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1.  Barriers to the Preclinical Development of Therapeutics that Target Aging Mechanisms 
Through the progress of basic science research, fundamental mechanisms that contribute to age-related decline are being described with increasing depth and detail. Although these efforts have identified new drug targets and compounds that extend life span in model organisms, clinical trials of therapeutics that target aging processes remain scarce. Progress in aging research is hindered by barriers associated with the translation of basic science discoveries into the clinic. This report summarizes discussions held at a 2014 Geroscience Network retreat focused on identifying hurdles that currently impede the preclinical development of drugs targeting fundamental aging processes. From these discussions, it was evident that aging researchers have varied perceptions of the ideal preclinical pipeline. To forge a clear and cohesive path forward, several areas of controversy must first be resolved and new tools developed. Here, we focus on five key issues in preclinical drug development (drug discovery, lead compound development, translational preclinical biomarkers, funding, and integration between researchers and clinicians), expanding upon discussions held at the Geroscience Retreat and suggesting areas for further research. By bringing these findings to the attention of the aging research community, we hope to lay the foundation for a concerted preclinical drug development pipeline.
doi:10.1093/gerona/glw112
PMCID: PMC5055650  PMID: 27535964
Aging; Geroscience network; Preclinical drug development
2.  IL-Y, a Synthetic Member of the IL-12 Cytokine Family, Suppresses the Development of Type 1 Diabetes in NOD Mice 
European journal of immunology  2015;45(11):3114-3125.
The IL-12 family of heterodimeric cytokines, consisting of IL-12, IL-23, IL-27, and IL-35, has important roles in regulating the immune response. IL-12 family members are comprised of a heterodimer consisting of α and β chains: IL-12 (p40 and p35), IL-23 (p40 and p19), IL-27 (Ebi3 and p28), and IL-35 (Ebi3 and p35). Given the combinatorial nature of the IL-12 family, we generated adenoviral vectors expressing two putative IL-12 family members not yet found naturally, termed IL-X (Ebi3 and p19) and IL-Y (p40 and p28), as single-chain molecules. scIL-Y, but not scIL-X, was able to stimulate significantly a unique cytokine/chemokine expression profile as well as activate STAT3 in mice, in part, through a pathway involving IL-27Rα in splenocytes. Adenoviral-mediated, intra-tumoral delivery of scIL-Y increased tumor growth in contrast to the anti-tumor effects of scIL-12 and scIL-23. Similarly, treatment of pre-diabetic non-obese diabetic (NOD) mice by intravenous injection of Ad.scIL-Y prevented the onset of hyperglycemia. Analysis of cells from Ad.scIL-Y-treated NOD mice demonstrated that scIL-Y reduced expression of inflammatory mediators such as IFN-γ. Our data demonstrates that a novel, synthetic member of the IL-12 family, termed IL-Y, confers unique immunosuppressive effects in two different disease models and thus could have therapeutic applications.
doi:10.1002/eji.201445403
PMCID: PMC4823813  PMID: 26260044
type 1 diabetes; regulatory T cells; interleukin-12; NOD mice; adenovirus
3.  Identification of a novel senolytic agent, navitoclax, targeting the Bcl‐2 family of anti‐apoptotic factors 
Aging Cell  2016;15(3):428-435.
Summary
Clearing senescent cells extends healthspan in mice. Using a hypothesis‐driven bioinformatics‐based approach, we recently identified pro‐survival pathways in human senescent cells that contribute to their resistance to apoptosis. This led to identification of dasatinib (D) and quercetin (Q) as senolytics, agents that target some of these pathways and induce apoptosis preferentially in senescent cells. Among other pro‐survival regulators identified was Bcl‐xl. Here, we tested whether the Bcl‐2 family inhibitors, navitoclax (N) and TW‐37 (T), are senolytic. Like D and Q, N is senolytic in some, but not all types of senescent cells: N reduced viability of senescent human umbilical vein epithelial cells (HUVECs), IMR90 human lung fibroblasts, and murine embryonic fibroblasts (MEFs), but not human primary preadipocytes, consistent with our previous finding that Bcl‐xl siRNA is senolytic in HUVECs, but not preadipocytes. In contrast, T had little senolytic activity. N targets Bcl‐2, Bcl‐xl, and Bcl‐w, while T targets Bcl‐2, Bcl‐xl, and Mcl‐1. The combination of Bcl‐2, Bcl‐xl, and Bcl‐w siRNAs was senolytic in HUVECs and IMR90 cells, while combination of Bcl‐2, Bcl‐xl, and Mcl‐1 siRNAs was not. Susceptibility to N correlated with patterns of Bcl‐2 family member proteins in different types of human senescent cells, as has been found in predicting response of cancers to N. Thus, N is senolytic and acts in a potentially predictable cell type‐restricted manner. The hypothesis‐driven, bioinformatics‐based approach we used to discover that dasatinib (D) and quercetin (Q) are senolytic can be extended to increase the repertoire of senolytic drugs, including additional cell type‐specific senolytic agents.
doi:10.1111/acel.12445
PMCID: PMC4854923  PMID: 26711051
ABT‐263; Bcl‐2 family; dasatinib; quercetin; senescent cells; TW‐37
4.  Regulation of Immune Responses by Extracellular Vesicles 
Nature reviews. Immunology  2014;14(3):195-208.
Extracellular vesicles (EVs) including exosomes, are small membrane vesicles derived from multivesicular bodies or from the plasma membrane. Most, if not all, cell types release EVs that then enter the bodily fluids. These vesicles contain a subset of proteins, lipids and nucleic acids that are derived from the parent cell. It is postulated that EVs have important roles in intercellular communication, both locally and systemically, by transferring their contents, including protein, lipids and RNAs, between cells. EVs are involved in numerous physiological processes, and vesicles from both non-immune and immune cells have important roles in immune regulation. Moreover, EV-based therapeutics are being developed and tested clinically for treatment of inflammatory and autoimmune diseases and cancer. Given the tremendous therapeutic potential of EVs this review focuses on the role of EVs in modulating immune responses and the therapeutic applications.
doi:10.1038/nri3622
PMCID: PMC4350779  PMID: 24566916
5.  Regulation of Substrate Oxidation Preferences in Muscle by the Peptide Hormone Adropin 
Diabetes  2014;63(10):3242-3252.
Rigorous control of substrate oxidation by humoral factors is essential for maintaining metabolic homeostasis. During feeding and fasting cycles, carbohydrates and fatty acids are the two primary substrates in oxidative metabolism. Here, we report a novel role for the peptide hormone adropin in regulating substrate oxidation preferences. Plasma levels of adropin increase with feeding and decrease upon fasting. A comparison of whole-body substrate preference and skeletal muscle substrate oxidation in adropin knockout and transgenic mice suggests adropin promotes carbohydrate oxidation over fat oxidation. In muscle, adropin activates pyruvate dehydrogenase (PDH), which is rate limiting for glucose oxidation and suppresses carnitine palmitoyltransferase-1B (CPT-1B), a key enzyme in fatty acid oxidation. Adropin downregulates PDH kinase-4 (PDK4) that inhibits PDH, thereby increasing PDH activity. The molecular mechanisms of adropin’s effects involve acetylation (suggesting inhibition) of the transcriptional coactivator PGC-1α, downregulating expression of Cpt1b and Pdk4. Increased PGC-1α acetylation by adropin may be mediated by inhibiting Sirtuin-1 (SIRT1), a PGC-1α deacetylase. Altered SIRT1 and PGC-1α activity appear to mediate aspects of adropin’s metabolic actions in muscle. Similar outcomes were observed in fasted mice treated with synthetic adropin. Together, these results suggest a role for adropin in regulating muscle substrate preference under various nutritional states.
doi:10.2337/db14-0388
PMCID: PMC4171656  PMID: 24848071
6.  The Achilles’ heel of senescent cells: from transcriptome to senolytic drugs 
Aging Cell  2015;14(4):644-658.
The healthspan of mice is enhanced by killing senescent cells using a transgenic suicide gene. Achieving the same using small molecules would have a tremendous impact on quality of life and the burden of age-related chronic diseases. Here, we describe the rationale for identification and validation of a new class of drugs termed senolytics, which selectively kill senescent cells. By transcript analysis, we discovered increased expression of pro-survival networks in senescent cells, consistent with their established resistance to apoptosis. Using siRNA to silence expression of key nodes of this network, including ephrins (EFNB1 or 3), PI3Kδ, p21, BCL-xL, or plasminogen-activated inhibitor-2, killed senescent cells, but not proliferating or quiescent, differentiated cells. Drugs targeting these same factors selectively killed senescent cells. Dasatinib eliminated senescent human fat cell progenitors, while quercetin was more effective against senescent human endothelial cells and mouse BM-MSCs. The combination of dasatinib and quercetin was effective in eliminating senescent MEFs. In vivo, this combination reduced senescent cell burden in chronologically aged, radiation-exposed, and progeroid Ercc1−/Δ mice. In old mice, cardiac function and carotid vascular reactivity were improved 5 days after a single dose. Following irradiation of one limb in mice, a single dose led to improved exercise capacity for at least 7 months following drug treatment. Periodic drug administration extended healthspan in Ercc1−/Δ mice, delaying age-related symptoms and pathology, osteoporosis, and loss of intervertebral disk proteoglycans. These results demonstrate the feasibility of selectively ablating senescent cells and the efficacy of senolytics for alleviating symptoms of frailty and extending healthspan.
doi:10.1111/acel.12344
PMCID: PMC4531078  PMID: 25754370
dasatinib; dependence receptors; ephrins; p21; PI3K delta; plasminogen-activated inhibitor; quercetin
7.  d-Amino Acid Substitution of Peptide-Mediated NF-κB Suppression in mdx Mice Preserves Therapeutic Benefit in Skeletal Muscle, but Causes Kidney Toxicity 
Molecular Medicine  2015;21(1):442-452.
In Duchenne muscular dystrophy (DMD) patients and the mdx mouse model of DMD, chronic activation of the classical nuclear factor-κB (NF-κB) pathway contributes to the pathogenesis that causes degeneration of muscle fibers, inflammation and fibrosis. Prior studies demonstrate that inhibition of inhibitor of κB kinase (IKK)-mediated NF-κB activation using l-isomer NF-κB essential modulator (NEMO)-binding domain (NBD) peptide-based approaches reduce muscle pathology in the mdx mouse. For our studies, the NBD peptide is synthesized as a fusion peptide with an eight-lysine (8K) protein transduction domain to facilitate intracellular delivery. We hypothesized that the d-isoform peptide could have a greater effect than the naturally occurring l-isoform peptide due to the longer persistence of the d-isoform peptide in vivo. In this study, we compared systemic treatment with low (1 mg/kg) and high (10 mg/kg) doses of l- and d-isomer 8K-wild-type-NBD peptide in mdx mice. Treatment with both l- or d-isoform 8K-wild-type-NBD peptide resulted in decreased activation of NF-κB and improved histology in skeletal muscle of the mdx mouse. However, we observed kidney toxicity (characterized by proteinuria), increased serum creatinine, activation of NF-κB and pathological changes in kidney cortex that were most severe with treatment with the d-isoform of 8K-wild-type-NBD peptide. The observed toxicity was also seen in normal mice.
doi:10.2119/molmed.2013.00141
PMCID: PMC4559531  PMID: 26018805
8.  Perspectives on the Use of Gene Therapy for Chronic Joint Diseases 
Current gene therapy  2008;8(4):273-286.
Advances in molecular and cellular biology have identified a wide variety of proteins including targeted cytokine inhibitors, immunomodulatory proteins, cytotoxic mediators, angiogenesis inhibitors, and intracellular signalling molecules that could be of great benefit in the treatment of chronic joint diseases, such as osteo- and rheumatoid arthritis. Unfortunately, protein-based drugs are difficult to administer effectively. They have a high rate of turnover, requiring frequent readministration, and exposure in non-diseased tissue can lead to serious side effects. Gene transfer technologies offer methods to enhance the efficacy of protein-based therapies, enabling the body to produce these molecules locally at elevated levels for extended periods. The proof of concept of gene therapies for arthritis has been exhaustively demonstrated in multiple laboratories and in numerous animal models. This review attempts to condense these studies and to discuss the relative benefits and limitations of the methods proposed and to discuss the challenges toward translating these technologies into clinical realities.
PMCID: PMC4350777  PMID: 18691023
Arthritis; gene therapy; adenovirus; adeno-associated virus; lentivirus; osteoarthritis; interleukin-1; tumor necrosis factor
9.  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
10.  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
11.  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
12.  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
13.  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
14.  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
15.  Mitochondrial-derived reactive oxygen species (ROS) play a causal role in aging-related intervertebral disc degeneration 
Oxidative damage is a well-established driver of aging. Evidence of oxidative stress exists in aged and degenerated discs, but it is unclear how it affects disc metabolism. In this study, we first determined whether oxidative stress negatively impacts disc matrix metabolism using disc organotypic and cell cultures. Mouse disc organotypic culture grown at atmospheric oxygen (20% O2) exhibited perturbed disc matrix homeostasis, including reduced proteoglycan synthesis and enhanced expression of matrix metalloproteinases, compared to discs grown at low oxygen levels (5% O2). Human disc cells grown at 20% O2 showed increased levels of mitochondrial-derived superoxide anions and perturbed matrix homeostasis. Treatment of disc cells with the mitochondria-targeted reactive oxygen species (ROS) scavenger XJB-5-131 blunted the adverse effects caused by 20% O2. Importantly, we demonstrated that treatment of accelerated aging Ercc1−/Δmice, previously established to be a useful in vivo model to study age-related intervertebral disc degeneration (IDD), also resulted in improved disc total glycosaminoglycan content and proteoglycan synthesis. This demonstrates that mitochondrial-derived ROS contributes to age-associated IDD in Ercc1−/Δmice. Collectively, these data provide strong experimental evidence that mitochondrial-derived ROS play a causal role in driving changes linked to aging-related IDD and a potentially important role for radical scavengers in preventing IDD.
doi:10.1002/jor.22320
PMCID: PMC3668354  PMID: 23389888
Aging; oxidative stress; reactive oxygen species (ROS); intervertebral discs; radical scavenger; nitroxide; matrix proteoglycan
16.  In Vivo Expression of HGF/NK1 and GLP-1 From dsAAV Vectors Enhances Pancreatic β-Cell Proliferation and Improves Pathology in the db/db Mouse Model of Diabetes 
Diabetes  2010;59(12):3108-3116.
OBJECTIVE
The purpose of the current study was to determine whether double-stranded adeno-associated virus (dsAAV)-mediated in vivo expression of β-cell growth factors, glucagon-like peptide-1 (GLP-1) and the NK1 fragment of hepatocyte growth factor (HGF/NK1) in β-cells, improves pathology in the db/db mouse model of type 2 diabetes.
RESEARCH DESIGN AND METHODS
The glucoregulatory actions of GLP-1 and full-length HGF are well characterized. Here, we test the ability of HGF/NK1 to induce proliferation of exogenous islets and MIN6 β-cells. In addition, we target both GLP-1 and HGF/NK1 to endogenous β-cells using dsAAV vectors containing the mouse insulin-II promoter. We compare the abilities of these gene products to induce islet proliferation in vitro and in vivo and characterize their abilities to regulate diabetes after AAV-mediated delivery to endogenous islets of db/db mice.
RESULTS
Recombinant HGF/NK1 induces proliferation of isolated islets, and dsAAV-mediated expression of both GLP-1 and HGF/NK1 induces significant β-cell proliferation in vivo. Furthermore, both GLP-1 and HGF/NK1 expressed from dsAAV vectors enhance β-cell mass and insulin secretion in vivo and significantly delay the onset of hyperglycemia in db/db mice.
CONCLUSIONS
A single treatment with dsAAV vectors expressing GLP-1 or HGF/NK1 enhances islet growth and significantly improves pathology in a mouse model of type 2 diabetes. This represents the first example of a successful use of HGF/NK1 for diabetes therapy, providing support for direct AAV-mediated in vivo delivery of β-cell growth factors as a novel therapeutic strategy for the treatment of type 2 diabetes.
doi:10.2337/db09-1886
PMCID: PMC2992772  PMID: 20841608
17.  The Roles of Tumor-Derived Exosomes in Cancer Pathogenesis 
Exosomes are endosome-derived, 30–100 nm small membrane vesicles released by most cell types including tumor cells. They are enriched in a selective repertoire of proteins and nucleic acids from parental cells and are thought to be actively involved in conferring intercellular signals. Tumor-derived exosomes have been viewed as a source of tumor antigens that can be used to induce antitumor immune responses. However, tumor-derived exosomes also have been found to possess immunosuppressive properties and are able to facilitate tumor growth, metastasis, and the development of drug resistance. These different effects of tumor-derived exosomes contribute to the pathogenesis of cancer. This review will discuss the roles of tumor-derived exosomes in cancer pathogenesis, therapy, and diagnostics.
doi:10.1155/2011/842849
PMCID: PMC3235485  PMID: 22190973
18.  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
19.  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
20.  B7-1/2, but not PD-L1/2 molecules, are required on IL-10-treated tolerogenic DC and DC-derived exosomes for in vivo function 
European journal of immunology  2009;39(11):3084-3090.
SUMMARY
Costimulatory molecules such as B7-1/2 and PD-L1/2 play an important role in the function of APC. The regulation of the surface levels of costimulatory molecules is one mechanism by which APC maintain the balance between tolerance and immunity. We examined the contributions of B7-1/2 and PD-L1/2 to the function of IL-10-treated, immunosuppressive DC as well as therapeutic exosomes derived from these DC. IL-10 treatment of DC significantly downregulated surface expression of MHC II, B7-1, B7-2, and decreased levels of MHC I and PD-L2. IL-10 treatment of DC resulted in a modified co-stimulatory profile of DC-secreted exosomes with a reduction in B7-1, PD-L1 and PD-L2. We further demonstrate that absence of B7-1 or B7-2 on donor DC results in a loss of ability of IL-10 treated DC and their exosomes to suppress the delayed-type hypersensitivity (DTH) response, whereas IL-10 treated DC deficient in PD-L1/2 as well as their secreted exosomes retained the ability to suppress DTH responses. We conclude that B7-1 and B7-2, but not PD-L1 and PD-L2, on IL-10 treated DC and DC-derived exosomes play a critical role in immunosuppressive functions of both DC and exosomes.
doi:10.1002/eji.200939407
PMCID: PMC3075967  PMID: 19757438
Dendritic cells; Exosomes; B7-1/2; PD-L1/2; Delayed-type hypersensitivity
21.  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
22.  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
23.  Adenoviral Mediated, Intra-Tumor Gene Transfer of Interleukin 23 Induces a Therapeutic Anti-tumor Response 
Cancer gene therapy  2009;16(10):776-785.
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. IL-23 has been shown to possess potent anti-tumor activities in several establishment models of cancer and a few therapeutic models, but the efficacy of local, adenoviral-mediated expression of IL-23 in established tumors has yet to be investigated. Here we have examined the anti-tumor activity of adenovirally-delivered IL-23 in a day 7 MCA205 murine fibrosarcoma tumor model. Three intratumoral injections of adenovirus expressing IL-23 (Ad.IL-23) significantly increased animal survival and resulted in complete rejection of 40 percent of tumors, with subsequent generation of protective immunity and MCA205-specific cytotoxic T-lymphocytes (CTLs). Additionally, we have shown that the anti-tumor activity of IL-23 is independent of IL-17, perforin and Fas ligand, but dependent on IFN-γ, CD4 and CD8 positive T-cells. These results demonstrate that direct intratumoral injection of adenovirus expressing IL-23 results in enhanced survival, tumor eradication and generation of protective immunity by generation of a Th1-type immune response.
doi:10.1038/cgt.2009.27
PMCID: PMC2745505  PMID: 19390568
Interleukin 23; adenovirus; cancer; gene therapy
24.  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
25.  Gene therapy of the rheumatic diseases: 1998 to 2008 
During the decade since the launch of Arthritis Research, the application of gene therapy to the rheumatic diseases has experienced the same vicissitudes as the field of gene therapy as a whole. There have been conceptual and technological advances and an increase in the number of clinical trials. However, funding has been unreliable and a small number of high-profile deaths in human trials, including one in an arthritis gene therapy trial, have provided ammunition to skeptics. Nevertheless, steady progress has been made in a number of applications, including rheumatoid arthritis and osteoarthritis, Sjögren syndrome, and lupus. Clinical trials in rheumatoid arthritis have progressed to phase II and have provided the first glimpses of possible efficacy. Two phase I protocols for osteoarthritis are under way. Proof of principle has been demonstrated in animal models of Sjögren syndrome and lupus. For certain indications, the major technological barriers to the development of genetic therapies seem to have been largely overcome. The translational research necessary to turn these advances into effective genetic medicines requires sustained funding and continuity of effort.
doi:10.1186/ar2563
PMCID: PMC2688220  PMID: 19232068

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