High-dose systemic adeno-associated virus vector administration causes liver and sinusoidal endothelial cell injury.

We analyzed retrospective data from toxicology studies involving administration of high doses of adeno-associated virus expressing different therapeutic transgenes to 21 cynomolgus and 15 rhesus macaques. We also conducted prospective studies to investigate acute toxicity following high-dose systemic administration of enhanced green fluorescent protein-expressing adeno-associated virus to 10 rhesus macaques. Toxicity was characterized by transaminitis, thrombocytopenia, and alternative complement pathway activation that peaked on post-administration day 3. Although most animals recovered, some developed ascites, generalized edema, hyperbilirubinemia, and/or coagulopathy that prompted unscheduled euthanasia. Study endpoint livers from animals that recovered and from unscheduled necropsies of those that succumbed to toxicity were analyzed via hypothesis-driven histopathology and unbiased single-nucleus RNA sequencing. All liver cell types expressed high transgene transcript levels at early unscheduled timepoints that subsequently decreased. Thrombocytopenia coincided with sinusoidal platelet microthrombi and sinusoidal endothelial injury identified via immunohistology and single-nucleus RNA sequencing. Acute toxicity, sinusoidal injury, and liver platelet sequestration were similarly observed with therapeutic transgenes and enhanced green fluorescent protein at doses ≥1 × 1014 GC/kg, suggesting it was the consequence of high-dose systemic adeno-associated virus administration, not green fluorescent protein toxicity. These findings highlight a potential toxic effect of high-dose intravenous adeno-associated virus on nonhuman primate liver microvasculature.

Immune transgene-dependent myocarditis in macaques after systemic administration of adeno-associated virus expressing human acid alpha-glucosidase.

Immune responses to human non-self transgenes can present challenges in preclinical studies of adeno-associated virus (AAV) gene therapy candidates in nonhuman primates. Although anti-transgene immune responses are usually mild and non-adverse, they can confound pharmacological readouts and complicate translation of results between species. We developed a gene therapy candidate for Pompe disease consisting of AAVhu68, a clade F AAV closely related to AAV9, that expresses an engineered human acid-alpha glucosidase (hGAA) tagged with an insulin-like growth factor 2 variant (vIGF2) peptide for enhanced cell uptake. Rhesus macaques were administered an intravenous dose of 1x1013 genome copies (GC)/kg, 5x1013 GC/kg, or 1 x 1014 GC/kg of AAVhu68.vIGF2.hGAA. Some unusually severe adaptive immune responses to hGAA presented, albeit with a high degree of variability between animals. Anti-hGAA responses ranged from absent to severe cytotoxic T-cell-mediated myocarditis with elevated troponin I levels. Cardiac toxicity was not dose dependent and affected five out of eleven animals. Upon further investigation, we identified an association between toxicity and a major histocompatibility complex class I haplotype (Mamu-A002.01) in three of these animals. An immunodominant peptide located in the C-terminal region of hGAA was subsequently identified via enzyme-linked immunospot epitope mapping. Another notable observation in this preclinical safety study cohort pertained to the achievement of robust and safe gene transfer upon intravenous administration of 5x1013 GC/kg in one animal with a low pre-existing neutralizing anti-capsid antibodies titer (1:20). Collectively, these findings may have significant implications for gene therapy inclusion criteria.

Fyn Kinase-Mediated PKCδ Y311 Phosphorylation Induces Dopaminergic Degeneration in Cell Culture and Animal Models: Implications for the Identification of a New Pharmacological Target for Parkinson's Disease.

Oxidative stress, neuroinflammation and apoptosis are some of the key etiological factors responsible for dopamin(DA)ergic degeneration during Parkinson's disease (PD), yet the downstream molecular mechanisms underlying neurodegeneration are largely unknown. Recently, a genome-wide association study revealed the FYN gene to be associated with PD, suggesting that Fyn kinase could be a pharmacological target for PD. In this study, we report that Fyn-mediated PKCδ tyrosine (Y311) phosphorylation is a key event preceding its proteolytic activation in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinsonism. MPP+/MPTP induced Fyn kinase activation in N27 DAergic neuronal cells and the mouse substantia nigra. PKCδ-Y311 phosphorylation by activated Fyn initiates the apoptotic caspase-signaling cascade during DAergic degeneration. Pharmacological attenuation of Fyn activity protected DAergic neurons from MPP+-induced degeneration in primary mesencephalic neuronal cultures. We further employed Fyn wild-type and Fyn knockout (KO) mice to confirm whether Fyn is a valid pharmacological target of DAergic neurodegeneration. Primary mesencephalic neurons from Fyn KO mice were greatly protected from MPP+-induced DAergic cell death, neurite loss and DA reuptake loss. Furthermore, Fyn KO mice were significantly protected from MPTP-induced PKCδ-Y311 phosphorylation, behavioral deficits and nigral DAergic degeneration. This study thus unveils a mechanism by which Fyn regulates PKCδ's pro-apoptotic function and DAergic degeneration. Pharmacological inhibitors directed at Fyn activation could prove to be a novel therapeutic target in the delay or halting of selective DAergic degeneration during PD.

The spatial transmission of SARS-CoV-2 in China under the prevention and control measures at the early outbreak.

BACKGROUND: Since severe acute respiratory syndrome coronavirus, 2 (SARS-CoV-2) was firstly reported in Wuhan City, China in December 2019, Novel Coronavirus Disease 2019 (COVID-19) that is caused by SARS-CoV-2 is predominantly spread from person-to-person on worldwide scales. Now, COVID-19 is a non-traditional and major public health issue the world is facing, and the outbreak is a global pandemic. The strict prevention and control measures have mitigated the spread of SARS-CoV-2 and shown positive changes with important progress in China. But prevention and control tasks remain arduous for the world. The objective of this study is to discuss the difference of spatial transmission characteristics of COVID-19 in China at the early outbreak stage with resolute efforts. Simultaneously, the COVID-19 trend of China at the early time was described from the statistical perspective using a mathematical model to evaluate the effectiveness of the prevention and control measures. METHODS: In this study, the accumulated number of confirmed cases publicly reported by the National Health Committee of the People's Republic of China (CNHC) from January 20 to February 11, 2020, were grouped into three partly overlapping regions: Chinese mainland including Hubei province, Hubei province alone, and the other 30 provincial-level regions on Chinese mainland excluding Hubei province, respectively. A generalized-growth model (GGM) was used to estimate the basic reproduction number to evaluate the transmissibility in different spatial locations. The prevention and control of COVID-19 in the early stage were analyzed based on the number of new cases of confirmed infections daily reported. RESULTS: Results indicated that the accumulated number of confirmed cases reported from January 20 to February 11, 2020, is well described by the GGM model with a larger correlation coefficient than 0.99. When the accumulated number of confirmed cases is well fitted by an exponential function, the basic reproduction number of COVID-19 of the 31 provincial-level regions on the Chinese mainland, Hubei province, and the other 30 provincial-level regions on the Chinese mainland excluding Hubei province, is 2.68, 6.46 and 2.18, respectively. The consecutive decline of the new confirmed cases indicated that the prevention and control measures taken by the Chinese government have contained the spread of SARS-CoV-2 in a short period. CONCLUSIONS: The estimated basic reproduction number thorough GGM model can reflect the spatial difference of SARS-CoV-2 transmission in China at the early stage. The strict prevention and control measures of SARS-CoV-2 taken at the early outbreak can effectively reduce the new confirmed cases outside Hubei and have mitigated the spread and yielded positive results since February 2, 2020. The research results indicated that the outbreak of COVID-19 in China was sustaining localized at the early outbreak stage and has been gradually curbed by China's resolute efforts.

MicroRNA-mediated inhibition of transgene expression reduces dorsal root ganglion toxicity by AAV vectors in primates.

Delivering adeno-associated virus (AAV) vectors into the central nervous system of nonhuman primates (NHPs) via the blood or cerebral spinal fluid is associated with dorsal root ganglion (DRG) toxicity. Conventional immune-suppression regimens do not prevent this toxicity, possibly because it may be caused by high transduction rates, which can, in turn, cause cellular stress due to an overabundance of the transgene product in target cells. To test this hypothesis and develop an approach to eliminate DRG toxicity, we exploited endogenous expression of microRNA (miR) 183 complex, which is largely restricted to DRG neurons, to specifically down-regulate transgene expression in these cells. We introduced sequence targets for miR183 into the vector genome within the 3' untranslated region of the corresponding transgene messenger RNA and injected vectors into the cisterna magna of NHPs. Administration of unmodified AAV vectors resulted in robust transduction of target tissues and toxicity in DRG neurons. Consistent with the proposal that immune system activity does not mediate this neuronal toxicity, we found that steroid administration was ineffective in alleviating this pathology. However, including miR183 targets in the vectors reduced transgene expression in, and toxicity of, DRG neurons without affecting transduction elsewhere in the primate's brain. This approach might be useful in reducing DRG toxicity and the associated morbidity and should facilitate the development of AAV-based gene therapies for many central nervous system diseases.

Dose Range Finding Studies with Two RPGR Transgenes in a Canine Model of X-Linked Retinitis Pigmentosa Treated with Subretinal Gene Therapy.

Recombinant adeno-associated viral (rAAV) vector-mediated gene therapy is being developed to treat X-linked retinitis pigmentosa (XLRP) in patients with mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. In preparation for a clinical gene therapy trial, we conducted dose range finding (DRF) studies with an AAV2 capsid with three surface tyrosine residues changed to phenylalanine (AAV2tYF) vector administered by subretinal injection in a naturally occurring RPGR-mutant canine model (XLPRA2) to compare two different human RPGR (hRPGR) transgenes and to establish a reasonable starting dose for a clinical trial. Different dose levels of two candidate vectors (0.15 mL at 1.2 × 1010-3.0 × 1012 vg/mL of rAAV2tYF-GRK1-hRPGRco or 4 × 1010-3.0 × 1012 vg/mL of rAAV2tYF-GRK1-hRPGRstb), 6.0 × 1011 vg/mL rAAV5-GRK1-hRPGRco reference vector or Vehicle were subretinally administered, and the dogs were followed for 8 weeks postdose. Ophthalmic examinations, analyses of retinal structure by in vivo imaging using confocal scanning laser ophthalmoscopy (cSLO)/optical coherence tomography (OCT) in the Lower (4.0 × 1010 vg/mL) and Lowest (1.2 × 1010 vg/mL) Doses, immunological responses by cell based assays or enzyme-linked immunosorbent assay, RPGR transgene expression, and reversal of opsin mislocalization by immunohistochemistry were performed. No sustained signs of ocular discomfort or ophthalmic complications were noted in any of the injected eyes except some in the High Dose group (3.0 × 1012 vg/mL), which showed signs of retinal detachment and inflammation. A change in fundus reflectivity suggestive of a rescue effect was seen in the High, Mid (6.0 × 1011 vg/mL), and Low (1.2 × 1011 vg/mL) Dose groups. cSLO/OCT demonstrated qualitative and quantitative evidence of rescue effect in eyes treated with the Lower Dose. Anti-hRPGR antibodies were absent, but neutralizing antibody titers against AAV2 were detected in all animals dosed with rAAV2tYF in an apparent dose-related pattern. RPGR expression was stronger for rAAV2tYF-GRK1-hRPGRco compared to rAAV2tYF-GRK1-hRPGRstb at all dose levels. Subretinal administration of rAAV2tYF-GRK1-hRPGRco and rAAV2tYF-GRK1-hRPGRstb both corrected rod and cone opsin mislocalization, two early markers of disease in the XLPRA2 canine model of RPGR-XLRP. These results support the selection and use of rAAV2tYF-GRK1-hRPGRco (AGTC-501) and guided the initial doses in clinical studies in patients with XLRP caused by RPGR mutations.

Toxicity and Efficacy Evaluation of an Adeno-Associated Virus Vector Expressing Codon-Optimized RPGR Delivered by Subretinal Injection in a Canine Model of X-linked Retinitis Pigmentosa.

Applied Genetic Technologies Corporation (AGTC) is developing a recombinant adeno-associated virus (rAAV) vector AGTC-501, also designated rAAV2tYF-GRK1-hRPGRco, to treat X-linked retinitis pigmentosa (XLRP) in patients with mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. The vector contains a codon-optimized human RPGR cDNA (hRPGRco) driven by a photoreceptor-specific promoter (G protein-coupled receptor kinase 1 [GRK1]), and is packaged in an AAV2 capsid variant with three surface tyrosine residues changed to phenylalanine (AAV2tYF). We conducted a toxicity and efficacy study of this vector administered by subretinal injection in the naturally occurring RPGR mutant (X-linked progressive retinal atrophy 2 [XLPRA2]) dog model. Sixteen RPGR mutant dogs divided into four groups of three to five animals each received either a subretinal injection of 0.07 mL of AGTC-501 at low (1.2 × 1011 vector genome [vg]/mL), mid (6 × 1011 vg/mL), or high dose (3 × 1012 vg/mL), or of vehicle control in the right eye at early-stage disease. The left eye remained untreated. Subretinal injections were well tolerated and were not associated with systemic toxicity. Electroretinography, in vivo retinal imaging, and histological analysis showed rescue of photoreceptor function and structure in the absence of ocular toxicity in the low- and mid-dose treatment groups when compared with the vehicle-treated group. The high-dose group showed evidence of both photoreceptor rescue and posterior segment toxicity. These results support the use of AGTC-501 in clinical studies with patients affected with XLRP caused by RPGR mutations and define the no-observed-adverse-effect level at 6 × 1011 vg/mL.

Ocular Inflammatory Response to Intravitreal Injection of Adeno-Associated Virus Vector: Relative Contribution of Genome and Capsid.

Both subretinal dosing and intravitreal (IVT) dosing of adeno-associated virus (AAV) in higher species induce mild and transient inflammatory responses that increase with dose. Foreign protein and foreign DNA are known inducers of inflammation, which is also true in the immune-privileged ocular environment. We explored which component(s) of AAV vectors, viral capsid, or viral DNA drive inflammatory responses. Recombinant AAV with three tyrosine to phenylalanine substitutions in the capsid of AAV serotype 2 (rAAV2tYF), and with a generic ubiquitous promoter (cytomegalovirus [CMV]) controlling the expression of humanized green fluorescent protein (hGFP), was processed to enrich for AAV capsids containing genome (full capsids), capsids without genome (empty capsids), and residual material. Nonhuman primate eyes were injected by IVT in both eyes. During in-life, ocular inflammation and development of neutralizing antibodies (NAb) were measured. Following termination, lymph node immunophenotyping was performed, vitreous was processed for cytokine and RNAseq analyses, and ocular sections were assessed for transgene expression (by in situ hybridization) and histopathology. IVT dosing of AAV vectors transiently raised cellular inflammation in the aqueous and induced a more sustained inflammation in the vitreous. Lowering the total capsid dose by removing empty AAV capsids reduced inflammation and improved viral transduction. IVT dosing of AAV induced systemic NAb to AAV irrespective of the vector preparation. Similarly, lymph node immunophenotyping revealed identical profiles irrespective of viral preparation used for dosing. Immune cells in the vitreous were identified based on RNAseq analysis. Three months postdose, cytokine levels were low, indicative of minimal levels of inflammation in agreement with histopathological assessment of the retina.

Mechanistic Interplay Between Autophagy and Apoptotic Signaling in Endosulfan-Induced Dopaminergic Neurotoxicity: Relevance to the Adverse Outcome Pathway in Pesticide Neurotoxicity.

Chronic exposure to pesticides is implicated in the etiopathogenesis of Parkinson's disease (PD). Previously, we showed that dieldrin induces dopaminergic neurotoxicity by activating a cascade of apoptotic signaling pathways in experimental models of PD. Here, we systematically investigated endosulfan's effect on the interplay between apoptosis and autophagy in dopaminergic neuronal cell models of PD. Exposing N27 dopaminergic neuronal cells to endosulfan rapidly induced autophagy, indicated by an increased number of autophagosomes and LC3-II accumulation. Prolonged endosulfan exposure (>9 h) triggered apoptotic signaling, including caspase-2 and -3 activation and protein kinase C delta (PKCδ) proteolytic activation, ultimately leading to cell death, thus demonstrating that autophagy precedes apoptosis during endosulfan neurotoxicity. Furthermore, inhibiting autophagy with wortmannin, a phosphoinositide 3-kinase inhibitor, potentiated endosulfan-induced apoptosis, suggesting that autophagy is an early protective response against endosulfan. Additionally, Beclin-1, a major regulator of autophagy, was cleaved during the initiation of apoptotic cell death, and the cleavage was predominantly mediated by caspase-2. Also, caspase-2 and caspase-3 inhibitors effectively blocked endosulfan-induced apoptotic cell death. CRISPR/Cas9-based stable knockdown of PKCδ significantly attenuated endosulfan-induced caspase-3 activation, indicating that the kinase serves as a regulatory switch for apoptosis. Additional studies in primary mesencephalic neuronal cultures confirmed endosulfan's effect on autophagy and neuronal degeneration. Collectively, our results demonstrate that a functional interplay between autophagy and apoptosis dictate pesticide-induced neurodegenerative processes in dopaminergic neuronal cells. Our study provides insight into cell death mechanisms in environmentally linked neurodegenerative diseases.

Toxicology and Pharmacology of an AAV Vector Expressing Codon-Optimized RPGR in RPGR-Deficient Rd9 Mice.

Applied Genetic Technologies Corporation (AGTC) is developing a recombinant adeno-associated virus (rAAV) vector AGTC-501, also designated AAV2tYF-GRK1-RPGRco, to treat retinitis pigmentosa (RP) in patients with mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. The vector contains a codon-optimized human RPGR cDNA (RPGRco) driven by a photoreceptor-specific promoter (G protein-coupled receptor kinase 1, GRK1) and is packaged in an AAV2 capsid with three surface tyrosine residues changed to phenylalanine (AAV2tYF). We conducted a safety and potency study of this vector administered by subretinal a injection in the naturally occurring RPGR-deficient Rd9 mouse model. Sixty Rd9 mice (20 per group) received a subretinal injection in the right eye of vehicle (control) or AAV2tYF-GRK1-RPGRco at one of two dose levels (4 × 108 or 4 × 109 vg/eye) and were followed for 12 weeks after injection. Vector injections were well tolerated, with no systemic toxicity. There was a trend towards reduced electroretinography b-wave amplitudes in the high vector dose group that was not statistically significant. There were no clinically important changes in hematology or clinical chemistry parameters and no vector-related ocular changes in life or by histological examination. Dose-dependent RPGR protein expression, mainly in the inner segment of photoreceptors and the adjacent connecting cilium region, was observed in all vector-treated eyes examined. Sequence integrity of the codon-optimized RPGR was confirmed by sequencing of PCR-amplified DNA, or cDNA reverse transcribed from total RNA extracted from vector-treated retinal tissues, and by sequencing of RPGR protein obtained from transfected HEK 293 cells. These results support the use of rAAV2tYF-GRK1-RPGRco in clinical studies in patients with XLRP caused by RPGR mutations.

Oxidative stress-mediated NFκB phosphorylation upregulates p62/SQSTM1 and promotes retinal pigmented epithelial cell survival through increased autophagy.

p62 is a scaffolding adaptor implicated in the clearance of protein aggregates by autophagy. Reactive oxygen species (ROS) can either stimulate or inhibit NFκB-mediated gene expression influencing cellular fate. We studied the effect of hydrogen peroxide (H2O2)-mediated oxidative stress and NFκB signaling on p62 expression in the retinal pigment epithelium (RPE) and investigated its role in regulation of autophagy and RPE survival against oxidative damage. Cultured human RPE cell line ARPE-19 and primary human adult and fetal RPE cells were exposed to H2O2-induced oxidative stress. The human apolipoprotein E4 targeted-replacement (APOE4) mouse model of AMD was used to study expression of p62 and other autophagy proteins in the retina. p62, NFκB p65 (total, phosphorylated, nuclear and cytoplasmic) and ATG10 expression was assessed by mRNA and protein analyses. Cellular ROS and mitochondrial superoxide were measured by CM-H2DCFDA and MitoSOX staining respectively. Mitochondrial viability was determined using MTT activity. qPCR-array system was used to investigate autophagic genes affected by p62. Nuclear and cytoplasmic levels of NFκB p65 were evaluated after cellular fractionation by Western blotting. We report that p62 is up-regulated in RPE cells under H2O2-induced oxidative stress and promotes autophagic activity. Depletion of endogenous p62 reduces autophagy by downregulation of ATG10 rendering RPE more susceptible to oxidative damage. NFκB p65 phosphorylation at Ser-536 was found to be critical for p62 upregulation in response to oxidative stress. Proteasome inhibition by H2O2 causes p62-NFκB signaling as antioxidant pre-treatment reversed p62 expression and p65 phosphorylation when RPE was challenged by H2O2 but not when by Lactacystin. p62 protein but not RNA levels are elevated in APOE4-HFC AMD mouse model, suggesting reduction of autophagic flux in disease conditions. Our findings suggest that p62 is necessary for RPE cytoprotection under oxidative stress and functions, in part, by modulating ATG10 expression. NFκB p65 activity may be a critical upstream initiator of p62 expression in RPE cells under oxidative stress.

Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD.

Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.

Oral delivery of Angiotensin-converting enzyme 2 and Angiotensin-(1-7) bioencapsulated in plant cells attenuates pulmonary hypertension.

Emerging evidences indicate that diminished activity of the vasoprotective axis of the renin-angiotensin system, constituting angiotensin-converting enzyme 2 (ACE2) and its enzymatic product, angiotensin-(1-7) [Ang-(1-7)] contribute to the pathogenesis of pulmonary hypertension (PH). However, long-term repetitive delivery of ACE2 or Ang-(1-7) would require enhanced protein stability and ease of administration to improve patient compliance. Chloroplast expression of therapeutic proteins enables their bioencapsulation within plant cells to protect against gastric enzymatic degradation and facilitates long-term storage at room temperature. Besides, fusion to a transmucosal carrier helps effective systemic absorption from the intestine on oral delivery. We hypothesized that bioencapsulating ACE2 or Ang-(1-7) fused to the cholera nontoxin B subunit would enable development of an oral delivery system that is effective in treating PH. PH was induced in male Sprague Dawley rats by monocrotaline administration. Subset of animals was simultaneously treated with bioencapsulaed ACE2 or Ang-(1-7) (prevention protocol). In a separate set of experiments, drug treatment was initiated after 2 weeks of PH induction (reversal protocol). Oral feeding of rats with bioencapsulated ACE2 or Ang-(1-7) prevented the development of monocrotaline-induced PH and improved associated cardiopulmonary pathophysiology. Furthermore, in the reversal protocol, oral ACE2 or Ang-(1-7) treatment significantly arrested disease progression, along with improvement in right heart function, and decrease in pulmonary vessel wall thickness. In addition, a combination therapy with ACE2 and Ang-(1-7) augmented the beneficial effects against monocrotaline-induced lung injury. Our study provides proof-of-concept for a novel low-cost oral ACE2 or Ang-(1-7) delivery system using transplastomic technology for pulmonary disease therapeutics.

Autophagy induction is a survival response against oxidative stress in bone marrow-derived mesenchymal stromal cells.

BACKGROUND AIMS: Bone marrow-derived mesenchymal stromal cells (BMSCs) are being extensively investigated as cellular therapeutics for many diseases, including cardiovascular diseases. Although preclinical studies indicated that BMSC transplantation into infarcted hearts improved heart function, there are problems to be resolved, such as the low survival rate of BMSCs during the transplantation process and in the ischemic region with extreme oxidative stress. Autophagy plays pivotal roles in maintaining cellular homeostasis and defending against environmental stresses. However, the precise roles of autophagy in BMSCs under oxidative stress remain largely uncharacterized. METHODS: BMSCs were treated with H2O2, and autophagic flux was examined by means of microtubule-associated protein 1A/1B-light chain 3 II/I ratio (LC3 II/I), autophagosome formation and p62 expression. Cytotoxicity and cell death assays were performed after co-treatment of BMSCs by autophagy inhibitor (3-methyladenine) or autophagy activator (rapamycin) together with H2O2. RESULTS: We show that short exposure (1 h) of BMSCs to H2O2 dramatically elevates autophagic flux (2- to 4-fold), whereas 6-h prolonged oxidative treatment reduces autophagy but enhances caspase-3 and caspase-6-associated apoptosis. Furthermore, we show that pre- and co-treatment with rapamycin ameliorates H2O2-induced caspase-3 and caspase-6 activation and cell toxicity but that 3-methyladenine exacerbates H2O2-induced cell apoptotic cell death. CONCLUSIONS: Our results demonstrate that autophagy is critical for the survival of BMSCs under oxidative conditions. Importantly, we also suggest that the early induction of autophagic flux is possibly a self-defensive mechanism common in oxidant-tolerant cells.

Diminazene aceturate enhances angiotensin-converting enzyme 2 activity and attenuates ischemia-induced cardiac pathophysiology.

Angiotensin-converting enzyme 2 (ACE2) plays a critical role against myocardial infarction (MI). We hypothesized that activation of intrinsic ACE2 would be protective against ischemia-induced cardiac pathophysiology. Diminazene aceturate (DIZE), a small molecule ACE2 activator, has been used to evaluate this hypothesis. DIZE (15 mg/kg per day, s.c.) was injected 2 days before MI surgery and continued throughout the study period. MI rats showed a 62% decrease in fractional shortening (%; control, 51.1±3.2; DIZE alone, 52.1±3.2; MI, 19.1±3.0), a 55% decrease in contractility (dP/dtmax mm Hg/s; control, 9480±425.3; DIZE alone, 9585±597.4; MI, 4251±657.7), and a 27% increase in ventricular hypertrophy (mg/mm; control, 26.5±1.5; DIZE alone, 26.9±1.4; MI, 33.4±1.1). DIZE attenuated the MI-induced decrease in fractional shortening by 89%, improved dP/dtmax by 92%, and reversed ventricular hypertrophy by 18%. MI also significantly increased ACE and angiotensin type 1 receptor levels but decreased ACE2 activity by 40% (control, 246.2±25.1; DIZE alone, 254.2±20.6; MI, 148.9±29.2; RFU/min), which was reversed by DIZE treatment. Thus, DIZE treatment decreased the infarct area, attenuated LV remodeling post-MI, and restored normal balance of the cardiac renin-angiotensin system. In addition, DIZE treatment increased circulating endothelial progenitor cells, increased engraftment of cardiac progenitor cells, and decreased inflammatory cells in peri-infarct cardiac regions. All of the beneficial effects associated with DIZE treatment were abolished by C-16, an ACE2 inhibitor. Collectively, DIZE and DIZE-like small molecules may represent promising new therapeutic agents for MI.

Vanadium induces dopaminergic neurotoxicity via protein kinase Cdelta dependent oxidative signaling mechanisms: relevance to etiopathogenesis of Parkinson's disease.

Environmental exposure to neurotoxic metals through various sources including exposure to welding fumes has been linked to an increased incidence of Parkinson's disease (PD). Welding fumes contain many different metals including vanadium typically present as particulates containing vanadium pentoxide (V2O5). However, possible neurotoxic effects of this metal oxide on dopaminergic neuronal cells are not well studied. In the present study, we characterized vanadium-induced oxidative stress-dependent cellular events in cell culture models of PD. V2O5 was neurotoxic to dopaminergic neuronal cells including primary nigral dopaminergic neurons and the EC50 was determined to be 37 microM in N27 dopaminergic neuronal cell model. The neurotoxic effect was accompanied by a time-dependent uptake of vanadium and upregulation of metal transporter proteins Tf and DMT1 in N27 cells. Additionally, vanadium resulted in a threefold increase in reactive oxygen species generation, followed by release of mitochondrial cytochrome c into cytoplasm and subsequent activation of caspase-9 (>fourfold) and caspase-3 (>ninefold). Interestingly, vanadium exposure induced proteolytic cleavage of native protein kinase Cdelta (PKCdelta, 72-74 kDa) to yield a 41 kDa catalytically active fragment resulting in a persistent increase in PKCdelta kinase activity. Co-treatment with pan-caspase inhibitor Z-VAD-FMK significantly blocked vanadium-induced PKCdelta proteolytic activation, indicating that caspases mediate PKCdelta cleavage. Also, co-treatment with Z-VAD-FMK almost completely inhibited V2O5-induced DNA fragmentation. Furthermore, PKCdelta knockdown using siRNA protected N27 cells from V2O5-induced apoptotic cell death. Collectively, these results demonstrate that vanadium can exert neurotoxic effects in dopaminergic neuronal cells via caspase-3-dependent PKCdelta cleavage, suggesting that metal exposure may promote nigral dopaminergic degeneration.

Chronic low-dose oxidative stress induces caspase-3-dependent PKCdelta proteolytic activation and apoptosis in a cell culture model of dopaminergic neurodegeneration.

Oxidative stress has been implicated as a key event in the degenerative process of dopaminergic neurons; however, the cellular mechanisms underlying chronic oxidative stress-induced neurodegeneration remain to be established. In this study, N27 cells, a dopaminergic neuronal cell line derived from rat mesencephalon, exposed to low doses of H(2)O(2) (0-30 muM for 12-24 hr) exhibited dose- and time-dependent increases in cytotoxicity and ROS generation. In addition, the H(2)O(2)-induced neurotoxicity was accompanied by increased caspase-3 activity and PKCdelta cleavage. Notably, treatment with antioxidants Trolox and MnTBAP or PKCdelta cleavage inhibitor z-DIPD-fmk significantly protected against oxidative stress-induced apoptotic cell death. These results demonstrate that the N27 cell line is a useful model for the study of the chronic low-dose oxidative stress-induced apoptotic cell death cascade and that caspase-3-dependent PKCdelta proteolytic activation may be important in the apoptotic process in dopaminergic neurons undergoing chronic oxidative insult.

Proteasome inhibitor-induced apoptosis is mediated by positive feedback amplification of PKCdelta proteolytic activation and mitochondrial translocation.

Emerging evidence implicates impaired protein degradation by the ubiquitin proteasome system (UPS) in Parkinson's disease; however cellular mechanisms underlying dopaminergic degeneration during proteasomal dysfunction are yet to be characterized. In the present study, we identified that the novel PKC isoform PKCdelta plays a central role in mediating apoptotic cell death following UPS dysfunction in dopaminergic neuronal cells. Inhibition of proteasome function by MG-132 in dopaminergic neuronal cell model (N27 cells) rapidly depolarized mitochondria independent of ROS generation to activate the apoptotic cascade involving cytochrome c release, and caspase-9 and caspase-3 activation. PKCdelta was a key downstream effector of caspase-3 because the kinase was proteolytically cleaved by caspase-3 following exposure to proteasome inhibitors MG-132 or lactacystin, resulting in a persistent increase in the kinase activity. Notably MG-132 treatment resulted in translocation of proteolytically cleaved PKCdelta fragments to mitochondria in a time-dependent fashion, and the PKCdelta inhibition effectively blocked the activation of caspase-9 and caspase-3, indicating that the accumulation of the PKCdelta catalytic fragment in the mitochondrial fraction possibly amplifies mitochondria-mediated apoptosis. Overexpression of the kinase active catalytic fragment of PKCdelta (PKCdelta-CF) but not the regulatory fragment (RF), or mitochondria-targeted expression of PKCdelta-CF triggers caspase-3 activation and apoptosis. Furthermore, inhibition of PKCdelta proteolytic cleavage by a caspase-3 cleavage-resistant mutant (PKCdelta-CRM) or suppression of PKCdelta expression by siRNA significantly attenuated MG-132-induced caspase-9 and -3 activation and DNA fragmentation. Collectively, these results demonstrate that proteolytically activated PKCdelta has a significant feedback regulatory role in amplification of the mitochondria-mediated apoptotic cascade during proteasome dysfunction in dopaminergic neuronal cells.