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She returned to Kansas City after completing a fellowship in reproductive endocrinology and infertility at the University of Michigan. Dr. Marsh specializes in treating women with infertility, PCOS, and obesity.",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"255491",title:"Dr.",name:"Courtney",middleName:null,surname:"Marsh",slug:"courtney-marsh",fullName:"Courtney Marsh",profilePictureURL:"https://mts.intechopen.com/storage/users/255491/images/system/255491.jpg",biography:"Dr. Courtney Marsh is Associate Professor of Obstetrics and Gynecology at the University of Kansas Health System and Chief of the Division of Reproductive Endocrinology and Infertility (REI). She attended the University of Kansas where she obtained both her undergraduate and medical school degrees. During medical school, she also completed a fellowship in Epidemiology at the Centers for Disease Control and Prevention at the National Center for Birth Defects and Developmental Disabilities. She completed her Obstetrics and Gynecology residency at Emory University and REI fellowship at the University of Michigan. Dr. Marsh\\'s research focuses on improving fertility outcomes. Currently, she is funded by Garmin International, the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), and the University of Kansas Medical Center.",institutionString:"University of Kansas Medical Center",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"1",totalChapterViews:"0",totalEditedBooks:"1",institution:{name:"University of Kansas Medical Center",institutionURL:null,country:{name:"United States of America"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"16",title:"Medicine",slug:"medicine"}],chapters:[{id:"75047",title:"Role of Sex Hormones in Human Body",slug:"role-of-sex-hormones-in-human-body",totalDownloads:26,totalCrossrefCites:0,authors:[null]},{id:"73546",title:"Androgen Signaling in the Placenta",slug:"androgen-signaling-in-the-placenta",totalDownloads:57,totalCrossrefCites:0,authors:[null]},{id:"75080",title:"Androgens and Cardiovascular Risk Factors in Polycystic Ovary Syndrome",slug:"androgens-and-cardiovascular-risk-factors-in-polycystic-ovary-syndrome",totalDownloads:0,totalCrossrefCites:0,authors:[null]},{id:"74803",title:"Neuroendocrinology of Pregnancy: Participation of Sex Hormones",slug:"neuroendocrinology-of-pregnancy-participation-of-sex-hormones",totalDownloads:25,totalCrossrefCites:0,authors:[null]},{id:"74544",title:"Non-Reproductive Effects of Estradiol: Hydromineral Homeostasis Control",slug:"non-reproductive-effects-of-estradiol-hydromineral-homeostasis-control",totalDownloads:40,totalCrossrefCites:0,authors:[null]},{id:"74462",title:"Serum Sex Hormone Profiles in Potentially Resectable Esophageal Cancer",slug:"serum-sex-hormone-profiles-in-potentially-resectable-esophageal-cancer",totalDownloads:8,totalCrossrefCites:0,authors:[null]},{id:"73469",title:"Testosterone",slug:"testosterone",totalDownloads:57,totalCrossrefCites:0,authors:[null]},{id:"74165",title:"Kisspeptin: Role in Female Infertility",slug:"kisspeptin-role-in-female-infertility",totalDownloads:43,totalCrossrefCites:0,authors:[null]},{id:"73669",title:"Small Molecules Inhibit Extranuclear Signaling by Estrogen: A Promising Strategy to Halt Breast Cancer Progression and Metastasis",slug:"small-molecules-inhibit-extranuclear-signaling-by-estrogen-a-promising-strategy-to-halt-breast-cance",totalDownloads:113,totalCrossrefCites:0,authors:[null]}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"304289",firstName:"Rebekah",lastName:"Pribetic",middleName:null,title:"Ms.",imageUrl:"https://mts.intechopen.com/storage/users/304289/images/13255_n.png",email:"rebekah@intechopen.com",biography:null}},relatedBooks:[{type:"book",id:"9785",title:"Endometriosis",subtitle:null,isOpenForSubmission:!1,hash:"f457ca61f29cf7e8bc191732c50bb0ce",slug:"endometriosis",bookSignature:"Courtney Marsh",coverURL:"https://cdn.intechopen.com/books/images_new/9785.jpg",editedByType:"Edited by",editors:[{id:"255491",title:"Dr.",name:"Courtney",surname:"Marsh",slug:"courtney-marsh",fullName:"Courtney Marsh"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"6550",title:"Cohort Studies in Health Sciences",subtitle:null,isOpenForSubmission:!1,hash:"01df5aba4fff1a84b37a2fdafa809660",slug:"cohort-studies-in-health-sciences",bookSignature:"R. 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Venkateswarlu",coverURL:"https://cdn.intechopen.com/books/images_new/371.jpg",editedByType:"Edited by",editors:[{id:"58592",title:"Dr.",name:"Arun",surname:"Shanker",slug:"arun-shanker",fullName:"Arun Shanker"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"878",title:"Phytochemicals",subtitle:"A Global Perspective of Their Role in Nutrition and Health",isOpenForSubmission:!1,hash:"ec77671f63975ef2d16192897deb6835",slug:"phytochemicals-a-global-perspective-of-their-role-in-nutrition-and-health",bookSignature:"Venketeshwer Rao",coverURL:"https://cdn.intechopen.com/books/images_new/878.jpg",editedByType:"Edited by",editors:[{id:"82663",title:"Dr.",name:"Venketeshwer",surname:"Rao",slug:"venketeshwer-rao",fullName:"Venketeshwer Rao"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"40970",title:"Efficient AAV Vector Production System: Towards Gene Therapy For Duchenne Muscular Dystrophy",doi:"10.5772/53023",slug:"efficient-aav-vector-production-system-towards-gene-therapy-for-duchenne-muscular-dystrophy",body:'Successful gene therapy requires an adequate level of long-term transgene expression in the target tissues. While various viral vectors have been considered for the delivery of genes in vivo, an adeno-associated virus (AAV)-based vector is emerging as the gene transfer vehicle with the most potential for use in the neuromuscular gene therapies. The advantages of the AAV vector include the lack of disease associated with a wild-type virus, the ability to transduce non-dividing cells, and the long-term expression of the delivered transgenes.[1] Some serotypes of recombinant AAV (rAAV) exhibit a potent tropism for striated muscles.[2] Therefore, a supplementation of secretory protein can be achieved with this vector to use intramuscular injection.[3] Since a 5-kb genome is considered to be the upper limit for a single AAV virion, various truncated genes could be provided to meet size capacity, if nessessarry.[4]
Due to ingenious cloning and preparation techniques, adenovirus vectors are efficient delivery systems of episomal DNA into eukaryotic cell nuclei.[5] The utility of adenovirus vectors has been increased by capsid modifications that alter tropism, and by the generation of hybrid vectors that promote chromosomal insertion.[6] Also, gutted adenovirus vectors devoid of all adenoviral genes allow for the insertion of large transgenes, and trigger fewer cytotoxic and immunogenic effects than do those only deleted in the E1 regions of the adenovirus early genes.[7] Human artificial chromosomes (HACs) have the capacity to deliver genes in any size into host cells without integrating the gene into the host genome, thereby preventing the possibility of insertional mutagenesis and genomic instability.[8]
Long-term correction of genetic diseases requires permanent integration of therapeutic genes into chromosomes of the affected cells. However, retrovirus vector integration can trigger deregulated premalignant cell proliferation with unexpected frequency, most likely driven by retrovirus enhancer activity on the LMO2 gene promoter. [9] A goal in clinical gene therapy is to develop gene transfer vehicles that can integrate exogenous therapeutic genes at specific chromosomal loci as a safe harbor, so that insertional oncogenesis is prevented. AAV can insert its genome into a specific locus, designated AAVS1, on chromosome 19 of the human genome.[10] The AAV Rep78/68 proteins and the Rep78/68-binding sequences are the trans- and cis-acting elements needed for this reaction. A dual high-capacity adenovirus-AAV hybrid vector with full-length human dystrophin-coding sequences flanked by AAV integration-enhancing elements was tested for targeted integration.[11]
AAV is a small (20-26nm) non-enveloped dependent parvovirus with a single-stranded linear genome that contains two open reading frames (rep and cap).[12] The viral genome is characterized by the inverted terminal repeats (ITRs) to flank these open reading frames (Figure 1A). The genome encodes four replication proteins (Rep78, Rep68, Rep52, and Rep40) and three capsid proteins (Cap: VP1, VP2, and VP3). The large Rep (Rep78 and Rep68) proteins regulate AAV gene expression and hold nicking activity at the terminal resolution site as well as binding activity at Rep binding elements to process AAV replication (Figure 1B). The small Rep proteins (Rep52 and Rep40) are used for the accumulation of single-stranded viral genome followed by packaging within AAV capsids.
The minimum sets of regions in helper adenovirus that mediate AAV vector replication are the E1, E2A, E4, and VA.[13] A human embryonic kidney cell line 293 encodes the E1 region of the Ad5 genome.[14] The helper plasmid assembling E2A, E4, and VA regions (Ad-helper plasmid) is cotransfected into the 293 cells, along with plasmids encoding the AAV vector genome (vector plasmid) as well as rep and cap genes (AAV-helper plasmid). AAV vector is produced as efficiently as when adenovirus infection is employed as a helper virus. Furthermore, contamination of most adenovirus proteins can be avoided in AAV vector stock made by this helper virus-free method.
The preparation of AAV vector for gene therapy study of neuromuscular diseases is greatly facilitated. Although AAV2 has been the serotype most extensively studied in preclinical and clinical trials, recently we have focused on the use of AAV vectors pseudotyped with capsid protein of alternative serotypes. A number of primate AAV serotypes have been characterized in the literature and are designated. There is divergence in homology and tropism for various AAV serotypes. For instance, the homology with capsid protein is only about 60% between AAV2 and AAV5[15], therefore the capsid structure could be responsible for the improved transduction efficiency.
A) The rep and cap genes flanked by ITRs. The large Rep proteins (Rep78 and Rep68) are produced from transcripts using p5 promoter, while small Rep (Rep52 and Rep40) are produced from p19 promoter. (B) Recombinant AAV production. AAV has productive infection in the presence of adenovirus helper regions (E1, E2A, E4, and VA). This process is characterized by genome replication, assembly of the capsid proteins (VP1, VP2, and VP3), and packaging leading to virion production along with exosome releasing.
We found that choice of AAV serotypes and promoters could be quite useful for targeted transgene expression. For instance, the transgene expression of rAAV5 with the Rous sarcoma virus (RSV) promoter was preferentially found in the granular cells of the gerbil hippocampus, whereas transgene expression of rAAV2 with the RSV promoter was found in the pyramidal and granular cells.[16] Since AAV3 vector can specifically transduce cochlear inner hair cells with high efficiency in vivo, rAAV-mediated transduction might be promising for gene replacement strategies to correct recessive genetic hearing loss due to monogenic mutation.[17] Also, there is a significant difference in transgene expression by various AAV serotypes transduced into muscle. We observed that intramuscular injection of AAV5-IL-10 promoted a much higher serum level of secreted transgene product, as compared to AAV2-mediated transfer.[18] We further demonstrated that AAV1 could more efficiently transduce the muscle than AAV5. Intramuscular single injection of modest doses of rAAV1 expressing IL-10 (6x1010 g.c. per rat) introduced therapeutic levels of the transgene expression over the long-term to treat pulmonary arterial hypertension.[3] rAAV1-mediated sustained IL-10 expression also significantly ameliorated hypertensive organ damage to improve survival rate of Dahl salt-sensitive rats.[19] Furthermore, this protein supplementation therapy by rAAV1-mediated muscle transduction was quite effective to prevent vascular remodeling and end-organ damage in the stroke-prone spontaneously hypertensive rat.[20] Interestingly, alpha-sarcoglycan expression with single intramuscular injection of rAAV8 was widely distributed in the hind limb muscle as well as cardiac muscle, and persisted for 7 months with a reversal of the muscle pathology and improvement in the contractile force in the alpha-sarcoglycan-deficient mice.[21] Intravenous administration of rAAV8 into the hind limb in dogs resulted in improved transgene expression in the skeletal muscles lasting over a period of 8 weeks.[22] Moreover, rAAV9 would be administered systemically with excellent cardiac tropism.[23] Further strategies have been attempted to discover novel AAV capsid sequences from primate tissue, which can be used to develop newer-generation rAAVs with a greater diversity of tissue tropism for clinical gene therapy.
Clinical gene therapy often requires rapid transduction with reasonable efficiency. In the case of AAV, second strand synthesis of the vector genome in the nucleus is the rate-limiting step for efficient transduction. Therefore, self-complementary AAV (scAAV) vector would be quite promising to promote efficient transduction regardless of DNA synthesis or annealing.[24] The scAAV vectors can bypass the inter-molecular annealing or second-strand synthesis by using intra-molecular annealing to immediately form transcriptionally active double-stranded DNA (Figure 2). Although immediate and efficient transduction could be observed with scAAV, the maximal insert size of the transgene cassette is reduced to 3.3 kb.[25]
To gain acceptance as a medical treatment with a dose of over 1x1013 genome copies (g.c.)/kg body weight, therapeutic strategies with AAV vectors require a scalable and provident production method. However, the production and purification of recombinant virus stocks with conventional techniques entails cumbersome procedures not suited to the clinical setting. Therefore, development of effective large-scale culture and purification steps are required to meet end-product specifications.
A production protocol of AAV vectors in the absence of a helper virus[13] is widely employed for triple plasmid transduction of human embryonic kidney 293 cells.[1] The adenovirus regions that mediate AAV vector replication (namely, the VA, E2A and E4 regions) were assembled into a helper plasmid. When this helper plasmid is co-transfected into 293 cells along with plasmids encoding the AAV vector genome and rep-cap genes, the AAV vector is produced as efficiently as when using adenovirus infection. Importantly, contamination of most adenovirus proteins can be avoided in AAV vector stock made by this helper virus-free method.
DNA rescue and transduction of a conventional single-stranded AAV (ssAAV) and a self-complementary AAV (scAAV) vector. Full-length ssAAV vector genome of both polarities are rescued from the vector plasmid and individually packaged into the AAV capsids. As a genome conversion in the transduced cell nucleus, the single-to-double stranded conversion of the DNA goes through the inter-molecular annealing or second strand synthesis. In contrast, a scAAV vector with half the size of the ssAAV genome has a mutation in the terminal resolution site (TRS) to form a vector genome with wild-type ITRs at the both ends and mutated ITR at the center of symmetry. After uncoating in the target cell nucleus, this DNA structure can readily fold into transcriptionally active double-stranded form through intra-molecular annealing.
Although various subtypes of the 293 cells harbor the E1 region of the adenovirus type 5 genome, to utilize a 293 cell stably expressing Bcl-xL (293B) has great advantage to support E1B19K function and protect cells from apoptosis.[26] Despite improvements in vector production, including the development of packaging cell lines expressing Rep/Cap or methods to regulate Rep/Cap,[27] maintaining such cell lines remains difficult, as the early expression of Rep proteins is toxic to cells.
We developed a large-scale transfection method of producing AAV vectors with an active gassing system that uses large culture vessels to process labor-effective transfection in a closed system.[28] This vector production system achieved reasonable production efficiency by improving gas exchange to prevent pH drop in the culture medium. Also, vector purification with the dual ion-exchange membrane adsorbers was effective and allowed higher levels of gene transfer in vivo.[29] Furthermore, the membrane adsorbers enabled the effective recovery of the AAV vector in the supernatant exosomes of the transduced cells culture. This rapid and scalable viral purification protocol is particularly promising for considerable in vivo experimentation and clinical investigations (Figure 3).
Recent developments also suggest that AAV vector production in insect cells would be compatible with current good manufacturing practice production on an industrial scale.[30]
A scalable triple plasmid transfection using active gassing. When (1) a vector plasmid encoding the transgene cassette flanked by ITRs is co-transfected into human embryonic kidney 293 cells with (2) an AAV packaging plasmid harboring rep-cap genes and (3) an adenovirus helper plasmid, the AAV vector is produced as efficiently as when using adenovirus infection. A large-scale transduction method to produce AAV vectors with an active gassing system makes use of large culture vessels for labor- and cost-effective vector production in a closed system. Samples containing vector particles are further purified with a quick two-tier CsCl gradient centrifugation and an ion-exchange chromatography to obtain highly purified vector stocks.
Our protocol utilizes the transfection of 293B cells in one 10-Tray flask (CF10; Nalge Nunc International, Rochester, NY) with a surface area of 6320 cm2 by using an active gassing at 500 ml/min. Typical transduction procedure is conducted with one or two CF10 to meet downstream purification protocol. Although previous protocols for recombinant virus production in a large culture vessel had the problem of insufficient transduction efficiency because of inadequate gas exchange, this method to use active gassing significantly improves productivity of the vectors and is linearly scalable from the small 225-cm2 flask.[3]
The 293B cells are cultured in Dulbecco’s modified Eagle’s medium and Nutrient Mixture F-12 (D-MEM/F-12, Invitrogen, Grand Island, NY) with 10% fetal bovine serum (SIGMA-ALDRICH, St Louis, MO), 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 oC in a 5% CO2 incubator. Cells are initially plated at 8 x 107 cells per CF10 to achieve a monolayer of 20 to 40% confluency when cells attached to surface of the flask. The volume of medium utilized per flask is 1120 ml. Subsequently, cells are grown for 48-72 h until reaching 70-90% confluence and are consequently transfected with appropriate triple plasmids. An aquarium pump (Nisso, Tokyo, Japan) should be used to circulate the gas through the CF10 with 5% CO2 and humidity in an incubator.
Half of the medium in the CF10 tissue culture flask are exchanged with fresh D-MEM/F-12 containing 10% FBS, 1 h before transfection of the 293 cells. Subsequently, the cells are co-transfected with 650 µg of each plasmid: a proviral vector plasmid, an AAV helper plasmid, as well as an adenoviral helper plasmid, using calcium phosphate co-precipitation. Each plasmid was added to 112 ml of 300 mM CaCl2. This solution was gently added to the same volume of 2 x HBS (290 mM NaCl, 50 mM HEPES buffer, 1.5 mM Na2HPO4, pH 7.0) and gently inverted 3 times to form a uniform solution. This solution was immediately mixed with fresh D-MEM/F-12 containing 10% FBS to produce a homogeneous plasmid solution mixture. Subsequently, the medium in the culture flask was replaced with this plasmid solution mixture. At the end of a 6-12 h incubation, the plasmid solution mixture in the culture flask was replaced with pre-warmed fresh D-MEM/F-12 containing 2% FBS.
The culture supernatant sample for the ion-exchange procedure is processed by centrifugation and filtration. The culture supernatant fluid 72-96 h after the transduction is sampled and then clarified with an appropriate amount of the activated charcoal (Wako Pure Chemical Industries, Osaka, Japan). Insoluble debris is removed by a centrifugation at 3,000 g for 15 min and filtration. The elucidated culture supernatant is enriched with a hollow fiber cross flow membrane (100,000 NMWC, GE Healthcare, Pittsburgh, PA). For the material obtained from a CF10, 5 mM MgCl2 (final concentration) with 2,500-5,000 units of Benzonase nuclease is added to incubate for 30 min at 37 oC. Sequentially, 5 mM EDTA (final concentration) is added to terminate the reaction. Place 38 ml of the sample solution in a semi-sterile ultracentrifuge tube (Ultrabottle #3430-3870; Nalge Nunc, Rochester, NY) and remove the cell debris by centrifugation at 10,000g for 15 minutes at 4 oC to achieve cleared lysates. The sample is quickly concentrated by the brief two-tier CsCl (1.25 and 1.60 g/cm3) step gradient centrifugation for 3 h and then the vector fraction is dialyzed in the MHA buffer (3.3 mM MES 3.3 mM HEPES [pH 8.0], 3.3 mM NaOAc).
Chromatography can be performed using an appropriate FPLC system, such as AKTA explorer 10S (Amersham Biosciences, Piscataway, NJ, USA) equipped with a 50 ml Superloop. The sample which passed through the MustangTM S membrane (optional treatment, PALL corporation, NY) is dialyzed against MHA buffer and further loaded onto an anion-exchange membrane (acrodisc unit with MustangTM Q membrane, PALL corporation, equilibrated with MHA buffer) at a rate of 3 ml/min. The membrane is then washed with 10 column volumes of MHA buffer. Bound virus on the MustangTM Q membrane is eluted over a 50 column volume span with a 0-2 M linear NaCl gradient in MHA buffer and 0.5-1 ml fractions are collected. Recombinant rAAV particle number is determined by quantitative PCR of DNase I-treated stocks with plasmid standards. The final titer of the purified vectors from a CF10 usually ranges around 5 x 1013 genome copies (g.c.), although it depends on the vector constructs and transgene.
Duchenne muscular dystrophy (DMD) is the most common form of childhood muscular dystrophy and is an X-linked recessive disorder with an incidence of one in 3500 live male births.[31] DMD causes progressive degeneration and regeneration of skeletal and cardiac muscles due to mutations in the dystrophin gene, which encodes a 427-kDa subsarcolemmal cytoskeletal protein.[32] DMD is associated with severe, progressive muscle weakness and typically leads to death between the ages of 20 and 35 years. Due to recent advances in respiratory care, much attention is now focused on treating the cardiac conditions suffered by DMD patients. The approximately 2.5-megabase dystrophin gene is the largest gene identified to date, and because of its size, it is susceptible to a high sporadic mutation rate. Absence of dystrophin and the dystrophin-glycoprotein complex (DGC) from the sarcolemma leads to severe muscle wasting. Whereas DMD is characterized by the absence of functional protein, Becker muscular dystrophy, which is commonly caused by in-frame deletions of the dystrophin gene, results in the synthesis of an incompletely functional protein.
Successful therapy for DMD requires the restoration of dystrophin protein in skeletal and cardiac muscles. While various viral vectors have been considered for the delivery of genes to muscle fibers, the AAV-based vector is emerging as an appropriate gene transfer vehicle with the most potential for use in DMD gene therapies. As for another candidate vehicle, the gutted adenovirus vector can package 14-kb of full-length dystrophin cDNA due to the large deletion in virus genome. Multiple proximal muscles of seven-day-old utrophin/dystrophin double knockout mice (dko mice), which typically show symptoms similar to human DMD, were effectively transduced with the gutted adenovirus bearing full-length murine dystrophin cDNA.[33] However, further improvements are needed to regulate the virus-associated host immune response before clinical trials can be performed.
A series of truncated dystrophin cDNAs containing rod repeats with hinge 1, 2, and 4 were constructed (Figure 4A).[4] Although AAV vectors are too small to package the full-length dystrophin cDNA, AAV vector-mediated gene therapy using a rod-truncated dystrophin gene provides a promising appraoch.[34] The structure and, particularly, the length of the rod are crucial for the function of micro-dystrophin.[35] An AAV type 2 vector expressing micro-dystrophin (DeltaCS1) under the control of a muscle-specific MCK promoter was injected into the tibialis anterior (TA) muscles of dystrophin-deficient mdx mice,[36] and resulted in extensive and long-term expression of micro-dystrophin that exhibited improved force generation. Likewise, AAV6 vector-mediated systemic micro-dystrophin gene transfer was effective in treating dko mice.[37] The potential for ameliorating the pathology of advanced-stage muscular dystrophy by systemic administration of AAV6 vectors encoding a micro-dystrophin expression construct was also demonstrated.[38] Furthermore, AAV9 vector-mediated micro-dystrophin transduction of mdx mice accomplished prevention of cardiac fibrosis as well as heart failure.[23] The transduction efficiency achieved with rAAV9 was nearly complete, with persistent expression for 74 weeks after transduction (Figure 4BC). Both the strong affinity of the rAAV9 for cardiac tissue and the therapeutic effect of the expressed micro-dystrophin might be involved in the prevention of the degeneration of the cardiomyocytes and cardiac fibrosis.
AAV9 vector-mediated cardiac transduction. (A) Structures of full-length and truncated dystrophin. Helper-dependent adenovirus vector can package 14-kb of full-length dystrophin cDNA because of the large-sized deletion in its genome. A mini-dystrophin is cloned from a patient with Becker muscular dystrophy, which is caused by in-frame deletions resulting in the synthesis of partially functional protein. A truncated micro-dystrophin cDNAs harboring only four rod repeats with hinge 1, 2, and 4 and a deleted C-terminal domain (delta CS1) is constructed to be packaged in the AAV vector. (B) Transverse section of mdx mouse heart at mid-ventricular level 24 weeks after transduction of micro-dystrophin, stained with anti-dystrophin antibody NCL-DysB. Scale bar, 500 µm. (C) Expression of dystrophin in C57BL10 hearts at the sarcolemma (a), while it is absent in mdx hearts (b). Magnified views of sections from the center of the left ventricle at 28 weeks (c-e) show micro-dystrophin expression in the areas indicated in B (scale bar, 100 µm). At 74 weeks after transduction, mdx mice still retain extensive expression of micro-dystrophin (f).
The impact of codon usage optimization on micro-dystrophin expression and function in the mdx mouse was demonstrated to compare the function of two different configurations of codon-optimized micro-dystrophin genes under the control of a muscle-restrictive promoter (Spc5-12).[39] Codon optimization of micro-dystrophin significantly increased micro-dystrophin mRNA and protein levels after intramuscular and systemic administration of plasmid DNA or rAAV8. By randomly assembling myogenic regulatory elements into synthetic promoter recombinant libraries, several artificial promoters were isolated whose transcriptional potencies greatly exceed those of natural myogenic and viral gene promoters.[40]
Although recent studies suggest that vectors based on AAV are capable of body-wide transduction in rodents,[21] translating the characteristics into large animals with advanced immune system remains a lot of challenges. Intravascular delivery can be performed as a form of limb perfusion, which might bypass the immune activation of DCs in the injected muscle.[41] We performed limb perfusion-assisted intravenous administration of rAAV8-lacZ into the hind limb of Beagle dogs (Figure 5A).[42] Administration of rAAV8 by limb perfusion demonstrated extensive transgene expression in the distal limb muscles of canine X-linked muscular dystrophy in Japan (CXMDJ) dogs without obvious immune responses for the duration of the experiment over four weeks after injection.
In comparison with fully dystrophin-deficient animals, targeted transgenic repair of skeletal muscle, but not cardiac muscle, paradoxically elicits a five-fold increase in cardiac injury and dilated cardiomyopathy.[43] Because the dystrophin-deficient heart is highly sensitive to increased stress, increased activity by the repaired skeletal muscle provides the stimulus for heightened cardiac injury and heart remodeling. In contrast, a single intravenous injection of AAV9 vector expressing micro-dystrophin efficiently transduces the entire heart in neonatal mdx mice, thereby ameliorating cardiomyopathy.[44]
Since a number of muscular dystrophy patients can be identified through newborn screening in future, neonatal transduction may lead to an effective early intervention in DMD patients. After a single intravenous injection, robust skeletal muscle transduction with AAV9 vector throughout the body was observed in neonatal dogs.[45] Systemic transduction was achieved in the absence of pharmacological intervention or immune suppression and lasted for at least six months, whereas rAAV9 was barely transduced into the cardiac muscle of dogs. Likewise, in utero gene delivery of full-length murine dystrophin to mdx mice using a high-capacity adenoviral vector resulted in effective protection from cycles of degeneration and regeneration.[46]
Neo-antigens introduced by AAV vectors evoke significant immune reactions in DMD muscle, since increased permeability of the DMD muscle allows leakage of the transgene products from the dystrophin-deficient sarcolemma of muscle fibers.[47] rAAV2 transfer into skeletal muscles of normal dogs resulted in low levels of transient expression, together with intense cellular infiltration, and the marked activation of cellular and humoral immune responses.[48] Furthermore, an in vitro interferon-gamma release assay showed that canine splenocytes respond to immunogens or mitogens more strongly than do murine splenocytes. Therefore, co-administration of immunosuppressants, cyclosporine (CSP) and mycophenolate mofetil (MMF) was attempted to improve rAAV2-mediated transduction. The AAV2 capsids can induce a cellular immune response via MHC class I antigen presentation with a cross-presentation pathway,[49] and rAAV2 could also stimulate human dendritic cells (DCs).[50] Whereas the non-immunogenic nature of AAV6 in murine studies, rAAV6 also elicited robust cellular immune responses in dogs.[51] In contrast, other serotypes, such as rAAV8, induce T-cell activation to a lesser degree.[42] The rAAV8-injected muscles showed lowed rates of infiltration of CD4+ and CD8+ T lymphocytes in the endomysium than the rAAV2-injected muscles.[42]
Resident antigen-presenting cells, such as DCs, myoblasts, myotubes and regenerating immature myofibers, should play a substantial role in the immune response against rAAV. Our study also showed that MyD88 and co-stimulating factors, such as CD80, CD86 and type I interferon, are up-regulated in both rAAV2- and rAAV8-transduced dog DCs (Figure 5B).[42]
rAAV-mediated transduction of dog. (A) Intravascular vector administration by limb perfusion. A blood pressure cuff is applied just above the knee of an anesthetized CXMDJ dog. A 24-gauge intravenous catheter is inserted into the lateral saphenous vein, connected to a three-way stopcock, and flushed with saline. With a blood pressure cuff inflated to over 300 mmHg, saline (2.6 ml/kg) containing papaverine (0.44 mg/kg, Sigma-Aldrich, St. Louis, MO) and heparin (16 U/kg) is injected by hand over a 10 second period. The three-way stopcock is connected to a syringe containing rAAV8 (1 x 1014 vg/kg, 3.8 ml/kg). The syringe is placed in a PHD 2000 syringe pump (Harvard Apparatus, Edenbridge, UK). Five minutes after the papaverine/heparin injection, rAAV8-LacZ is injected at a rate of 0.6 ml/sec. Two minutes after the rAAV injection, the blood pressure cuff is released and the catheter is removed. Four weeks after the transduction, the expression slightly fell off. (B) AAV-mediated stimulation of innate immune response via TLR9/MyD88 pathway. Bone marrow (BM)-derived dendritic cells (DCs) were obtained from humerus bones and cultured in RPMI (10% FCS, p/s) for 7 days with canine GM-CSF and IL-4. DCs were transduced with rAAV2- or rAAV8-lacZ (1x106 vg/cell for 4 hours, and mRNA levels of MyD88 and IFN-ß were analyzed. Untransduced cells were used as a normalization standard to demonstrate relative value of expression. Results are representative of two independent experiments. Error bars represent s.e.m., n = 3.
While low immunogenicity was considered a major strength supporting the use of rAAV in clinical trials, a number of observations have recently provided a more balanced view of this procedure.[52] An obvious barrier to AAV transduction is the presence of circulating neutralizing antibodies that prevent the virion from binding to its cellular receptor.[53] This potential threat can be reduced by prescreening patients for AAV serotype-specific neutralizing antibodies or by performing therapeutic procedures such as plasmapheresis before gene transfer. Another challenge recently revealed is the development of a cell-mediated cytotoxic T-cell (CTL) response to AAV capsid peptides. In the human factor IX gene therapy trial in which rAAV was delivered to the liver, only short-term transgene expression was achieved and levels of therapeutic protein declined to baseline levels 10 weeks after vector infusion.[52] This was accompanied by elevation of serum transaminase levels and a CTL response toward specific AAV capsid peptides. To overcome this response, transient immunosuppression may be required until AAV capsids are completely cleared. Additional findings suggest that T-cell activation requires AAV2 capsid binding to the heparan sulfate proteoglycan (HSPG) receptor, which would permit virion shuttling into a DC pathway, as cross-presentation.[54] Exposure to vectors from other AAV clades, such as AAV8, did not activate capsid-specific T-cells.
The initial clinical studies lay the foundation for future studies, providing important information about vector dose, viral serotype selection, and immunogenicity in humans. The first virus-mediated gene transfer for muscle disease was carried out for limb-girdle muscular dystrophy type 2D using rAAV1. The study, consisting of intramuscular injection of virus into a single muscle, was limited in scope and the main conclusion was to establish the safety of this procedure in phase I clinical trials. The first clinical gene therapy trial for DMD began in March 2006.[55] This was a Phase I/IIa study in which an AAV vector was used to deliver micro-dystrophin to the biceps of boys with DMD. The study was conducted on six boys with DMD, each of whom received an injection of mini-dystrophin-expressing rAAV2.5 in a muscle of one arm and a placebo in the other arm. Dystrophin-specific T cells were detected after treatment, providing evidence of transgene expression even when the functional protein was not visualized in skeletal muscle.[56] The potential for T-cell immunity to self and non-self dystrophin epitopes should be considered in designing and monitoring experimental therapies for this disease. Basically, this issue is in common with the treatment of genetic diseases. Although concerns regarding risk of an immune response to the transgene product limited the ability to achieve therapeutic efficacy, rAAV2-mediated gene transfer to human skeletal muscle can persist for up to a decade.[57]
After more than two decades of expectations, the field of gene therapy appears close to reaching a regulatory approval by proposing rAAV-mediated muscle transduction. European medicine agency eventually recommends first gene therapy medicine for approval. (http://www.ema.europa.eu/ema) The European Medicines Agency’s Committee for Medicinal Products for Human Use has recommended the authorization of Glybera (rAAV1-expressing LPL S447X variant) for marketing in the European Union. It is intended to treat lipoprotein lipase deficiency in patients with severe or multiple pancreatitis attacks, despite dietary fat restrictions.
To regulate host immune response against vectors and transgene products, treatments involving immunosuppressants and other strategies have been attempted in the animal models. A brief course of immunosuppression with a combination of anti-thymocyte globulin (ATG), CSP and MMF was effective in permitting AAV6-mediated, long-term and robust expression of a canine micro-dystrophin in the skeletal muscle of a dog DMD model.[58] To establish the feasibility of multiple AAV1 injections for extending the treatment to whole body muscles, the dystrophic mdx mouse was repeatedly transduced with AAV1 vector, and the immune response was characterized.[59] By blocking the T-B crosstalk with anti-CD40 Abs and CTLA4/Fc fusion protein, a five-day-long immunomodulation treatment was found to be sufficient for totally abrogating the formation of anti-AAV1 antibodies.
There have been numerous reports to develop the therapeutic potential of mesenchymal stem cells (or mesenchymal multipotent stromal cells MSCs).[60] Because of their immunomodulatory properties, increasing experimental and early clinical observations indicate that allogeneic, and even xenogeneic, MSCs may be useful for tissue transplantation.[61] In fact, the immune tolerance with MSCs is well investigated in various animal studies. Infusion of syngeneic MSCs into a sensitized mouse model of kidney transplantation resulted in the expansion of donor-specific T- regulatory cells into lymphoid organs, prolonged allograft survival and promoted the development of tolerance.[62]
The use of a histone deacetylase (HDAC) inhibitor depsipeptide effectively enhances the utility of rAAV-mediated gene therapy.[63] In contrast to adenovirus-mediated transduction, the improved transduction with rAAV induced by the depsipeptide is due to enhanced transgene expression rather than to increased viral entry. The enhanced transduction is related to the histone-associated chromatin form of the rAAV concatemer in the transduced cells. Since various HDAC inhibitors are approved in clinical usage for many diseases to achieve therapeutic benefits, the application of such inhibitors to the rAAV-mediated gene therapy is theoretically and practically reasonable.
Transplantation of genetically modified vector-producing cells is a possible future treatment for genetic diseases as an in situ gene therapy. MSCs are known to accumulate at the site of inflammation or tumors, and therefore can be utilized as a platform for the targeted delivery of therapeutic agents.[64] The MSCs-based targeted gene therapy should enhance the therapeutic efficacy, since MSCs would deliver therapeutic molecules in a concentrated fashion. This targeted therapy can also reduce systemic adverse side effects, because the reagents act locally without elevating their systemic concentrations. We developed the genetically-modified MSCs that produce viral vectors to augment therapeutic efficacy of systemic gene therapy.[65] MSCs isolated from the SD rats bone marrow were transfected with retroviral vector components by nucleofection. As a result, the injection of luciferase-expressing vector-producing MSCs caused significantly stronger signal of bioluminescence at the site of subcutaneous tumors in mice compared with luciferease-expressing non-vector-producing MSCs.[66] Furthermore, tumor-bearing nude mice were treated with the vector-producing MSCs combined with HSV-tk/GCV system to demonstrate improved anti-tumor effects. This study suggests the effectiveness of vector-producing MSCs in systemic gene therapy. The therapeutic benefit of this strategy should be further examined by using rAAV-producing MSCs in the various animal models of inflammatory diseases including neuromuscular disorders.
A DNA shuffling-based approach for developing cell type-specific vectors is an intriguing possibility to achieve altered tropism. Capsid genomes of AAV serotypes 1-9 were randomly reassembled using PCR to generate a chimeric capsid library.[67] A single infectious clone (chimeric-1829) containing genome fragments from AAV1, 2, 8, and 9 was isolated from an integrin minus hamster melanoma cell line previously shown to have low permissiveness to AAV. Molecular modeling studies suggest that AAV2 contributes to surface loops at the icosahedral threefold axis of symmetry, while AAV1 and 9 contribute to two-fold and five-fold symmetry interactions, respectively.
A versatile rAAV targeting system to redirect rAAV-mediated transduction to specific cell surface receptors would be useful. Insertion of an IgG binding domain of protein A into the AAV2 capsid at amino acid position 587 could permit antibody-mediated vector retargeting, although producing mosaic particles is required to avoid low particle yields.[68] Alternatively, a targeting system using the genetic fusion of short biotin acceptor peptide along with the metabolic biotinylation via a biotin ligase was developed for the purification and targeting of multiple AAV serotypes.[69]
Although an increasing number of scalable methods for purification of rAAV have been described, in order to generate sufficient clinical-grade vector to support clinical trials we need to further improve a large-scale GMP-compatible system for production and purification. To translate gene transduction technologies into clinical practice, development of an effective delivery system with improved vector constructs as well as efficient immunological modulation must be established. A novel protocol that considers all of these issues would help improve the therapeutic benefits of clinical gene therapy.
This work was supported by the Grant for Research on Nervous and Mental Disorders, Health Science Research Grants for Research on the Human Genome and Gene Therapy; and the Grant for Research on Brain Science from the Ministry of Health, Labor and Welfare of Japan. This work was also supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT). We would like to thank Dr. James M. Wilson for providing p5E18-VD2/8 and pAAV2/9.
Air pollution is a worldwide concern because of the health problems associated with its uncontrolled emissions that affect all the biological systems. Within the wide range of pollutants, the suspended particles or particulate matter (PM) are of particular interest, which became more important since IARC listed them as carcinogens. The toxicity of PM is the consequence of the elements adhered to its surface [1]. An example of this are the particles generated by the combustion of fossil fuels and its derivatives, these particles usually consist of a carbon core on which complex mixtures of compounds are adhered, such as: polyaromatic hydrocarbons, toxins, sulfates, nitrates, and especially transition metals like vanadium, manganese, chromium, among others [2]. Metals are considered to play an important role in the induction of toxic effects reported in the literature [3].
Metals are the largest category of globally distributed pollutants with a tendency to accumulate in some human tissues and with a high toxic potential at relatively low concentrations. Constant exposure to certain metals has been linked to inflammation, cell damage, and cancer [4]. Each metal has its own mechanisms of action [5]. Some of them produce its adverse effects alone, while others interact with various factors resulting in greater damage in different organs and systems [4]. It is known that metals, including vanadium, have different toxic pathways, and oxidative stress is the most frequent mechanism [5].
Oxidative stress is the consequence of an imbalance between the production of free radicals and the antioxidant capacity of an organism [6]. It may result from the increase in exposure to oxidants, due to the decrease in the protection against oxidants, or because both events occur simultaneously [7].
A free radical represents any chemical species of independent existence that has one or more missing electrons spinning in its external atomic orbitals. This electrochemical configuration is unstable and gives them property of being a highly reactive and short-lived chemical species [8]. Most of the free radicals of biological interest are usually extremely reactive and have a very short life span (microsecond fractions). When a radical reacts with a non-radical compound, it results in other free radicals, thus generating chain reactions that produce biological effects [9], coupled with the fact that when they collide with a biomolecule and subtract an electron (oxidizing it), it loses its specific function in the cell [8].
Regardless of the origin, free radicals can interact with the biomolecules found in the cell such as nucleic acids [10], proteins, lipids, and carbohydrates [9], thereby causing potentially serious modifications and consequences in the cell [10].
Vanadium is an element that is find in various oxidation states and participates in reactions that lead to the production of free radicals such as superoxide, peroxovanadyl, and the highly reactive radical hydroxyl [8].
The increasing production of free radicals leads the cell to an imbalance in its redox state and thus generating oxidative stress; therefore, the cellular dysfunction will be reflected in the failure of organs and systems.
The cell is the basic functional unit of life and its dysfunction induced by oxidative stress might produce DNA damage and cell death.
The International Agency for Research on Cancer lists vanadium pentoxide (V2O5) as “a possible carcinogen for humans” in group 2B. The above was based on evidence of lung cancer generation in mice that was published by the National Toxicology Program [11]. However, evidence on the carcinogenicity of vanadium has been widely questioned by Duffus in 2007 [12] and Starr et al. [13]. Information related to the carcinogenic and genotoxic potential of vanadium pentoxide (V2O5) is limited [14]. In both animal and human models, the effects on the DNA caused by vanadium include single strand breaks, micronuclei, chromosomal aberrations (structural and numerical), and oxidation of nitrogenous bases [15, 16]. The spectrum of alterations that DNA might have as a consequence of free radicals interaction, in this case caused by vanadium, are: deoxyribose oxidation, modification of nitrogen bases, chain cross-linking, and ruptures [6]. The double or single chain breaks that are generated by the interaction of free radicals with DNA are produced by the fragmentation of the sugar-phosphate skeleton or indirectly by the cleavage of oxidized bases [17].
The above indicates that vanadium is an element with mutagenic potential, because its genotoxic, aneugenic, and clastogenic effects, although there are not strongly data supporting that vanadium is carcinogenic, this possibility should not be eliminated, because the DNA damage caused by the exposure and therefore genetic instability, processes closely related to the generation of malignancy [18].
Cell death is central to physiological homeostasis; the balance between cellular differentiation, proliferation, and death support aspects of biology, including embryogenesis, organ function, tissue remodeling, immune regulation, and carcinogenesis. Cell death was once believed to be the result of one of three different processes: apoptosis, autophagy or necrosis; however, in the last decade about 15 different types have been reported, highlighting that a cell can die via different pathways which depends on the intensity of the stimuli [19]. Reactive oxygen species (ROS) activates cell death and plays different roles in the biological systems which can be either injurious or beneficial. Generation of ROS might be caused by metals such as: arsenic, cadmium, chromium, cobalt, copper, gold, iron, nickel, rhodium titanium or vanadium [8]. Vanadium compounds can interconvert into different species (vanadyl and vanadate) event which is constantly occurring inside the cell in the presence of ROS [20].
Studies in vivo and in vitro showed that vanadium compounds induce cell death in leukemia [21], lung cancer [22] cervical and breast carcinoma [23], neuroblastoma [24], liver carcinoma [25], osteosarcoma [26], and pancreatic ductal adenocarcinoma [27]. In vitro studies demonstrated that the cell lines stimulated with vanadium compounds produce H2O2 and O2 and induce autophagy, necroptosis, and mitotic catastrophe [27]. Apoptosis is the main type of cell death associated with vanadium compounds, reporting the release of cytochrome c from mitochondria [21] and the disruption of the mitochondrial membrane potential [25]. This type of cell death is mediated through the activation of p53 and p21 [27], which modulate the activation or inactivation of phosphorylation of some proteins such as MEK, ERK 1/2, PI3K, p38, JNK, TNF-alpha, and NFkB [28].
The systemic vanadium effects observed in vivo and in vitro are briefly described below.
The reprotoxic effects of vanadium in male reproductive system in laboratory animals include interruption of spermatogenesis [29], morphological and biochemical changes in spermatogenic cells [30], abnormalities in the shape and movement of sperm, as well as decrease in the weight of the testis, epididymis, and prostate [31].
One of the mechanisms of vanadium toxicity includes imbalance in the cellular redox state [30]; testicular cells are highly susceptible to free radical actions because its membranes are rich in polyunsaturated fatty acids, which limits the fluidity of the membrane and alters the functioning of integral membrane proteins [32].
In rat’s testis, after given sodium metavanadate (NaVO3), an increase in malondialdehyde (MDA) was found, which is a product of lipid peroxidation, as well as a decrease in the activity of superoxide dismutase (SOD) and catalase [33]. Intraperitoneal administration of NaVO3 caused in the testis a decrease in the number of germ cells, the presence of degenerated cells, and necrosis of the seminiferous tubules, associated to the increase in testicular lipid peroxidation and inhibition of the activity of antioxidant enzymes (SOD and catalase) [34]; alteration in spermatogenesis, decrease in serum testosterone, LH and FSH levels, inhibition of steroidogenic enzyme activity, increase in testicular vanadium concentration, inhibition of antioxidant enzymes (SOD, catalase and GPx), increased levels of lipid peroxidation [29], and abnormalities in the form of sperm have also been reported [35].
During female reproductive processes, such as ovarian follicle development, ovarian steroid synthesis, ovulation, fertilization, and implantation, require certain amounts of ROS [36]; however, due to the oxidizing effects of vanadium, the delicate balance between ROS generation and the cellular antioxidant system can be altered.
In the case of the female reproductive system of rats, it has been observed that the administration of vanadium sulfate (VOSO3) causes oxidative stress and biochemical alterations in uterine cells, such as the decrease in the activity of alkaline phosphatase and adenosine triphosphatase; while in the ovary, the damage of the oocyte and ovarian follicles was observed, as well as stromal fibrosis [37]. In an inhalation model of vanadium in non-pregnant females, histological alterations were found in the ovary and uterus and lipid peroxidation, indicated by the increase in the levels of 4-hydroxynonenal (4-HNE) a marker of oxidative stress [30].
Vanadium crosses the placental barrier and exerts its toxic effects on embryos and fetuses; in rats, it has been observed that fetal weight decreases and the number of implants and fetuses, while the number of resorptions, malformations, and dead fetus increases [31]. The fetotoxic and embryotoxic effects of vanadium have also been associated with oxidative stress since both in fetuses and in mothers exposed to vanadyl sulfate (VOSO4), and lipid peroxidation was observed in the liver [37].
Kidney chronic disease (CKD) has increased worldwide. The main risk factors for the development of this disease are diabetes, metabolic syndrome, and hypertension. However, there are a segment of population that has none of these risk factors and there are other factors that are being studied as a possible cause of renal injury. One of them is the environmental pollution, particularly pollution by metals in atmosphere and water. Arsenic, cadmium, mercury, lead, and vanadium have been reported as nephrotoxic metals because of the production of ROS and the induction of oxidative stress. These metals enter the body by oral or inhaled exposure, then they are absorbed, enter into the systemic circulation, and distributed into the organs where they may accumulate. Finally, most of them are eliminated by the kidney, reason why this organ is one of the most affected structures by metals [38]. Also, there are reports that in CKD when there is a problem to eliminate pollutant metals, these can concentrate into kidney cells and the damage worsened when it occurs in humans, both in children and adults [39]. Oxidative stress and inflammation are the principal mechanisms of renal injury; in addition, arsenic, cadmium, mercury, and lead are associated to hyperglycemia that may aggravate the oxidative stress and the renal damage. Vanadium is an exception because it has a hypoglycemic effect, but this does not ameliorate its toxicity [40].
Vanadium is nephrotoxic, as it has been proved mainly in animal models, but also in humans [41]. In a report of human acute poisoning by oral ammonium metavanadate, hypoglycemia, bronchoconstriction, and acute renal insufficiency were the causes of death; in a chronic model of vanadium exposure reported glomerulonephritis, glomerular and tubular necrosis that lead to renal insufficiency and hypertension [42]. The reported findings in other study with ammonium metavanadate p.o. at doses of 30, 45, and 60 mg/kg were edema, vacuolization, and degeneration of epithelial tubular cells at 21st day of exposure [43]. Another research group, using different compounds and doses of vanadium (45 and 90 mg/kg) reported thickening of glomerular basement membrane, pyknotic nuclei, cellular vacuolization, and pyelonephritis [44]. In our group, in a subchronic model of vanadium inhalation, we found foci of inflammatory cells, vacuolation, loss of microvilli of epithelial tubular cells, and changes in urine parameters as proteinuria and hematuria associated to the increase, in a time dependent manner, of 4-hidroxynonenal (4-HNE) [45] (Figure 1A and B). Oxidative stress is also the toxic vanadium mechanism reported by other groups, for example, Marouane et al. [46] found lipid peroxidation, protein denaturation, DNA degradation, and cell membrane disintegration; in addition, Scibior et al. [47] reported elevated malonaldehyde (MDA) as a marker of oxidative stress and enhanced total antioxidant status in a rat model of 12-week oral sodium metavanadate exposure.
4-hydrxynonenal (4-HNE) in kidney and liver as a marker of oxidative stress. (A) Kidney tubules in control group with a basal 4-HNE immunoreactivity. (B) In vanadium group, 4-HNE immunoreactivity increased in microvilli of proximal tubules after 8th-week exposure. (C) Liver of control group with a basal 4-HNE immunoreactivity. (D) Liver of vanadium group after 8th-week exposure with increase in 4-HNE immunoreactivity in hepatocytes, some of them with a very intense mark.
The immune system is an interactive network of lymphoid organs, cells, humoral factors, and cytokines whose function is to distinguish between self and non-self-antigens in the host system, thus providing mechanisms against infections and tolerance to the components of the host. When an antigen attacks the host, two distinct, yet interrelated, branches of the immune system are activated, the nonspecific/innate and specific/adaptive immune response. Both of these systems have certain physiological mechanisms, which enable the host to recognize foreign materials as foreign materials and to neutralize, eliminate, or metabolize them [48]. The immune system is a target of air pollutants, such as vanadium that might impair its function and induce oxidative stress.
In previous studies, effects from vanadium inhalation on the immune system have been demonstrated. Changes in the spleen morphology and a decrease in humoral immune responses have been reported [49], as well as a decrease in the number of thymic dendritic cells, its expression of CD11c and MHC-II biomarkers, and an increase of thymic medullar epithelial cells [50]. Oxidative stress could be an important mechanism involved in these effects and some mechanisms are described as follows:
Sodium metavanadate (NaVO3) induced oxidative stress through generation of ROS and depletion of the antioxidant defense systems. When the exposure is chronic, the oxidative stress turns out in severe damage [51].
The effect of vanadyl sulfate (VOSO4) in blood glucose and in the spleen, in streptozotocin (STZ)-induced diabetic rats was evaluated. The levels of lipid peroxidation (LPO) and glutathione (GSH) in the spleen were analyzed. After 60 days of treatment, spleen LPO significantly increased, but spleen GSH levels significantly decreased in the diabetic group. On the other hand, treatment with VOSO4 reversed these effects in STZ diabetic animals [52]. These studies show that vanadium induced oxidative stress in the spleen, which might disrupt the immune response.
The liver as the major site for metabolism, biotransformation and detoxification of drugs and foreign compounds, is constantly exposed to ROS resulting in oxidative stress and frequently, permanent and irreversible tissue damage [53]. Studies have shown that liver is one of the most important target tissues for vanadium toxicity with its capacity to form ROS by interacting with mitochondrial redox centers, mainly in mitochondrial respiratory processes I, II, and III [54]. Studies with HepG2 cell line have shown that exposure to vanadium causes damage to nuclear and mitochondrial DNA, as well as decreased cell viability [55]. In vivo studies from our group demonstrate that vanadium increases lipid peroxidation in V-exposed animals [56]. Figure 1C and D show the oxidative marker 4-HNE in liver parenchyma.
As a heavily irrigated, highly connected organ with neural, endocrine, digestive, absorptive, and immune functions, the gut can enter oxidative cycles mostly by two well-defined mechanisms:
Ambient-polluting microparticle swallowing: especially in heavily polluted areas (industrial centers, cities, mines, etc.), the air is charged with carbon PM, whose size varies between 10 and 2.5 (or even less) micrometers. Such particles are normally covered by metals (vanadium, for instance), which get trapped via natural defense mechanisms in the nasal and oral mucosa, slowly, descending into the pharynx and into the digestive tract carried on through saliva [30].
Direct toxic ingestion: recent research relates ingestion of food ingredients—especially sugar (sucrose or high fructose) present mostly in sugar-sweetened beverages (SSB)—with tissue damage. Although there is no specific data on gut tissue damage, it has been reported in other bodily systems—e.g., kidney [45]. This represents a particularly severe problem in a world where no matter the country, the SSB consumption increases steadily year after year [57].
Research on this matter has still a long path to walk. However, preliminary results from ongoing protocols at our laboratory show a significant rise in 4-HNE levels in the gut epithelium in response to air pollution and SSB consumption mice models, which indicate higher oxidative stress levels vs. control groups.
Air pollution has been associated to thrombosis and cardiovascular risk. Pollutants, including PM and metals may induce oxidative stress and inflammation predisposing to endothelial dysfunction, platelet activation, and procoagulant state [58]. There is epidemiological evidence that elevated concentrations of pollutants, e.g., vanadium, are associated to an increase in ER visits for acute cardiovascular effects or exacerbations of preexisting cardiovascular diseases [59].
Vanadium induces oxidative stress, and there is evidence of their toxic effects on endothelium, platelets, and myocardium. An in vitro study using HUVEC (human umbilical vein endothelial cells) exposed to different V2O5 concentrations reported an increase in ROS that damaged endothelial cells leading to apoptosis and diminished proliferation. This might be involved in endothelial dysfunction and increased cardiovascular risk associated to metals [60]. An in vivo vanadium inhalation mice model, from our group, reported thrombocytosis that is an increase in platelet number, but also the presence of giant platelets that are associated to increase reactivity [61]. Also, we found a megakaryocytosis with an increase in megakaryocytes size and granularity because of the activation of JAK/STAT pathway [40, 62, 63]. Platelet aggregation after subchronic vanadium inhalation diminished, but activation markers of platelets P-selectin or CD-62p were increased after the 4th week of exposure, maybe because of the slow elimination of vanadium, so it is possible that this metal has on platelet aggregation a long-term effects [64]. Another effect of vanadium on cardiovascular system is arrhythmia; in our group, we studied its effect on myocardium N-cadherin and connexin-43, important proteins in the intercalated discs. The reduction of both proteins and its effect on the electric stimuli conduction was proposed to explain the pathophysiology of the arrhythmias induced by vanadium [65]. Vanadium and other metals induce oxidative stress that may damage several cells of cardiovascular system.
The lung is one of the main targets of air pollution damage because it is the first site in contact with the pollutants suspended in the air. After reaching the alveolar epithelium, the pollutants can cross the alveoli-capillary barrier. There are various reports that demonstrate the damage caused to this organ by exposure to specific contaminants, such as vanadium that is part of the suspended particles.
In vivo, it has been reported that inhaled exposure to vanadium, mainly in the form of pentoxide induces histopathological changes in the lung, such as fibrosis [66], inflammation [30, 66, 67], hyperplasia and epithelial metaplasia [30, 67] and apoptotic cell death [68], among others.
Experimental evidence supports that exposure to V2O5 increases the production of ROS in lung cells. Wang et al. [68] reported increase in ROS production in mice bronchoalveolar lavage cells treated with a concentration of 10 μm of sodium metavanadate (NaVO3), in a time-exposure dependent manner (3, 10, 30, and 60 minutes) through a spin trapping essay.
On the other hand, other evidence shows that exposure to V modifies in the lung glutathione concentrations, both in its oxidized (GSSG) and reduced (GSH) forms. It is known that oxidative stress results in the depletion of GSH and the increase in GSS; so, the determination of their respective concentrations in blood and other tissues is considered a measure of intracellular oxidative stress [69].
Schuler et al. reported that in their inhalation model of V2O5 at exposure concentrations of 0.25, 1, and 4 mg/m3, there was an increase in the levels of oxidized glutathione (GSSG) in lung tissue, with the consequent reduction in the ratio between reduced and oxidized glutathione (GSH/GSSG) concentrations [70]. Kulkarni and colleagues reported the same finding in relation to GSH concentration in lung tissue in a model of exposure to V2O5 nanoparticles [66]. In addition to this finding in the same study, the significant increase in MDA levels in plasma was identified. The MDA is a final product of lipid peroxidation.
Another biomarker of oxidative damage that has been identified is the 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxoGuo) in the DNA. Schuler demonstrated the increase in the formation of 8-oxoGuoin at exposure concentrations of 1 and 4 mg/m3 of V2O5 in lung cells [70].
Neurotoxic metals as vanadium can induce oxidative damage in the brain and develop blood brain barrier disruption, neuropathology, and neuronal damage that can trigger central nervous system alterations as depression, increase in anger, fatigue, and tremors between other clinical features [71]. Also, a decrease in tyrosine hydroxylase and dopamine levels has been reported after vanadium exposure [72]. Chronical exposure to NaVO3 can cause, in mice, metal accumulation in the olfactory bulb, brain stem, and cerebellum, as well as histopathological alterations like nuclear shrinkage in the prefrontal cortex and cell death of the hippocampal pyramidal cells and cerebellum Purkinje cells [71]. The accumulation of vanadium in the brain depends more on the exposure time than on the concentration of the metal. In fact, it is reported that disruption of ependymal cells is observed after long periods of vanadium inhalation [73].
Recently, behavioral alterations due to vanadium occupational exposure have been reported. Vanadium exposed workers exhibited poor performance in the simple reaction time, digit span memory, and Benton visual retention tests [74]. Memory loss in mice exposed to vanadium for 3 months was observed; nevertheless, in these animals, memory was recovered 9 months after vanadium was removal [75]. Increased incidence of Parkinson’s disease is related to environmental metal exposure. It has been reported that vanadium pentoxide (V2O5) is neurotoxic to dopaminergic neurons via caspase-3-dependent PKCδ cleavage, so maybe vanadium can promote nigral dopaminergic degeneration [76].
The cells exposed continuously to oxidative stress are not defenseless against free radicals. All aerobic organisms count with a series of mechanisms protecting them against oxidative damage; among them are antioxidant molecules which represent a first line of defense. If the antioxidant mechanisms fail, the cell uses others such as: transient cell arrest, biomolecular repair systems or apoptosis death processes [7].
An antioxidant is any substance that when is present in low concentrations, compared to the oxidizable substrate, decreases or prevents the substrate oxidation. Oxidizable substrates comprise everything that is found in living tissues including proteins, lipids, carbohydrates, and nucleic acids [77].
Cells use a series of antioxidant compounds that react directly with oxidizing agents, functioning as “sweepers” or chemical shields [7]; these molecules have enzymatic or non-enzymatic actions. Non-enzymatic antioxidants carry out the reduction of free radicals through electron donation, thus avoiding oxidative reactions. Glutathione (GSH), alpha-tocopherol (vitamin E), ascorbic acid (vitamin C), carnosine, bilirubin, and uric acid are the main molecules performing this function.
Ascorbate is an important water-soluble antioxidant in biological fluids, because it eliminates reactive oxygen species and radicals such as: alkoxy, hydroxyl, peroxyl, and hydroperoxyl radicals, singlet oxygen, superoxide anion, and ozone. It also eliminates reactive species and radicals derived from nitrogen and chlorine and even radicals that come from other antioxidants [78].
In general, a large number of studies have been carried out to show the beneficial effects of ascorbate. Evidence indicates that supplementation with this compound protects against lipid oxidation in vivo, particularly in individuals exposed to exacerbated conditions of oxidative stress, such as smokers [79].
Epidemiological studies of treatment with this antioxidant have shown consistently favorable effects in patients with cardiovascular disease or coronary risk. In addition, it has been suggested that the increase in ascorbate consumption significantly decreases the incidence and mortality from cardiovascular diseases. Even in pathologies related to free radicals and the inability of the organism defenses against them, as is the case of cancer, epidemiological studies show that increased consumption of ascorbate decreases the incidence and mortality from cancer [79].
Experimental evidence indicates that ascorbic acid works as an antidote against acute vanadium poisoning. In mice, Jones and Basinger [80] examined several compounds and concluded that ascorbate was the most promising for human use.
Domingo et al. [81] administered NaVO3 to mice intraperitoneally and observed, as did Jones and Basinger, that ascorbate proved to be the most effective antidote against vanadium poisoning. In another study, Domingo et al. [82] showed that ascorbate stimulates urinary excretion of vanadium when mice are injected intramuscularly with VOSO4.
Another water-soluble antioxidant is carnosine which is a dipeptide composed of β-alanine and L-histidine; it is found naturally in many mammalian species, mainly in the skeletal muscle. It is estimated that 99% of the carnosine in the organism is found in muscular tissue [83].
It has been reported that carnosine may form complexes with transition metals and has antioxidant activity, which implies mechanisms such as chelation of metals, scavenging of ROS, and peroxyl radicals [83].
The antioxidant efficiency of carnosine in the nervous system, when mice are exposed to vanadium inhalation was successfully tested by our group. It was observed that in those groups with carnosine treatment, a larger size granulose cells with a greater number of dendritic spines, and in general less adverse ultrastructural morphological changes, as well as less lipid peroxidation were observed [84].
Air pollution has been continuously mentioned as one of the problems that decrease the quality and life expectancy of all living organisms, included humankind. It is true that not all the sources of pollution are from anthropological origin; however, a great deal of it are generated by humans and can be prevented or controlled by those who generate it.
The use of fossil fuels as the quasi unique source of energy and limited use of other sources of energy will maintain the air pollutant levels high enough to keep its deleterious health effects.
As it is revised in this chapter, metals are one of the air pollutants that enter through the respiratory tract, reaching by the systemic circulation every cell in living organisms. Vanadium is one of the elements adhered to PM which results from the incomplete combustion of fossil fuels. PM generates ROS, mainly those that contains transition metals (e.g., Fe, V, and Mn).
Reported previously in this chapter, one of the main toxic mechanisms of metals is oxidative stress which affects all biomolecules. DNA oxidative damage may conduct the cell to genotoxic and mutagenic changes and further to cell death or cancer.
When proteins are oxidized: cell structure, cell signaling modification, and/or disruption of cellular enzymatic processes could be noticed. The reactive molecules which results from these interactions with proteins and ROS may interplay with specific peptide residues such as: lysines, arginines, histidines, and cysteines. The result of these actions causes the formation of reactive carbonyls and protein carbonylation, and its accumulations have been related with chronic diseases and aging.
If lipids are in contact with ROS, peroxidation occurs producing MDA, a biomarker of oxidative stress that could interact with proteins forming protein adducts and inactivating the protein. Another lipid peroxidation product is 4-HNE with cytotoxic effects and the induction of pro-inflammatory cytokines, which could result in cellular dysfunction and death [85].
If the sources of V or other pollutants are not reduced and the oxidative insults prevail, we can supplement our system with antioxidants such as vitamin C. This water-soluble molecule is not synthesized by humans, and its supplementation is obtained by different dietary sources such as fruits and vegetables or by vitamin C supplements. One of the benefits of vitamin C is its antioxidant action, scavenging ROS and NOS species. In addition, it helps to regenerate alpha-tocopherol and coenzyme Q; also, vitamin C inhibits NAD(P)H oxidase decreasing ROS formation [86]. Another less known endogenous and exogenous antioxidant is carnosine that in our laboratory showed promising antioxidant effects in the nervous system [84].
The systems and organs affected by the oxidative potential of vanadium and the protective effect of antioxidants are summarized in Figure 2.
Oxidative stress by vanadium and antioxidants protective effects (this figure was created by Biorender software in www.biorender.com).
While humankind decide to work together in order to find a common solution for controlling air pollution, scientist should be working in finding more and better antioxidants to prevent and ameliorate the effects that metals, such as V adhered to PM, have on living organisms, that meanwhile might reduce oxidative stress, its injurious effects and improves the quality of life on the planet.
This work was partially supported by project PAPIIT-DGAPA UNAM IN200418.
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