulation of the tumour suppressor von Hippel-Lindau protein (pVHL) and is positively controlled by NADPH-oxidase-dependent production of reactive oxygen species, indicating a specific O(2)-independent post-transcriptional control of HIF in mobilized HSCs. This novel finding is discussed in the context of the proposed role of HIF as a mediator of progenitor cell recruitment to injured ischemic tissues and/or in the control of the maintenance of the undifferentiated state.


Subject(s)
Hematopoietic Stem Cells/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Oxygen Consumption/physiology , Antigens, CD34/metabolism , Blood Circulation , Cell Hypoxia , Cells, Cultured , Hematopoietic Stem Cells/drug effects , Humans , Models, Biological , NADPH Oxidases/metabolism , Protein Processing, Post-Translational , Reactive Oxygen Species/metabolism , Reactive Oxygen Species/pharmacology , Von Hippel-Lindau Tumor Suppressor Protein/metabolism
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