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1.
EMBO Mol Med ; 15(6): e17144, 2023 06 07.
Article in English | MEDLINE | ID: mdl-37158379

ABSTRACT

In the practice of medicine, many fundamental biological pathways that require tight on/off control, such as inflammation and circulatory homeostasis, are regulated by serine proteinases, but we rarely consider the unique protease inhibitors that, in turn, regulate these proteases. The serpins are a family of proteins with a shared tertiary structure, whose members largely act as serine protease inhibitors, found in all forms of life, ranging from viruses, bacteria, and archaea to plants and animals. These proteins represent up to 2-10% of proteins in the human blood and are the third most common protein family.


Subject(s)
Serpins , Animals , Humans , Serpins/genetics , Serpins/chemistry , Serpins/metabolism , Serine Proteinase Inhibitors/pharmacology , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/metabolism , Serine Proteases/metabolism , Inflammation
2.
Biomolecules ; 12(10)2022 Sep 22.
Article in English | MEDLINE | ID: mdl-36291555

ABSTRACT

Human alpha 1 antitrypsin (hAAT) is a multifunctional protein that has been shown to have anti-inflammatory and cellular protective properties. While previous studies demonstrated the antiaging potential of hAAT, the mechanism(s) underlying the antiaging effect remain elusive. In this study, we performed a detailed analysis of transcriptomic data that indicated that NF-κB-targeted genes and NF-κB-regulated pathways were selectively inhibited by hAAT treatment. We further showed that the first detectable impact of hAAT treatment was the inhibition of the nuclear activity of NF-κB. Subsequently, hAAT treatment suppressed the mRNA levels of NF-κB-targeted genes, as well as NF-κB itself (P65 and P50), in human senescent cells. Using Drosophila models, we further examined the impact of hAAT on locomotor activity and endurance. Finally, using an adult-specific promotor, we demonstrated that overexpression of hAAT in the late stage of life significantly extended the lifespan of transgenic flies. These results extend the current understanding of the anti-inflammatory function of hAAT.


Subject(s)
Longevity , alpha 1-Antitrypsin , Animals , Humans , alpha 1-Antitrypsin/metabolism , Longevity/genetics , NF-kappa B/genetics , Drosophila/metabolism , RNA, Messenger
3.
Biomolecules ; 13(1)2022 12 29.
Article in English | MEDLINE | ID: mdl-36671451

ABSTRACT

Hypertension may develop before or after the onset of diabetes and it is known to increase the risk of developing diabetic nephropathy. Alpha-1 antitrypsin (AAT) is a multi-functional protein with beneficial effects in various diseases but its role in reducing blood pressure in the diabetic kidney has not been thoroughly studied. Like blood pressure, epithelial sodium channels (ENaC) and its adaptor protein myristoylated alanine-rich C-kinase substrate (MARCKS) are regulated by circadian rhythms. Our hypothesis is that administration of human AAT (hAAT) reduces blood pressure in hypertensive diabetic mice by attenuating membrane expression of ENaC and its association with the actin cytoskeleton. First, we show hAAT administration results in reduced blood pressure in diabetic db/db mice compared to vehicle treatment in both the inactive and active cycles. Western blotting and immunohistochemistry analyses showed a reduction of ENaC and the actin cytoskeleton protein, MARCKS in the kidneys of diabetic db/db mice treated with hAAT compared to vehicle. hAAT treatment resulted in elevated amounts of extracellular vesicles present in the urine of diabetic db/db mice compared to vehicle treatment both in the inactive and active cycles. Multiple hexosylceramides, among other lipid classes increased in urinary EVs released from hAAT treated hypertensive diabetic mice compared to vehicle treated mice. Taken together, these data suggest hAAT treatment could normalize blood pressure in the diabetic kidney in a mechanism involving attenuation of renal ENaC and MARCKS protein expression and possibly ceramide metabolism to hexosylceramide in kidney cells.


Subject(s)
Diabetes Mellitus, Experimental , Diabetic Nephropathies , Hypertension , Animals , Humans , Mice , Blood Pressure , Diabetes Mellitus, Experimental/drug therapy , Diabetic Nephropathies/drug therapy , Hypertension/drug therapy , Mice, Inbred Strains , Myristoylated Alanine-Rich C Kinase Substrate , Epithelial Sodium Channels/metabolism , Receptors, Adrenergic, alpha-1/metabolism
4.
Front Physiol ; 12: 710313, 2021.
Article in English | MEDLINE | ID: mdl-34630137

ABSTRACT

Human alpha-1 antitrypsin (hAAT) is a versatile protease inhibitor, but little is known about its targets in the aldosterone-sensitive distal nephron and its role in electrolyte balance and blood pressure control. We analyzed urinary electrolytes, osmolality, and blood pressure from hAAT transgenic (hAAT-Tg) mice and C57B/6 wild-type control mice maintained on either a normal salt or high salt diet. Urinary sodium, potassium, and chloride concentrations as well as urinary osmolality were lower in hAAT-Tg mice maintained on a high salt diet during both the active and inactive cycles. hAAT-Tg mice showed a lower systolic blood pressure compared to C57B6 mice when maintained on a normal salt diet but this was not observed when they were maintained on a high salt diet. Cathepsin B protein activity was less in hAAT-Tg mice compared to wild-type controls. Protein expression of the alpha subunit of the sodium epithelial channel (ENaC) alpha was also reduced in the hAAT-Tg mice. Natriuretic peptide receptor C (NPRC) protein expression in membrane fractions of the kidney cortex was reduced while circulating levels of atrial natriuretic peptide (ANP) were greater in hAAT-Tg mice compared to wild-type controls. This study characterizes the electrolyte and blood pressure phenotype of hAAT-Tg mice during the inactive and active cycles and investigates the mechanism by which ENaC activation is inhibited in part by a mechanism involving decreased cathepsin B activity and increased ANP levels in the systemic circulation.

5.
J Clin Med ; 8(9)2019 Aug 29.
Article in English | MEDLINE | ID: mdl-31470606

ABSTRACT

Diffuse alveolar hemorrhage (DAH) is a fatal complication in patients with lupus. DAH can be induced in B6 mice by an intraperitoneal injection of pristane. Since human alpha-1-antitrypsin (hAAT) is an anti-inflammatory and immuno-regulatory protein, we investigated the protective effect of hAAT against pristane-induced DAH in B6 mice and hAAT transgenic (hAAT-Tg) mice. We first showed that hAAT Tg expression lowers TNF-α production in B cells, as well as CD4+ T cells in untreated mice. Conversely, the frequency of regulatory CD4+CD25+ and CD4+CD25-IL-10+ cells was significantly higher in hAAT-Tg than in B6 mice. This confirmed the anti-inflammatory effect of hAAT that was observed even at steady state. One week after a pristane injection, the frequency of peritoneal Ly6Chi inflammatory monocytes and neutrophils in hAAT-Tg mice was significantly lower than that in B6 mice. Importantly, pristane-induced DAH was completely prevented in hAAT-Tg mice and this was associated with a modulation of anti- to pro-inflammatory myeloid cell ratio/balance. We also showed that treatment with hAAT decreased the severity of DAH in B6 mice. These results showed for the first time that hAAT has a therapeutic potential for the treatment of DAH.

6.
J Clin Med ; 8(9)2019 Aug 28.
Article in English | MEDLINE | ID: mdl-31466263

ABSTRACT

We, and others, have previously achieved high and sustained levels of transgene expression from viral vectors, such as recombinant adeno-associated virus (rAAV). However, regulatable transgene expression may be preferred in gene therapy for diseases, such as type 1 diabetes (T1D) and rheumatoid arthritis (RA), in which the timing and dosing of the therapeutic gene product play critical roles. In the present study, we generated a positive feedback regulation system for human alpha 1 antitrypsin (hAAT) expression in the rAAV vector. We performed quantitative kinetics studies in vitro and in vivo demonstrating that this vector system can mediate high levels of inducible transgene expression. Transgene induction could be tailored to occur rapidly or gradually, depending on the dose of the inducing drug, doxycycline (Dox). Conversely, after withdrawal of Dox, the silencing of transgene expression occurred slowly over the course of several weeks. Importantly, rAAV delivery of inducible hAAT significantly prevented T1D development in non-obese diabetic (NOD) mice. These results indicate that this Dox-inducible vector system may facilitate the fine-tuning of transgene expression, particularly for hAAT treatment of human autoimmune diseases, including T1D.

7.
Mol Ther Methods Clin Dev ; 11: 131-142, 2018 Dec 14.
Article in English | MEDLINE | ID: mdl-30547047

ABSTRACT

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease characterized by high levels of pathogenic autoantibodies and tissue damage. Multiple studies showed that dendritic cell (DC) activation plays a critical role in SLE pathogenesis. Human alpha 1 antitrypsin (hAAT) is a serine proteinase inhibitor with potent anti-inflammatory and cytoprotective properties. In this study, we first examined the effects of hAAT on the functions of DCs from lupus-prone mice, and we showed that hAAT treatment efficiently inhibited CpG- (TLR9 agonist) induced activation of bone marrow-derived conventional and plasmacytoid DCs as well as the production of pro-inflammatory cytokines. The hAAT treatment also attenuated DC help for B cell proliferation and immunoglobulin M (IgM) production. We next tested the protective effect of hAAT protein and gene therapy using recombinant adeno-associated virus 8 (rAAV8-CB-hAAT) in a spontaneous lupus mouse model, and we showed that both treatments decreased autoantibody levels. Importantly, rAAV8-CB-hAAT did not induce an immune response to its transgene product (hAAT), but it showed more pronounced therapeutic effects in reducing urine protein levels and extending the lifespan of these mice. These results indicate that AAT has therapeutic potential in the treatment of SLE in humans.

8.
Methods Mol Biol ; 1826: 143-155, 2018.
Article in English | MEDLINE | ID: mdl-30194599

ABSTRACT

Alpha-1-antitrypsin (AAT) is a circulating protein, a serpin, with multiple protective functions. Beside the well-known proteinase inhibitory function, which protects the lungs from chronic obstructive pulmonary disease (COPD), many studies have shown that AAT inhibits pro-inflammatory cytokine gene expression and functions. These anti-inflammatory and immune-regulatory properties have led to studies testing the therapeutic effect of AAT in autoimmune disease models. Initially, a study using recombinant adeno-associated viral (rAAV) vector showed that AAT gene therapy prevented type 1 diabetes (T1D) development in a nonobese diabetic (NOD) mouse model. Consequently, several studies confirmed that AAT therapy prevented and reversed T1D. AAT therapy has also been tested and has demonstrated protective effects in a collagen-induced arthritis model and a systemic lupus erythematosus (SLE) mouse model. This chapter describes methods that evaluate AAT functions in autoimmune mouse models.


Subject(s)
Arthritis, Experimental , Dependovirus , Diabetes Mellitus, Type 1 , Genetic Therapy , Lupus Erythematosus, Systemic , alpha 1-Antitrypsin , Animals , Arthritis, Experimental/genetics , Arthritis, Experimental/metabolism , Arthritis, Experimental/pathology , Arthritis, Experimental/therapy , Diabetes Mellitus, Type 1/genetics , Diabetes Mellitus, Type 1/metabolism , Diabetes Mellitus, Type 1/pathology , Diabetes Mellitus, Type 1/therapy , Disease Models, Animal , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/metabolism , Lupus Erythematosus, Systemic/pathology , Lupus Erythematosus, Systemic/therapy , Mice , Mice, Inbred NOD , Pulmonary Disease, Chronic Obstructive/genetics , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Disease, Chronic Obstructive/pathology , Pulmonary Disease, Chronic Obstructive/therapy , Transduction, Genetic , alpha 1-Antitrypsin/biosynthesis , alpha 1-Antitrypsin/genetics
9.
Methods Mol Biol ; 1826: 183-196, 2018.
Article in English | MEDLINE | ID: mdl-30194601

ABSTRACT

The challenge for alpha-1-antitrypsin (AAT also known as SERPINA1) gene therapy is to achieve long term and high levels of AAT production. Recombinant adeno-associated virus (rAAV) vector has several advantages for AAT gene delivery including no viral genes in the vector, no requirement of integration for long-term transgene expression, low immunogenicity, and wide tropism. AAV-mediated AAT gene therapy has been developed and tested in animal models for AAT deficiency, type 1 diabetes, rheumatoid arthritis, and osteoporosis. AAV-mediated AAT gene therapy has also been tested in clinical studies and has shown promising results. Here we describe the methods of rAAV-AAT vector construction and production as well as AAT gene delivery through (1) liver-directed, (2) muscle-directed, and (3) mesenchymal stem cell (MSC)-mediated routes. We will also describe methods for the evaluation of AAT expression for each delivery approach.


Subject(s)
Arthritis, Rheumatoid , Dependovirus , Genetic Therapy/methods , Osteoporosis , Transduction, Genetic/methods , alpha 1-Antitrypsin Deficiency , alpha 1-Antitrypsin , Animals , Arthritis, Rheumatoid/genetics , Arthritis, Rheumatoid/metabolism , Arthritis, Rheumatoid/pathology , Arthritis, Rheumatoid/therapy , Humans , Mice , Osteoporosis/genetics , Osteoporosis/metabolism , Osteoporosis/pathology , Osteoporosis/therapy , alpha 1-Antitrypsin/biosynthesis , alpha 1-Antitrypsin/genetics , alpha 1-Antitrypsin Deficiency/genetics , alpha 1-Antitrypsin Deficiency/metabolism , alpha 1-Antitrypsin Deficiency/pathology , alpha 1-Antitrypsin Deficiency/therapy
10.
Aging Cell ; 17(1)2018 02.
Article in English | MEDLINE | ID: mdl-29045001

ABSTRACT

Inflammaging plays an important role in most age-related diseases. However, the mechanism of inflammaging is largely unknown, and therapeutic control of inflammaging is challenging. Human alpha-1 antitrypsin (hAAT) has immune-regulatory, anti-inflammatory, and cytoprotective properties as demonstrated in several disease models including type 1 diabetes, arthritis, lupus, osteoporosis, and stroke. To test the potential anti-inflammaging effect of hAAT, we generated transgenic Drosophila lines expressing hAAT. Surprisingly, the lifespan of hAAT-expressing lines was significantly longer than that of genetically matched controls. To understand the mechanism underlying the anti-aging effect of hAAT, we monitored the expression of aging-associated genes and found that aging-induced expressions of Relish (NF-ĸB orthologue) and Diptericin were significantly lower in hAAT lines than in control lines. RNA-seq analysis revealed that innate immunity genes regulated by NF-kB were significantly and specifically inhibited in hAAT transgenic Drosophila lines. To confirm this anti-inflammaging effect in human cells, we treated X-ray-induced senescence cells with hAAT and showed that hAAT treatment significantly decreased the expression and maturation of IL-6 and IL-8, two major factors of senescence-associated secretory phenotype. Consistent with results from Drosophila,RNA-seq analysis also showed that hAAT treatment significantly inhibited inflammation related genes and pathways. Together, our results demonstrated that hAAT significantly inhibited inflammaging in both Drosophila and human cell models. As hAAT is a FDA-approved drug with a confirmed safety profile, this novel therapeutic potential may make hAAT a promising candidate to combat aging and aging-related diseases.


Subject(s)
Aging/physiology , Inflammation/drug therapy , Osteoporosis/drug therapy , alpha 1-Antitrypsin/pharmacology , Animals , Drosophila , Genetic Therapy/methods , Longevity/drug effects
11.
Chronic Obstr Pulm Dis ; 5(4): 289-301, 2018 Oct 05.
Article in English | MEDLINE | ID: mdl-30723786

ABSTRACT

Autoimmune diseases are conditions caused by an over reactive immune system that attacks self-tissues and organs. Although the pathogenesis of autoimmune disease is complex and multi-factorial, inflammation is commonly involved. Therefore, anti-inflammatory therapies hold potential for the treatment of autoimmune diseases. However, long-term control of inflammation is challenging and most of the currently used drugs have side effects. Alpha-1 antitrypsin (AAT) is an anti-inflammatory protein with a well-known safety profile. The therapeutic potential of AAT has been tested in several autoimmune disease models. The first study using a recombinant adeno-associated viral (rAAV) vector showed that AAT gene transfer prevented the development of type 1 diabetes (T1D) in the non-obese diabetic (NOD) mouse model. Subsequent studies showed that treatment with AAT protein prevented and reversed type 1 diabetes. The beneficial effects of AAT treatment have also been observed in other autoimmune disease models such as rheumatoid arthritis and systemic lupus erythematosus. This paper reviews the therapeutic application of AAT and discusses possible mechanisms of action in various autoimmune diseases.

12.
J Neurochem ; 142(6): 908-919, 2017 Sep.
Article in English | MEDLINE | ID: mdl-28640931

ABSTRACT

Neuroinflammation, especially activation of microglia, the key immune cells in the brain, has been proposed to contribute to the pathogenesis of ischemic stroke. However, the dynamics and the potential mediators of microglial activation following ischemic neuronal injury are not well understood. In this study, using oxygen/glucose deprivation and reoxygenation with neuronal and microglial cell cultures as an in vitro model of ischemic neuronal injury, we set out to identify neuronal factors released from injured neurons that are capable of inducing microglial activation. Conditioned media (CM) from hippocampal and cortical neurons exposed to oxygen/glucose deprivation and reoxygenation induced significant activation of microglial cells as well as primary microglia, evidenced by up-regulation of inducible nitric oxide synthase, increased production of nitrite and reactive oxygen species, and increased expression of microglial markers. Mechanistically, neuronal ischemia-responsive protein 94 (Irp94) was a key contributor to microglial activation since significant increase in Irp94 was detected in the neuronal CM following ischemic insult and immunodepletion of Irp94 rendered ischemic neuronal CM ineffective in inducing microglial activation. Ischemic insult-augmented oxidative stress was a major facilitator of neuronal Irp94 release, and pharmacological inhibition of NADPH oxidase significantly reduced the ischemic injury-induced neuronal reactive oxygen species production and Irp94 release. Taken together, these results indicate that neuronal Irp94 may play a pivotal role in the propagation of ischemic neuronal damage. Continued studies may help identify Irp94 and/or related proteins as potential therapeutic targets and/or diagnostic/prognostic biomarkers for managing ischemia-associated brain disorders.

13.
Mol Med ; 23: 57-69, 2017 05.
Article in English | MEDLINE | ID: mdl-28332697

ABSTRACT

Osteoporosis is a global public health problem affecting more than 200 million people worldwide. We previously showed that treatment with alpha-1 antitrypsin (AAT), a multifunctional protein with anti-inflammatory properties, mitigated bone loss in an ovariectomized mouse model. However, the underlying mechanisms of the protective effect of AAT on bone tissue are largely unknown. In this study, we investigated the effect of AAT on osteoclast formation and function in vitro. Our results showed that AAT dose-dependently inhibited the formation of RANKL (receptor activator of nuclear factor κB ligand) induced osteoclasts derived from mouse bone marrow macrophages/monocyte (BMM) lineage cells and the murine macrophage cell line, RAW 264.7 cells. In order to elucidate the possible mechanisms underlying this inhibition, we tested the effect of AAT on the gene expression of cell surface molecules, transcription factors, and cytokines associated with osteoclast formation. We showed that AAT inhibited M-CSF (macrophage colony-stimulating factor) induced cell surface RANK expression in osteoclast precursor cells. In addition, AAT inhibited RANKL-induced TNF-α production, cell surface CD9 expression, and dendritic cell-specific transmembrane protein (DC-STAMP) gene expression. Importantly, AAT treatment significantly inhibited osteoclast-associated mineral resorption. Together, these results uncovered new mechanisms for the protective effects of AAT and strongly support the notion that AAT has therapeutic potential for the treatment of osteoporosis.


Subject(s)
Osteoclasts/drug effects , alpha 1-Antitrypsin/pharmacology , Animals , Bone Marrow Cells/cytology , Cytokines/metabolism , Macrophage Colony-Stimulating Factor , Male , Mice , Mice, Inbred C57BL , Osteoclasts/metabolism , Osteoporosis/drug therapy , RANK Ligand , RAW 264.7 Cells
14.
Neurosci Lett ; 642: 43-50, 2017 03 06.
Article in English | MEDLINE | ID: mdl-28147225

ABSTRACT

Electroacupuncture (EA) has been shown to alleviate the symptoms associated with major depressive disorder; however, the underlying mechanisms remain unclear. While the mainstay treatment for depression are pharmacological agents that modulate serotonergic and/or noradrenergic activity of the brain, recent data suggest that, neurotrophins may play a larger role in the pathogenesis of depression and may offer better therapeutic potential in alleviating symptoms associated with depression. One downstream target of neurotrophins is the extracellular signal-regulated kinase (ERK)/Mitogen-activated protein kinase (MAPK) cascade, a major mediator of cellular stress often associated with clinical depression. In this study, we assessed whether the efficacy of EA is due to regulation of these novel pathways using an animal model of depression induced by chronic unpredictable mild stress (CUMS). We found that EA stimulation at specific locations, Baihui (GV20), and Yintang (GV29) ameliorated the behavioral responses of CUMS, which included reduced locomotion, decreased sucrose intake and weight loss. Furthermore, EA increased the activation of ERK and ribosomal s6 kinase (RSK) levels under stress. Both the behavioral and biochemical responses to EA were attenuated with administration of ERK inhibitor, suggesting that EA improves depression-like symptoms in stressed rats, in part, by activation of ERK signaling.


Subject(s)
Behavior, Animal/physiology , Depression/therapy , Electroacupuncture , MAP Kinase Signaling System/physiology , Stress, Physiological/physiology , Stress, Psychological/therapy , Animals , Annexin A5/metabolism , Apoptosis/physiology , Depression/metabolism , Disease Models, Animal , Hippocampus/metabolism , Male , Rats , Rats, Sprague-Dawley , Stress, Psychological/metabolism , Treatment Outcome
15.
Hum Gene Ther ; 28(2): 179-189, 2017 02.
Article in English | MEDLINE | ID: mdl-27802778

ABSTRACT

Osteoporosis is a common health problem severely affecting the quality of life of many people, especially women. Current treatment options for osteoporosis are limited due to their association with several side-effects and moderate efficacy. Therefore, novel therapies for osteoporosis are needed. This study tested the feasibility of adipose tissue-derived mesenchymal stem cell (ATMSC)-based human alpha-1 antitrypsin (hAAT, SERPINA1) gene therapy for the prevention of bone loss in an ovariectomized (OVX) mouse model. Eight-week-old female C57BL6 mice underwent ovariectomy and were treated with hAAT (protein therapy), ATMSC (stem-cell therapy), ATMSC + hAAT (combination of ATMSC and hAAT therapy), and ATMSCs infected with lentiviral vectors expressing hAAT (ATMSC-based hAAT gene therapy). The study showed that lenti-hAAT vector-infected ATMSCs (ATMSC-LV-hAAT) produced high levels of hAAT. Transplantation of these cells significantly decreased OVX-induced serum levels of interleukin 6 and interleukin 1 beta, and receptor activator of nuclear factor kappa B gene expression levels in bone tissue. Immunohistological analysis revealed that transplanted cells migrated to the bone tissue and secreted hAAT. Importantly, bone microstructure analysis by microcomputerized tomography showed that this treatment significantly protected against OVX-induced bone loss. The results suggest a novel strategy for the treatment of osteoporosis in humans.


Subject(s)
Adipose Tissue/cytology , Genetic Therapy , Genetic Vectors/administration & dosage , Lentivirus/genetics , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Osteoporosis/therapy , alpha 1-Antitrypsin/genetics , Animals , Bone Density , Combined Modality Therapy , Disease Models, Animal , Mice , Osteoporosis/etiology , Ovariectomy/adverse effects
16.
Hum Gene Ther ; 27(9): 679-86, 2016 09.
Article in English | MEDLINE | ID: mdl-27158796

ABSTRACT

Osteoporosis is a major healthcare burden affecting mostly postmenopausal women characterized by compromised bone strength and increased risk of fragility fracture. Although pathogenesis of this disease is complex, elevated proinflammatory cytokine production is clearly involved in bone loss at menopause. Therefore, anti-inflammatory strategies hold a great potential for the prevention of postmenopausal osteoporosis. In this study, we investigated the effect of gene therapy of recombinant adeno-associated virus (rAAV)-mediated human alpha-1 antitrypsin (hAAT), a multifunctional protein that has anti-inflammatory property, on bone loss in an ovariectomy-induced osteoporosis mouse model. Adult ovariectomized (OVX) mice were intraperitoneally (i.p.) injected with hAAT (protein therapy), rAAV8-CB-hAAT (gene therapy), or phosphate buffer saline (PBS). Age-matched and sham-operated animals were used as controls. Eight weeks after the treatment, animals were sacrificed and bone-related biomarkers and vertebral bone structure were evaluated. Results showed that hAAT gene therapy significantly decreased serum IL-6 level and receptor activator of NF-κB (RANK) gene expression in bone. Importantly, hAAT gene therapy increased bone volume/total volume and decreased structure model index (SMI) compared to PBS injection in OVX mice. These results demonstrate that hAAT gene therapy by rAAV vector efficiently mitigates bone loss possibly through inhibition of proinflammatory cytokine IL-6 and RANK gene expression. Considering the safety profile of hAAT and rAAV vector in humans, our results provide a new alternative for the treatment of osteoporosis.


Subject(s)
Dependovirus/genetics , Genetic Therapy , Genetic Vectors/administration & dosage , Osteoporosis/prevention & control , Ovariectomy/adverse effects , alpha 1-Antitrypsin/genetics , Animals , Disease Models, Animal , Female , Mice , Mice, Inbred C57BL , Osteoporosis/etiology
17.
PLoS One ; 11(5): e0156583, 2016.
Article in English | MEDLINE | ID: mdl-27232337

ABSTRACT

Systemic lupus erythematosus (SLE) is an autoimmune disorder with a worldwide distribution and considerable mortality and morbidity. Although the pathogenesis of this disease remains elusive, over-reactive dendritic cells (DCs) play a critical role in the disease development. It has been shown that human alpha-1 antitrypsin (hAAT) has protective effects in type 1 diabetes and rheumatoid arthritis mouse models. In the present study, we tested the effect of AAT on DC differentiation and functions, as well as its protective effect in a lupus-prone mouse model. We showed that hAAT treatment significantly inhibited LPS (TLR4 agonist) and CpG (TLR9 agonist) -induced bone-marrow (BM)-derived conventional and plasmacytoid DC (cDC and pDC) activation and reduced the production of inflammatory cytokines including IFN-I, TNF-α and IL-1ß. In MRL/lpr mice, hAAT treatment significantly reduced BM-derived DC differentiation, serum autoantibody levels, and importantly attenuated renal pathology. Our results for the first time demonstrate that hAAT inhibits DC activation and function, and it also attenuates autoimmunity and renal damage in the MRL/lpr lupus model. These results imply that hAAT has a therapeutic potential for the treatment of SLE in humans.


Subject(s)
Dendritic Cells/cytology , Dendritic Cells/drug effects , Lupus Erythematosus, Systemic/immunology , alpha 1-Antitrypsin/pharmacology , Animals , Autoantibodies/biosynthesis , Bone Marrow Cells/cytology , Cell Differentiation/drug effects , Cytokines/biosynthesis , Cytokines/metabolism , Disease Models, Animal , Disease Progression , Female , Gene Expression Regulation/drug effects , Humans , Lupus Erythematosus, Systemic/metabolism , Male , Mice
18.
PLoS One ; 10(9): e0138184, 2015.
Article in English | MEDLINE | ID: mdl-26372641

ABSTRACT

OBJECTIVE: Adipose tissue derived stem cells (ADSCs) transplantation has recently gained widespread enthusiasm, particularly in the perspective to use them as potential alternative cell sources for hepatocytes in cell based therapy, mainly because of their capability of hepatogenic differentiation in vitro and in vivo. But some challenges remain to be addressed, including whether ADSCs can be provided effectively to the target organ and whether subsequent proliferation of transplanted cells can be achieved. To date, intrasplenic injection is the conventional method to deliver ADSCs into the liver; however, a number of donor cells retained in the spleen has been reported. In this study, our objective is to evaluate a novel route to transplant ADSCs specifically to the liver. We aimed to test the feasibility of in situ transplantation of ADSCs by injecting bioencapsulated ADSCs into the liver in mouse model. METHODS: The ADSCs isolated from human alpha 1 antitrypsin (M-hAAT) transgenic mice were used to allow delivered ADSCs be readily identified in the liver of recipient mice, and alginate was selected as a cell carrier. We first evaluated whether alginate microspheres are implantable into the liver tissue by injection and whether ADSCs could migrate from alginate microspheres (study one). Once proven, we then examined the in vivo fate of ADSCs loaded microspheres in the liver. Specifically, we evaluated whether transplanted, undifferentiated ASDCs could be induced by the local microenvironment toward hepatogenic differentiation and the distribution of surviving ADSCs in major tissue organs (study two). RESULTS: Our results indicated ADSCs loaded alginate microspheres were implantable into the liver. Both degraded and residual alginate microspheres were observed in the liver up to three weeks. The viable ADSCs were detectable surrounding degraded and residual alginate microspheres in the liver and other major organs such as bone marrow and the lungs. Importantly, transplanted ADSCs underwent hepatogenic differentiation to become cells expressing albumin in the liver. These findings improve our understanding of the interplay between ADSCs (donor cells), alginate (biomaterial), and local microenvironment in a hepatectomized mouse model, and might improve the strategy of in situ transplantation of ADSCs in treating liver diseases.


Subject(s)
Adipose Tissue/cytology , Alginates/chemistry , Liver/cytology , Stem Cell Transplantation/methods , Stem Cells/chemistry , Stem Cells/cytology , Animals , Capsules , Cell Differentiation , Feasibility Studies , Glucuronic Acid/chemistry , Hepatocytes/cytology , Hexuronic Acids/chemistry , Humans , Injections , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microspheres , alpha 1-Antitrypsin/genetics
19.
J Stroke Cerebrovasc Dis ; 23(5): e355-63, 2014.
Article in English | MEDLINE | ID: mdl-24582784

ABSTRACT

Our objective is to develop a new therapy for the treatment of stroke. Currently, the only effective therapy for acute ischemic stroke is the thrombolytic agent recombinant tissue plasminogen activator. α1-Antitrypsin (AAT), a serine proteinase inhibitor with potent anti-inflammatory, anti-apoptotic, antimicrobial, and cytoprotective activities, could be beneficial in stroke. The goal of this study is to test whether AAT can improve ischemic stroke outcome in an established rat model. Middle cerebral artery occlusion was induced in male rats via intracranial (i.c.) microinjection of endothelin-1. Five to 10 minutes after stroke induction, rats received either i.c. or intravenous delivery of human AAT. Cylinder and vibrissae tests were used to evaluate sensorimotor function before and 72 hours after middle cerebral artery occlusion. Infarct volumes were examined via either 2,3,5-triphenyltetrazolium chloride assay or magnetic resonance imaging 72 hours after middle cerebral artery occlusion. Despite equivalent initial strokes, at 72 hours, the infarct volumes of the human AAT treatment groups (local and systemic injection) were statistically significantly reduced by 83% and 63% (P < .0001 and P < .05, respectively) compared with control rats. Human AAT significantly limited sensory motor system deficits. Human AAT could be a potential novel therapeutic drug for the protection against neurodegeneration after ischemic stroke, but more studies are needed to investigate the protective mechanisms and efficacy in other animal models.


Subject(s)
Brain/drug effects , Infarction, Middle Cerebral Artery/drug therapy , Neuroprotective Agents/pharmacology , alpha 1-Antitrypsin/pharmacology , Animals , Brain/blood supply , Brain/pathology , Brain/physiopathology , Cytoprotection , Disease Models, Animal , Endothelin-1 , Humans , Infarction, Middle Cerebral Artery/chemically induced , Infarction, Middle Cerebral Artery/diagnosis , Infarction, Middle Cerebral Artery/physiopathology , Injections, Intravenous , Magnetic Resonance Imaging , Male , Microinjections , Motor Activity/drug effects , Neuroprotective Agents/administration & dosage , Rats, Sprague-Dawley , Sensory Thresholds/drug effects , Time Factors , alpha 1-Antitrypsin/administration & dosage
20.
Hum Gene Ther Methods ; 25(1): 72-82, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24191859

ABSTRACT

Induced pluripotent stem (iPS) cells have great potential for personalized regenerative medicine. Although several different methods for generating iPS cells have been reported, improvement of safety and efficiency is imperative. In this study, we tested the feasibility of using a triple tyrosine mutant AAV2 (Y444+500+730F) vector, designated AAV2.3m, to generate iPS cells. We developed a polycistronic rAAV2.3m vector expressing three reprogramming factors, Klf4, Oct4, and Sox2, and then used this vector to infect mouse adipose-derived mesenchymal stem cells (AT-MSCs) to induce the generation of iPS cells. We demonstrated that (1) the triple tyrosine mutant AAV2 vector is able to reprogram mouse adult adipose tissue-derived stem cells into the pluripotent state. Those rAAV2.3m-derived iPS (rAAV2.3m-iPS) cells express endogenous pluripotency-associated genes including Oct4, Sox2, and SSEA-1, and form teratomas containing multiple tissues in vivo; (2) c-myc, an oncogene, is dispensable in rAAV2.3m-mediated cellular reprogramming; and (3) transgene expression is undetectable after reprogramming, whereas vector DNA is detectable, indicating that transgenes are silenced. These results indicated the rAAV vector may have some advantages in generating iPS cells.


Subject(s)
Adipose Tissue/cytology , Cellular Reprogramming , Dependovirus/genetics , Genetic Vectors/metabolism , Mesenchymal Stem Cells/cytology , Pluripotent Stem Cells/cytology , Animals , Genetic Vectors/genetics , Humans , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Lewis X Antigen/genetics , Lewis X Antigen/metabolism , Mesenchymal Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism , Teratoma/pathology , Transduction, Genetic
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