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1.  An overview of underlying causes and animal models for the study of age-related degenerative disorders of the spine and synovial joints 
As human lifespan increases so does the incidence of age-associated degenerative joint diseases, resulting in significant negative socioeconomic consequences. Osteoarthritis (OA) and intervertebral disc degeneration (IDD) are the most common underlying causes of joint-related chronic disability and debilitating pain in the elderly. Current treatment methods are generally not effective and involve either symptomatic relief with non-steroidal anti-inflammatory drugs and physical therapy or surgery when conservative treatments fail. The limitation in treatment options is due to our incomplete knowledge of the molecular mechanism of degeneration of articular cartilage and disc tissue. Basic understanding of the age-related changes in joint tissue is thus needed to combat the adverse effects of aging on joint health. Aging is caused at least in part by time-dependent accumulation of damaged organelles and macromolecules, leading to cell death and senescence and the eventual loss of multipotent stem cells and tissue regenerative capacity. Studies over the past decades have uncovered a number of important molecular and cellular changes in joint tissues with age. However, the precise causes of damage, cellular targets of damage, and cellular responses to damage remain poorly understood. The objectives of this review are (1) to provide an overview of the current knowledge about the sources of endogenous and exogenous damaging agents and how they contribute to age-dependent degenerative joint disease, and (2) highlight animal models of accelerated aging that could potentially be useful for identifying causes of and therapies for degenerative joint diseases.
doi:10.1002/jor.22204
PMCID: PMC3628921  PMID: 23483579
Synovial joints; intervertebral disc; DNA repair; aging; oxidative damage
2.  DNA damage drives accelerated bone aging via an NF-κB-dependent mechanism 
Advanced age is one of the most important risk factors for osteoporosis. Accumulation of oxidative DNA damage has been proposed to contribute to age-related deregulation of osteoblastic and osteoclastic cells. ERCC1 (Excision Repair Cross Complementary group 1)-XPF (Xeroderma Pigmentosum Group F) is an evolutionarily conserved structure-specific endonuclease that is required for multiple DNA repair pathways. Inherited mutations affecting expression of ERCC1-XPF cause a severe progeroid syndrome in humans, including early onset of osteopenia and osteoporosis, or anomalies in skeletal development. Herein, we used progeroid ERCC1-XPF deficient mice, including Ercc1-null (Ercc1−/−) and hypomorphic (Ercc1−/Δ) mice, to investigate the mechanism by which DNA damage leads to accelerated bone aging. Compared to their wild-type littermates, both Ercc1−/− and Ercc1−/Δ mice display severe, progressive osteoporosis caused by reduced bone formation and enhanced osteoclastogenesis. ERCC1 deficiency leads to atrophy of osteoblastic progenitors in the bone marrow stromal cell (BMSC) population. There is increased cellular senescence of BMSCs and osteoblastic cells, as characterized by reduced proliferation, accumulation of DNA damage and a senescence-associated secretory phenotype (SASP). This leads to enhanced secretion of inflammatory cytokines known to drive osteoclastogenesis, such as IL-6, TNFα, and RANKL and thereby induces an inflammatory bone microenvironment favoring osteoclastogenesis. Furthermore, we found that the transcription factor NF-κB is activated in osteoblastic and osteoclastic cells of the Ercc1 mutant mice. Importantly, we demonstrated that haploinsufficiency of the p65 NF-κB subunit partially rescued the osteoporosis phenotype of Ercc1−/Δ mice. Finally, pharmacological inhibition of the NF-κB signaling via an IKK inhibitor reversed cellular senescence and SASP in Ercc1−/Δ BMSCs. These results demonstrate that DNA damage drives osteoporosis through an NF-κB-dependent mechanism. Therefore, the NF-κB pathway represents a novel therapeutic target to treat aging-related bone disease.
doi:10.1002/jbmr.1851
PMCID: PMC3662975  PMID: 23281008
osteoporosis; osteoblasts; osteoclasts; bone; nucleotide excision repair; progeria; aging; NF-κB transcription factor; ERCC1-XPF endonuclease
3.  Pharmacologic IKK/NF-κB inhibition causes antigen presenting cells to undergo TNFα dependent ROS-mediated programmed cell death 
Scientific Reports  2014;4:3631.
Monocyte-derived antigen presenting cells (APC) are central mediators of the innate and adaptive immune response in inflammatory diseases. As such, APC are appropriate targets for therapeutic intervention to ameliorate certain diseases. APC differentiation, activation and functions are regulated by the NF-κB family of transcription factors. Herein, we examined the effect of NF-κB inhibition, via suppression of the IκB Kinase (IKK) complex, on APC function. Murine bone marrow-derived macrophages and dendritic cells (DC), as well as macrophage and DC lines, underwent rapid programmed cell death (PCD) after treatment with several IKK/NF-κB inhibitors through a TNFα-dependent mechanism. PCD was induced proximally by reactive oxygen species (ROS) formation, which causes a loss of mitochondrial membrane potential and activation of a caspase signaling cascade. NF-κB-inhibition-induced PCD of APC may be a key mechanism through which therapeutic targeting of NF-κB reduces inflammatory pathologies.
doi:10.1038/srep03631
PMCID: PMC3887388  PMID: 24406986
4.  Genotoxic stress accelerates age-associated degenerative changes in intervertebral discs 
Intervertebral disc degeneration (IDD) is the leading cause of debilitating spinal disorders such as chronic lower back pain. Aging is the greatest risk factor for IDD. Previously, we demonstrated IDD in a murine model of a progeroid syndrome caused by reduced expression of a key DNA repair enzyme. This led us to hypothesize that DNA damage promotes IDD. To test our hypothesis, we chronically exposed adult wild-type (Wt) and DNA repair-deficient Ercc1−/Δ mice to the cancer therapeutic agent mechlorethamine (MEC) or ionization radiation (IR) to induce DNA damage and measured the impact on disc structure. Proteoglycan, a major structural matrix constituent of the disc, was reduced 3-5x in the discs of MEC- and IR-exposed animals compared to untreated controls. Expression of the protease ADAMTS4 and aggrecan proteolytic fragments were significantly increased. Additionally, new PG synthesis was reduced 2-3x in MEC- and IR-treated discs compared to untreated controls. Both cellular senescence and apoptosis were increased in discs of treated animals. The effects were more severe in the DNA repair-deficient Ercc1−/Δ mice than in Wt littermates. Local irradiation of the vertebra in Wt mice elicited a similar reduction in PG. These data demonstrate that genotoxic stress drives degenerative changes associated with IDD.
doi:10.1016/j.mad.2012.11.002
PMCID: PMC3558562  PMID: 23262094
Intervertebral disc; aging; DNA damage; genotoxic stress; matrix proteoglycan
5.  Plasma-derived MHCII+ exosomes from tumor-bearing mice suppress tumor antigen-specific immune responses 
European journal of immunology  2012;42(7):1778-1784.
Tumor-specific immunosuppression is frequently observed in tumor-bearing hosts. Exosomes are nano-sized, endosomal-derived membrane vesicles secreted by most tumor and hematopoietic cells and have been shown to actively participate in immune regulation. We previously demonstrated that antigen-specific immunosuppressive exosomes could be isolated from the blood plasma of antigen-immunized mice. Here we demonstrate that plasma-derived exosomes isolated from mice bearing OVA-expressing tumors were able to suppress OVA-specific immune response in a mouse delayed-type hypersensitivity model. Enrichment of tumor-derived exosomes in the plasma of mice bearing subcutaneous melanoma was not detected using an exosome-tagging approach. Instead, depletion of MHC Class II+ vesicles from plasma-derived exosomes or using plasma-derived exosomes isolated from MHC Class II deficient mice resulted in significant abrogation of the suppressive effect. These results demonstrate that circulating host-derived, MHC Class II+ exosomes in tumor-bearing hosts are able to suppress the immune response specific to tumor antigens.
doi:10.1002/eji.201141978
PMCID: PMC3471134  PMID: 22585706
6.  ISSLS PRIZE WINNER: INHIBITION OF NF-κB ACTIVITY AMELIORATES AGE-ASSOCIATED DISC DEGENERATION IN A MOUSE MODEL OF ACCELERATED AGING 
Spine  2012;37(21):1819-1825.
Study Design
NF-κB activity was pharmacologically and genetically blocked in an accelerated aging mouse model to mitigate age-related disc degenerative changes.
Objective
To study the mediatory role of NF-κB signaling pathway in age-dependent intervertebral disc degeneration.
Summary of Background Data
Aging is a major contributor to intervertebral disc degeneration (IDD), but the molecular mechanism behind this process is poorly understood. NF-κB is a family of transcription factors which play a central role in mediating cellular response to damage, stress, and inflammation. Growing evidence implicates chronic NF-κB activation as a culprit in many aging-related diseases, but its role in aging-related IDD has not been adequately explored. We studied the effects of NF-κB inhibition on IDD using a DNA repair-deficient mouse model of accelerated aging (Ercc1-/Δ mice) previously been reported to exhibit age-related IDD.
Methods
Systemic inhibition of NF-κB activation was achieved either genetically by deletion of one allele of the NF-κB subunit p65 (Ercc1-/Δp65+/- mice) or pharmacologically by chronic intra-peritoneal administration of the Nemo Binding Domain (8K-NBD) peptide to block the formation of the upstream activator of NF-κB, IκB Inducible Kinase (IKK), in Ercc1-/Δ mice. Disc cellularity, total proteoglycan content and proteoglycan synthesis of treated mice and untreated controls were assessed.
Results
Decreased disc matrix proteoglycan content, a hallmark feature of IDD, and elevated disc NF-κB activity were observed in discs of progeroid Ercc1-/Δ mice and naturally aged wild-type compared to young WT mice. Systemic inhibition of NF-κB by the 8K-NBD peptide in Ercc1-/Δ mice increased disc proteoglycan synthesis and ameriolated loss disc cellularity and matrix proteoglycan. These results were confirmed genetically by using the p65 haploinsufficient Ercc1-/Δp65+/- mice.
Conclusion
These findings demonstrate that the IKK/NF-κB signaling pathway is a key mediator of age-dependent IDD and represents a therapeutic target for mitigating disc degenerative diseases associated with aging.
doi:10.1097/BRS.0b013e31824ee8f7
PMCID: PMC3395770  PMID: 22343279
NF-kB;aging; proteoglycan; disc degeneration; DNA damage repair; ERCC1-deficient mice
7.  AAV8-Mediated Gene Transfer of Interleukin-4 to Endogenous β-Cells Prevents the Onset of Diabetes in NOD Mice 
We previously demonstrated that intra-peritoneal delivery of adeno-associated virus serotype 8 (AAV8) stably transduces the pancreas, including the β-cells in the endogenous islets. We also demonstrated the ability to deliver and express genes specifically in β-cells for at least 6 months using a murine insulin promoter (mIP) in a double-stranded, self-complementary AAV vector (dsAAV8-mIP). Here we evaluated the effects of dsAAV8-mIP mediated delivery of interleukin 4 (mIL-4) to endogenous β-cells in NOD mice. In 4 week old NOD mice, the extent of gene transfer and expression in endogenous β-cells following i.p. delivery of dsAAV8-mIP-eGFP was comparable to normal BALB/c mice. Furthermore, following i.p. delivery of dsAAV8-mIP-IL4, expression of mIL-4 was detected in islets isolated and cultured from the treated mice. AAV8-mIP mediated gene expression of mIL-4 to endogenous β-cells of 4 and 8 week old NOD mice prevented the onset of hyperglycemia in NOD mice and reduced the severity of insulitis. Moreover, expression of mIL-4 also maintained the level of CD4+CD25+FoxP3+ cells and adoptive co-transfer of splenocytes from diabetes-free IL-4 vector recipients and splenocytes from wild type diabetic NOD mice prevented the onset diabetes. Taken together, these results demonstrate that local expression of mIL-4 in islets prevents islet destruction and blocks autoimmunity, in part, through regulation of T cell function. These results also demonstrate the utility of using dsAAV8-mIP gene transfer to endogenous NOD β-cells to examine the role of specific gene products in preventing or exacerbating the onset of type 1 diabetes.
doi:10.1038/mt.2008.116
PMCID: PMC3560428  PMID: 18560422
8.  The Anti-tumor Effects of Adenoviral-Mediated, Intra-tumoral delivery Interleukin 23 require Endogenous IL-12 
Cancer gene therapy  2011;19(2):135-143.
IL-23 is a member of the IL-12 family of heterodimeric cytokines, comprised of p19 and p40 subunits, which exhibits immunostimulatory properties similar to IL-12. We have demonstrated previously that adenoviral-mediated, intra-tumoral delivery of IL-23 (Ad.IL-23) was able to induce systemic anti-tumor immunity. Here we demonstrate that Ad.IL-23 requires endogenous IL-12 for conferring an anti-tumor effect after adenoviral-mediated intra-tumoral delivery. In contrast, Ad.IL-12 does not require IL-23 for its anti-tumor effects although endogenous IL-23 appears important for induction of systemic anti-tumor immunity by IL-12. However, despite the requirement for endogenous IL-12, co-delivery of IL-23 and IL-12 does not provide even an additive local or systemic anti-tumor effect, regardless of the dose. We further demonstrate that although the use of a single chain IL-23 (scIL-23) results in higher level of expression and a more pronounced IL-23-mediated anti-tumor effect, there is still no synergy with IL-12. These results demonstrate that whereas significant anti-tumor effects are achieved by intratumoral injection of adenovirus expressing either scIL-23 or IL-12 alone and that IL-23 requires endogenous IL-12 for maximum anti-tumor benefit, the combined use of these cytokines provides no additive or synergistic effect.
doi:10.1038/cgt.2011.78
PMCID: PMC3508719  PMID: 22158522
Interleukin 23; Interleukin 12; adenovirus; cancer; gene therapy
9.  Exosomes from IDO+ DC are therapeutic in CIA and DTH disease models 
Arthritis and rheumatism  2009;60(2):380-389.
Objective
We have demonstrated previously that dendritic cells (DC), modified with immunosuppressive cytokines, and exosomes derived from the DC can suppress the onset of murine CIA and reduce the severity of established arthritis. Indoleamine 2,3-dioxygenase (IDO) is a tryptophan degrading enzyme important for immune regulation and tolerance maintenance. DC expressing functional IDO can inhibit T cells by either depleting them of essential tryptophan and/or by producing toxic metabolites, as well as by generating regulatory T cells. In this study, we examined the immunosuppressive effects of bone marrow derived DC, genetically modified to express IDO, and IDO+-DC-derived exosomes.
Methods
Bone marrow derived DC were adenovirally transduced with IDO or CTLA4-Ig (an inducer of IDO), and the resulting DC and exosomes were tested for their immunosuppressive ability in the collagen-induced arthritis and delayed type hypersensitivity murine models.
Results
We demonstrate that both DC and exosomes derived from DC overexpressing IDO are anti-inflammatory in collagen-induced arthritis and delayed type hypersensitivity murine models. The suppressive effects were partially dependent on B7 costimulatory molecules. In addition, gene transfer of CTLA4-Ig to DC resulted in induction of IDO in the DC and exosomes able to reduce inflammation in an IDO-dependent manner.
Conclusion
These results demonstrate that both IDO expressing DC and DC-derived exosomes are immunosuppressive and anti-inflammatory, and are able to reverse established arthritis. Therefore, exosomes from IDO+ DC may represent a novel therapy for rheumatoid arthritis.
doi:10.1002/art.24229
PMCID: PMC3491653  PMID: 19180475
Dendritic cells; Exosomes; IDO; Arthritis; Inflammatory disease
10.  Isolation of Muscle-Derived Stem/Progenitor Cells based on adhesion characteristics to collagen-coated surfaces 
Summary
Our lab developed and optimized a method, known as the modified pre-plate technique, to isolate stem/progenitor cells from skeletal muscle. This method separates different populations of myogenic cells based on their propensity to adhere to collagen I-coated surface. Based on their surface markers and stem-like properties, including self-renewal, multi-lineage differentiation, and ability to promote tissue regeneration, the last cell fraction or slowest to adhere to the collagen-coated surface (pre-plate 6; pp6) appear to be early, quiescent progenitor cells termed muscle-derived stem/progenitor cells (MDSPCs). The cell fractions preceding pp6 (pp1–5) are likely populations of more committed (differentiated) cells, including fibroblast- and myoblastlike cells. This technique may be used to isolate MDSPCs from skeletal muscle of humans or mice regardless of age, sex or disease state, although the yield of MDSPCs varies with age and health. MDSPCs can be used for regeneration of a variety of tissues including bone, articular cartilage, skeletal and cardiac muscle and nerve. MDSPCs are currently being tested in clinical trials for treatment of urinary incontinence and myocardial infarction. MDSPCs from young mice have also been demonstrated to extend lifespan and healthspan in mouse models of accelerated aging through an apparent paracrine/endocrine mechanism. Here we detail methods for isolation and characterization of MDSPCs.
doi:10.1007/978-1-62703-317-6_5
PMCID: PMC3731996  PMID: 23400434
Pre-plate technique; muscle-derived stem/progenitor cells; muscle; adult stem cells; regenerative medicine
11.  The oxidative DNA lesions 8,5′-cyclopurines accumulate with aging in a tissue-specific manner 
Aging Cell  2012;11(4):714-716.
Summary
Accumulation of DNA damage is implicated in aging. This is supported by the fact that inherited defects in DNA repair can cause accelerated aging of tissues. However, clear-cut evidence for DNA damage accumulation in old age is lacking. Numerous studies report measurement of DNA damage in nuclear and mitochondrial DNA from tissues of young and old organisms, with variable outcomes. Variability results from genetic differences between specimens or the instability of some DNA lesions. To control these variables and test the hypothesis that elderly organisms have more oxidative DNA damage than young organisms, we measured 8,5′-cyclopurine-2′-deoxynucleosides (cPu), which are relatively stable, in tissues of young and old wild-type and congenic progeroid mice. We found that cPu accumulate spontaneously in the nuclear DNA of wild-type mice with age and to a greater extent in DNA repair-deficient progeroid mice, with a similar tissue-specific pattern (liver>kidney>brain). These data, generated under conditions where genetic and environmental variables are controlled, provide strong evidence that DNA repair mechanisms are inadequate to clear endogenous lesions over the lifespan of mammals. The similar, although exaggerated, results obtained from progeroid, DNA repair-deficient mice and old normal mice support the conclusion that DNA damage accumulates with, and likely contributes to aging.
doi:10.1111/j.1474-9726.2012.00828.x
PMCID: PMC3399950  PMID: 22530741
ageing; progeria; DNA damage; nucleotide excision repair; oxidative DNA lesion
12.  LIM DOMAIN PROTEIN-3 (LMP3) COOPERATES WITH BMP7 TO PROMOTE TISSUE REGENERATION BY LIGAMENT PROGENITOR CELLS 
Gene therapy  2012;20(1):1-6.
Gene transfer of key regulators of osteogenesis for mesenchymal stem cells (MSCs) represents a promising strategy to regenerate bone. It has been reported that LMP3, a transcription variant of LIM domain mineralization protein (LMP) lacking LIM domains, can induce osteogenesis in vitro and in vivo. Since little is known about the effects of LMP3 gene therapy on periodontal ligament (PDL) cell osteogenic differentiation, this study sought to explore whether gene delivery of LMP3 can promote PDL cell mineralization and bone formation. Our results showed that adenoviral mediated gene transfer of LMP3 (AdLMP3) significantly upregulated ALP, BSP, and BMP2 gene expression and increased in vitro matrix mineralization in human PDL. Although AdLMP3 gene delivery to PDL cells did not induce ectopic bone formation in vivo, we found that AdLMP3 augments new bone formation, which co-delivered with AdBMP7 gene transfer. Our study provides evidence that there is a synergistic effect between LMP3 and BMP-7 in vivo, suggesting that LMP3 delivery may be used to augment BMP-mediated osteogenesis. LMP3 and BMP-7 combinatory gene therapy may also have specific applications for oral and periodontal regenerative medicine.
doi:10.1038/gt.2011.203
PMCID: PMC3330200  PMID: 22241179
gene therapy; LIM domain mineralization protein; bone morphogenetic protein; osteogenesis; regenerative medicine
13.  Synovial fibroblasts spread rheumatoid arthritis to unaffected joints 
Nature medicine  2009;15(12):1414-1420.
Active rheumatoid arthritis is characterized by originating from few but affecting subsequently the majority of joints. Thus far, the pathways of the progression of the disease are largely unknown. As rheumatoid arthritis synovial fibroblasts (RASFs) are key players in joint destruction and migrate in vitro, the current study evaluated the potential of RASFs to spread the disease in vivo. To simulate the primary joint of origin, healthy human cartilage was co-implanted subcutaneously into SCID mice together with RASFs. At the contralateral flank, healthy cartilage was implanted without cells. RASFs showed an active movement to the naïve cartilage via the vasculature independent of the site of application of RASFs into the SCID mouse, leading to a strong destruction of the target cartilage. These findings support the hypothesis that the characteristic clinical phenomenon of destructive arthritis spreading between joints is mediated, at least in part, by the transmigration of activated RASFs.
doi:10.1038/nm.2050
PMCID: PMC3678354  PMID: 19898488
14.  NF-κB inhibition delays DNA damage–induced senescence and aging in mice 
The Journal of Clinical Investigation  2012;122(7):2601-2612.
The accumulation of cellular damage, including DNA damage, is thought to contribute to aging-related degenerative changes, but how damage drives aging is unknown. XFE progeroid syndrome is a disease of accelerated aging caused by a defect in DNA repair. NF-κB, a transcription factor activated by cellular damage and stress, has increased activity with aging and aging-related chronic diseases. To determine whether NF-κB drives aging in response to the accumulation of spontaneous, endogenous DNA damage, we measured the activation of NF-κB in WT and progeroid model mice. As both WT and progeroid mice aged, NF-κB was activated stochastically in a variety of cell types. Genetic depletion of one allele of the p65 subunit of NF-κB or treatment with a pharmacological inhibitor of the NF-κB–activating kinase, IKK, delayed the age-related symptoms and pathologies of progeroid mice. Additionally, inhibition of NF-κB reduced oxidative DNA damage and stress and delayed cellular senescence. These results indicate that the mechanism by which DNA damage drives aging is due in part to NF-κB activation. IKK/NF-κB inhibitors are sufficient to attenuate this damage and could provide clinical benefit for degenerative changes associated with accelerated aging disorders and normal aging.
doi:10.1172/JCI45785
PMCID: PMC3386805  PMID: 22706308
15.  Regenerating the injured kidney with human umbilical cord mesenchymal stem cell-derived exosomes 
Transplantation of adult stem cells is being used to facilitate repair or regeneration of damaged or diseased tissues. However, in many cases, the therapeutic effects of the injected stem cells are mediated by factors secreted by stem cells and not by differentiation of the transplanted stem cells. Recent reports have identified a class of microvesicles, termed exosomes, released by stem cells that are able to confer therapeutic effects on injured renal and cardiac tissue. In this issue of Stem Cell Research & Therapy, Zhou and colleagues demonstrate the ability of exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs), but not non-stem cell-derived exosomes, to improve acute kidney injury induced by cisplatin in rats. The authors demonstrate that hucMSC exosomes can reduce cisplatin-mediated renal oxidative stress and apoptosis in vivo and increase renal epithelial cell proliferation in culture. These results suggest that stem cell-derived exosomes, which are easy to isolate and safer to use than the parental stem cells, could have significant clinical utility.
doi:10.1186/scrt187
PMCID: PMC3706756  PMID: 23680102
16.  Immunosuppressive Exosomes: A New Approach for Treating Arthritis 
Rheumatoid arthritis (RA) is a chronic autoimmune disease and one of the leading causes of disability in the USA. Although certain biological therapies, including protein and antibodies targeting inflammatory factors such as the tumor necrosis factor, are effective in reducing symptoms of RA, these treatments do not reverse disease. Also, although novel gene therapy approaches have shown promise in preclinical and clinical studies to treat RA, it is still unclear whether gene therapy can be readily and safely applied to treat the large number of RA patients. Recently, nanosized, endocytic-derived membrane vesicles “exosomes” were demonstrated to function in cell-to-cell communication and to possess potent immunoregulatory properties. In particular, immunosuppressive DC-derived exosomes and blood plasma- or serum-derived exosomes have shown potent therapeutic effects in animal models of inflammatory and autoimmune disease including RA. This paper discusses the current knowledge on the production, efficacy, mechanism of action, and potential therapeutic use of immunosuppressive exosomes for arthritis therapy.
doi:10.1155/2012/573528
PMCID: PMC3324137  PMID: 22548070
17.  Targeting of XJB-5-131 to mitochondria suppresses oxidative DNA damage and motor decline in a mouse model of Huntington’s disease 
Cell reports  2012;2(5):1137-1142.
SUMMARY
Oxidative damage and mitochondrial dysfunction are implicated in aging and age-related neurodegenerative diseases, including Huntington’s disease (HD). Many naturally occurring antioxidants have been tested to correct for deleterious effects of reactive oxygen species, but often they lack specificity, are tissue variable, and the efficacy is marginal in human clinical trials. To increase specificity and efficacy, we have designed a synthetic antioxidant, XJB-5-131, to target mitochondria. We demonstrate in a mouse model of HD that XJB-5-131 has remarkably beneficial effects. XJB-5-131 reduces oxidative damage to mitochondrial DNA, maintains mitochondrial DNA copy number, suppresses motor decline and weight loss, enhances neuronal survival, and improves mitochondrial function. The findings poise XJB-5-131 as a promising therapeutic compound.
doi:10.1016/j.celrep.2012.10.001
PMCID: PMC3513647  PMID: 23122961
18.  Safety Assessment of Intradiscal Gene Therapy II: Effect of Dosing and Vector Choice 
Spine  2008;33(14):1509-1517.
Study Design
Clinical, biochemical, and histological analysis was performed following in vivo delivery of cDNA encoding various anabolic cytokines and marker genes to the lumbar epidural space of New Zealand white rabbits, using both adenoviral and adeno-associated viral vectors.
Objective
To mimic errant or misplaced doses of gene therapy in order to better ascertain the potential risks associated with alternative vectors and transgene products with regard to their application to problems of the intervertebral disc.
Summary of Background Data
Work done with several anabolic cytokines including bone morphogenic proteins and transforming growth factors, has demonstrated the potential of gene therapy. Recently, data has been published demonstrating that improperly dosed or delivered adenoviral-mediated gene therapy within the subarachnoid space can result in significant morbidity in rabbits. There are currently no studies examining the effect of these errors within the epidural space or using an adeno-associated viral (AAV) vector.
Methods
Using either adenoviral or adeno-associated viral vectors, complementary DNA (cDNA) encoding anabolic cytokines bone morphogenic protein-2 (BMP-2) and transforming growth factor-beta 1 (TGF-β1) and marker proteins LacZ and green fluorescent protein (GFP) were injected into the epidural space of 37 New Zealand white rabbits at the L5/6 level. Rabbits were then observed clinically for up to six weeks, after which the rabbits were sacrificed in order to perform a comprehensive biochemical and histological analysis.
Results
Following adenoviral-mediated delivery of anabolic cytokine cDNA, up to eighty percent of rabbits suffered significant clinical, biochemical, and histological morbidity. Conversely, AAV-mediated delivery of any cDNA and adenoviral-mediated delivery of marker protein cDNA resulted in no clinical, histological, or biochemical morbidity.
Conclusion
Properly dosed and directed gene therapy seems to be both safe and potentially efficacious. This study suggests that side effects of gene therapy may be due to a combination of dosing, transgene product, and vector choice, and that newer AAV vectors may reduce these side-effects and decrease the risk of this technology.
doi:10.1097/BRS.0b013e318178866c
PMCID: PMC3510668  PMID: 18520636
19.  Biologic Response of Human Intervertebral Disc Cells to Gene Therapy Cocktail 
Spine  2008;33(17):1850-1855.
Study Design
In vitro experiment using human intervertebral disc (IVD) cells and adenovirus-therapeutic gene constructs.
Objectives
To examine the biologic effect of “cocktail” therapeutic gene transfer to human IVD cells in three dimensional cultures.
Summary of Background Data
. Gene therapy is regarded as a potential option for the treatment of degenerative disc disease. Although various anabolic genes have previously been introduced for this purpose, cocktail gene transfer of anabolic genes to IVD cells has never been attempted.
Materials and Methods
Human IVDs were harvested during surgical disc procedures and cultured. We prepared recombinant adenovirus constructs bearing the TGF-β1 gene (Ad/TGF-β1), the IGF-1 gene (Ad/IGF-1), and the BMP-2 gene (Ad/BMP-2). Transgene expression was detected by luciferase assays, enzyme linked immuno-sorbent assays, and Western blot analysis. Newly synthesized proteoglycan was measured by 35S-sulfate incorporation on Sephadex G-25M in PD 10 columns. Human IVD cells were transduced by single, double, and triple combination of Ad/TGF-β1, Ad/IGF-1, Ad/BMP-2 with an MOI of 75, then cultured three-dimensionally in alginate beads.
Results
Transgene expression was detected at 18 hours after viral transduction. IVD cultures with Ad/TGF-β1, Ad/IGF-1, Ad/BMP-2 (MOI of 75) showed 2.9, 1.8, and 1.9 fold increases, respectively, in proteoglycan synthesis compared to control. Human IVD cultures with double gene combination (MOI of 75) showed 3.2 to 3.9 fold increases of proteoglycan synthesis. Lastly, Human IVD cultures with triple gene combination (TGF-β1+IGF-1+BMP-2 genes with an MOI of 75) transfer demonstrated 4.7 fold increase in proteoglycan synthesis compared control.
Conclusion
Combination or “cocktail” gene therapy offers a promising mechanism for maximizing matrix synthesis with low dose of adenoviral mixtures, circumventing systemic, local toxic effect, and immune response.
doi:10.1097/BRS.0b013e31817e1cd7
PMCID: PMC3508786  PMID: 18622355
Gene Therapy; Intervertebral Disc; Proteoglycan
20.  Ex-vivo transduced autologous skin fibroblasts expressing human Lim Mineralization Protein-3 efficiently form new bone in animal models 
Gene therapy  2008;15(19):1330-1343.
Local gene transfer of the human LIM Mineralization Protein (LMP), a novel intracellular positive regulator of the osteoblast differentiation program, can induce efficient bone formation in rodents. In order to develop a clinically relevant gene therapy approach to facilitate bone healing, we have used primary dermal fibroblasts transduced ex vivo with Ad.LMP3 and seeded on an hydroxyapatite/collagen matrix prior to autologous implantation. Here we demonstrate that genetically modified autologous dermal fibroblasts expressing Ad.LMP-3 are able to induce ectopic bone formation following implantation of the matrix into the mouse triceps and paravertebral muscles. Moreover, implantation of the Ad.LMP-3-modified dermal fibroblasts into a rat mandibular bone critical size defect model results in efficient healing as determined by X-ray, histology and three dimensional micro computed tomography (3DμCT). These results demonstrate the effectiveness of the non-secreted intracellular osteogenic factor LMP-3, in inducing bone formation in vivo. Moreover, the utilization of autologous dermal fibroblasts implanted on a biomaterial represents a promising approach for possible future clinical applications aimed at inducing new bone formation.
doi:10.1038/gt.2008.116
PMCID: PMC3488859  PMID: 18633445
LMP; autologous transplantation; skin fibroblasts; gene therapy; bone formation; animal models
21.  Accelerated Aging of Intervertebral Discs in a Mouse Model of Progeria 
Intervertebral disc degeneration (IDD) is a common and debilitating disorder that results in reduced flexibility of the spine, pain, and reduced mobility. Risk factors for IDD include age, genetic predisposition, injury, and other environmental factors such as smoking. Loss of proteoglycans (PGs) contributes to IDD with advancing age. Currently there is a lack of a model for rapid investigation of disc aging and evaluation of therapeutic interventions. Here we examined progression of disc aging in a murine model of a human progeroid syndrome caused by deficiency of the DNA repair endonuclease, ERCC1–XPF (Ercc1−/Δ mice). The ERCC1-deficient mice showed loss of disc height and degenerative structural changes in their vertebral bodies similar to those reported for old rodents. Compared to their wild-type littermates, Ercc1−/Δ mice also exhibit other age-related IDD characteristics, including premature loss of disc PG, reduced matrix PG synthesis, and enhanced apoptosis and cell senescence. Finally, the onset of age-associated disc pathologies was further accelerated in Ercc1−/Δ mice following chronic treatment with the chemotherapeutic agent mechlorethamine. These results demonstrate that Ercc1−/Δ mice represent an accurate and rapid model of disc aging and provide novel evidence that DNA damage negatively impacts PG synthesis.
doi:10.1002/jor.21153
PMCID: PMC3477798  PMID: 20973062
intervertebral disc degeneration; aging; DNA repair; proteoglycan; mouse models
22.  Inhibition of the IKK/NF-κB Pathway by AAV Gene Transfer Improves Muscle Regeneration in Older mdx Mice 
Gene therapy  2010;17(12):1476-1483.
The IκB kinase (IKKα, β and the regulatory subunit IKKγ) complex regulates nuclear factor of κB (NF-κB) transcriptional activity, which is upregulated in many chronic inflammatory diseases. NF-κB signaling promotes inflammation and limits muscle regeneration in Duchenne muscular dystrophy (DMD), resulting in fibrotic and fatty tissue replacement of muscle that exacerbates the wasting process in dystrophic muscles. Here we examined whether dominant-negative forms of IKKα (IKKα-dn) and IKKβ (IKKβ-dn) delivered by adeno-associated viral (AAV) vectors to the gastrocnemius (GAS) and tibialis anterior (TA) muscles of 1, 2, and 11 month-old mdx mice, a murine DMD model, block NF-κB activation and increase muscle regeneration. At one month post-treatment, the levels of nuclear NF-κB in locally treated muscle were decreased by gene transfer with either AAV-CMV-IKKα-dn or AAV-CMV-IKKβ-dn, but not by IKK wild-type controls (IKKα and β) or PBS. Although treatment with AAV-IKKα-dn or AAV-IKKβ-dn vectors had no significant effect on muscle regeneration in young mdx mice treated at 1 and 2 months of age and collected 1 month later, treatment of old (11 month) mdx with AAV-CMV-IKKα-dn or AAV-CMV-IKKβ-dn significantly increased levels of muscle regeneration. In addition, there was a significant decrease in myofiber necrosis in the AAV-IKKα-dn and AAV-IKKβ-dn treated mdx muscle in both young and old mice. These results demonstrate that inhibition of IKKα or IKKβ in dystrophic muscle reduces the adverse effects of NF-κB signaling, resulting in a therapeutic effect. Moreover, these results clearly demonstrate the therapeutic benefits of inhibiting NF-κB activation by AAV gene transfer in dystrophic muscle to promote regeneration, particularly in older mdx mice, and block necrosis.
doi:10.1038/gt.2010.110
PMCID: PMC3471137  PMID: 20720575
AAV; NF-kappa B; DMD; and muscle regeneration
23.  Lim Mineralization Protein 3 Induces the Osteogenic Differentiation of Human Amniotic Fluid Stromal Cells through Kruppel-Like Factor-4 Downregulation and Further Bone-Specific Gene Expression 
Multipotent mesenchymal stem cells with extensive self-renewal properties can be easily isolated and rapidly expanded in culture from small volumes of amniotic fluid. These cells, namely, amniotic fluid-stromal cells (AFSCs), can be regarded as an attractive source for tissue engineering purposes, being phenotypically and genetically stable, plus overcoming all the safety and ethical issues related to the use of embryonic/fetal cells. LMP3 is a novel osteoinductive molecule acting upstream to the main osteogenic pathways. This study is aimed at delineating the basic molecular events underlying LMP3-induced osteogenesis, using AFSCs as a cellular model to focus on the molecular features underlying the multipotency/differentiation switch. For this purpose, AFSCs were isolated and characterized in vitro and transfected with a defective adenoviral vector expressing the human LMP3. LMP3 induced the successful osteogenic differentiation of AFSC by inducing the expression of osteogenic markers and osteospecific transcription factors. Moreover, LMP3 induced an early repression of the kruppel-like factor-4, implicated in MSC stemness maintenance. KLF4 repression was released upon LMP3 silencing, indicating that this event could be reasonably considered among the basic molecular events that govern the proliferation/differentiation switch during LMP3-induced osteogenic differentiation of AFSC.
doi:10.1155/2012/813894
PMCID: PMC3471036  PMID: 23097599
24.  Getting arthritis gene therapy into the clinic 
Nature reviews. Rheumatology  2010;7(4):244-249.
Gene transfer technologies enable the controlled, targeted and sustained expression of gene products at precise anatomical locations, such as the joint. In this way, they offer the potential for more-effective, less-expensive treatments of joint diseases with fewer extra-articular adverse effects. A large body of preclinical data confirms the utility of intra-articular gene therapy in animal models of rheumatoid arthritis and osteoarthritis. However, relatively few clinical trials have been conducted, only one of which has completed phase II. This article summarizes the status in 2010 of the clinical development of gene therapy for arthritis, identifies certain constraints to progress and suggests possible solutions.
doi:10.1038/nrrheum.2010.193
PMCID: PMC3460537  PMID: 21135882
25.  Systemic delivery of NEMO binding domain/IKKγ inhibitory peptide to young mdx mice improves dystrophic skeletal muscle histopathology 
Neurobiology of disease  2011;43(3):598-608.
The activation of nuclear factor κB (NF-κB) contributes to muscle degeneration that results from dystrophin deficiency in human Duchenne muscular dystrophy (DMD) and in the mdx mouse. In dystrophic muscle, NF-κB participates in inflammation and failure of muscle regeneration. Peptides containing the NF-κB Essential Modulator (NEMO) binding domain (NBD) disrupt the IκB kinase complex, thus blocking NF-κB activation. The NBD peptide, which is linked to a protein transduction domain to achieve in vivo peptide delivery to muscle tissue, was systemically delivered to mdx mice for 4 or 7 weeks to study NF-κB activation, histological changes in hind limb and diaphragm muscle and ex vivo function of diaphragm muscle. Decreased NF-κB activation, decreased necrosis and increased regeneration were observed in hind limb and diaphragm muscle in mdx mice treated systemically with NBD peptide, as compared to control mdx mice. NBD peptide treatment resulted in improved generation of specific force and greater resistance to lengthening activations in diaphragm muscle ex vivo. Together these data support the potential of NBD peptides for the treatment of DMD by modulating dystrophic pathways in muscle that are downstream of dystrophin deficiency.
doi:10.1016/j.nbd.2011.05.008
PMCID: PMC3145633  PMID: 21624467
Duchenne Muscular Dystrophy; mdx mouse; histopathology; muscle necrosis; muscle regeneration; specific force; lengthening activation; protein transduction domain; NEMO binding domain peptide; NF-κB

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