Chemical Modulators for Targeting Autism Spectrum Disorders: From Bench to Clinic.

Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by diverse behavioral symptoms such as repetitive behaviors, social deficits, anxiety, hyperactivity, and irritability. Despite their increasing incidence, the specific pathological mechanisms of ASD are still unknown, and the degree and types of symptoms that vary from patient to patient make it difficult to develop drugs that target the core symptoms of ASD. Although various atypical antipsychotics and antidepressants have been applied to regulate ASD symptoms, these drugs can only alleviate the symptoms and do not target the major causes. Therefore, development of novel drugs targeting factors directly related to the onset of ASD is required. Among the various factors related to the onset of ASD, several chemical modulators to treat ASD, focused on serotonin (5-hydroxytryptamine, 5-HT) and glutamate receptors, microbial metabolites, and inflammatory cytokines, are explored in this study. In particular, we focus on the chemical drugs that have improved various aspects of ASD symptoms in animal models and in clinical trials for various ages of patients with ASD.

Nanoparticle-Mediated Blocking of Excessive Inflammation for Prevention of Heart Failure Following Myocardial Infarction.

Severe cardiac damage following myocardial infarction (MI) causes excessive inflammation, which sustains tissue damage and often induces adverse cardiac remodeling toward cardiac function impairment and heart failure. Timely resolution of post-MI inflammation may prevent cardiac remodeling and development of heart failure. Cell therapy approaches for MI are time-consuming and costly, and have shown marginal efficacy in clinical trials. Here, nanoparticles targeting the immune system to attenuate excessive inflammation in infarcted myocardium are presented. Liposomal nanoparticles loaded with MI antigens and rapamycin (L-Ag/R) enable effective induction of tolerogenic dendritic cells presenting the antigens and subsequent induction of antigen-specific regulatory T cells (Tregs). Impressively, intradermal injection of L-Ag/R into acute MI mice attenuates inflammation in the myocardium by inducing Tregs and an inflammatory-to-reparative macrophage polarization, inhibits adverse cardiac remodeling, and improves cardiac function. Nanoparticle-mediated blocking of excessive inflammation in infarcted myocardium may be an effective intervention to prevent the development of post-MI heart failure.

Senolytic Therapy for Cerebral Ischemia-Reperfusion Injury.

Ischemic stroke is one of the leading causes of death, and even timely treatment can result in severe disabilities. Reperfusion of the ischemic stroke region and restoration of the blood supply often lead to a series of cellular and biochemical consequences, including generation of reactive oxygen species (ROS), expression of inflammatory cytokines, inflammation, and cerebral cell damage, which is collectively called cerebral ischemia-reperfusion (IR) injury. Since ROS and inflammatory cytokines are involved in cerebral IR injury, injury could involve cellular senescence. Thus, we investigated whether senolytic therapy that eliminates senescent cells could be an effective treatment for cerebral IR injury. To determine whether IR induces neural cell senescence in vitro, astrocytes were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). OGD/R induced astrocyte senescence and senescent cells in OGD/R-injured astrocytes were effectively eliminated in vitro by ABT263, a senolytic agent. IR in rats with intraluminal middle cerebral artery occlusion induced cellular senescence in the ischemic region. The senescent cells in IR-injured rats were effectively eliminated by intravenous injections of ABT263. Importantly, ABT263 treatment significantly reduced the infarct volume and improved neurological function in behavioral tests. This study demonstrated, for the first time, that senolytic therapy has therapeutic potential for cerebral IR injury.

Umbilical Cord Mesenchymal Stem Cell-Derived Nanovesicles Potentiate the Bone-Formation Efficacy of Bone Morphogenetic Protein 2.

Recombinant human bone morphogenetic protein 2 (rhBMP-2) is one of the most potent osteogenic factors used to treat bone loss. However, at higher doses, rhBMP-2 does not necessarily increase bone formation but rather increases the incidence of adverse side effects. Here, we investigated whether umbilical cord mesenchymal stem cell (UCMSC)-derived nanovesicles (NVs) further increase the in vivo bone formation at high doses of rhBMP-2. In the presence of UCMSC-derived NVs, proliferation, migration, and tube formation of human umbilical vein endothelial cells were stimulated in vitro. Furthermore, migration and osteogenesis of human bone marrow-derived mesenchymal stem cells were stimulated. To examine the efficacy of UCMSC-derived NVs on in vivo bone formation, collagen sponges soaked with rhBMP-2 and UCMSC-derived NVs were used in athymic nude mice with calvarial defects. At a high rhBMP-2 dosage (500 ng/mL), UCMSC-derived NVs significantly promoted bone formation in calvarial defects; however, the UCMSC-derived NVs alone did not induce in vivo bone formation. Our results indicate that UCMSC-derived NVs can potentiate the bone formation efficacy of rhBMP-2 at a high dosage.

Microtubule-destabilizing agents enhance STING-mediated innate immune response via biased mechanism in human monocyte cells.

The stimulator of the interferon gene (STING) signaling pathway acts as a primary defense system against DNA pathogens. Because of the crucial role of STING in type I interferon (IFN) response and innate immunity, extensive research has been conducted to elucidate the roles of various effector molecules involved in STING-mediated signal transduction. However, despite the substantial contribution of microtubules to the immune system, the association between the STING signaling pathway and microtubules remains unclear. In this study, we revealed that the modulation of STING via microtubule-destabilizing agents (MDAs) specifically induced type I IFN responses rather than inflammatory responses in human monocytes. Co-treatment of MDAs with STING agonists induced the elevation of phospho-TANK-binding kinase 1 (TBK1), amplifying the innate immune response. However, during the deficiency of TBK1, the non-canonical signaling pathway through nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) contributed to MDA-induced STING activation in type I IFN response which suggested the versatile regulation of MDA in STING-mediated immunity.

A Senolytic-Eluting Coronary Stent for the Prevention of In-Stent Restenosis.

The vast majority of drug-eluting stents (DES) elute either sirolimus or one of its analogues. While limus drugs stymie vascular smooth muscle cell (VSMC) proliferation to prevent in-stent restenosis, their antiproliferative nature is indiscriminate and limits healing of the endothelium in stented vessels, increasing the risk of late-stent thrombosis. Oxidative stress, which is associated with vascular injury from stent implantation, can induce VSMCs to undergo senescence, and senescent VSMCs can produce pro-inflammatory cytokines capable of inducing proliferation of neighboring nonsenescent VSMCs. We explored the potential of senolytic therapy, which involves the selective elimination of senescent cells, in the form of a senolytic-eluting stent (SES) for interventional cardiology. Oxidative stress was modeled in vitro by exposing VSMCs to H2O2, and H2O2-mediated senescence was evaluated by cytochemical staining of senescence-associated ß-galactosidase activity and qRT-PCR. Quiescent VSMCs were then treated with the conditioned medium (CM) of H2O2-treated VSMCs. Proliferative effects of CM were analyzed by staining for proliferating cell nuclear antigen. Senolytic effects of the first-generation senolytic ABT263 were observed in vitro, and the effects of ABT263 on endothelial cells were also investigated through an in vitro re-endothelialization assay. SESs were prepared by dip coating. Iliofemoral arteries of hypercholesteremic rabbits were implanted with SES, everolimus-eluting stents (EESs), or bare-metal stents (BMSs), and the area of stenosis was measured 4 weeks post-implantation using optical coherence tomography. We found that a portion of H2O2-treated VSMCs underwent senescence, and that CM of H2O2-treated senescent VSMCs triggered the proliferation of quiescent VSMCs. ABT263 reverted H2O2-mediated senescence and the proliferative capacity of senescent VSMC CM. Unlike everolimus, ABT263 did not affect endothelial cell migration and/or proliferation. SES, but not EES, significantly reduced stenosis area in vivo compared with bare-metal stents (BMSs). This study shows the potential of SES as an alternative to current forms of DES.

Local Delivery of Senolytic Drug Inhibits Intervertebral Disc Degeneration and Restores Intervertebral Disc Structure.

Intervertebral disc (IVD) degeneration (IVDD) is a leading cause of chronic low back pain. There is a strong clinical demand for more effective treatments for IVDD as conventional treatments provide only symptomatic relief rather than arresting IVDD progression. This study shows that senolytic therapy with local drug delivery can inhibit IVDD and restore IVD integrity. ABT263, a senolytic drug, is loaded in poly(lactic-co-glycolic acid) nanoparticles (PLGA-ABT) and intradiscally administered into injury-induced IVDD rat models. The single intradiscal injection of PLGA-ABT may enable local delivery of the drug to avascular IVD, prevention of potential systemic toxicity caused by systemic administration of senolytic drug, and morbidity caused by repetitive injections of free drug into the IVD. The strategy results in the selective elimination of senescent cells from the degenerative IVD, reduces expressions of pro-inflammatory cytokines and matrix proteases in the IVD, inhibits progression of IVDD, and even restores the IVD structure. This study demonstrates for the first time that local delivery of senolytic drug can effectively treat senescence-associated IVDD. This approach can be extended to treat other types of senescence-associated degenerative diseases.

Local delivery of a senolytic drug in ischemia and reperfusion-injured heart attenuates cardiac remodeling and restores impaired cardiac function.

Myocardial ischemia-reperfusion (IR) generates stress-induced senescent cells (SISCs) that play an important role in the pathophysiology of adverse cardiac remodeling and heart failure via secretion of pro-inflammatory molecules and matrix-degrading proteases. Thus, removal of senescent cells using a senolytic drug could be a potentially effective treatment. However, clinical studies on cancer treatment with a senolytic drug have revealed that systemic administration of a senolytic drug often causes systemic toxicity. Herein we show for the first time that local delivery of a senolytic drug can effectively treat myocardial IR injury. We found that biodegradable poly(lactic-co-glycolic acid) nanoparticle-based local delivery of a senolytic drug (ABT263-PLGA) successfully eliminated SISCs in the IR-injured rat hearts without systemic toxicity. Consequently, the treatment ameliorated inflammatory responses and attenuated adverse remodeling. Surprisingly, the ABT263-PLGA treatment restored the cardiac function over time, whereas the cardiac function decreased over time in the no treatment group. Mechanistically, the ABT263-PLGA treatment not only markedly reduced the expression of pro-inflammatory molecules and matrix-degrading proteases, but also induced macrophage polarization from the inflammatory phase to the reparative phase via efferocytosis of apoptotic SISCs by macrophages. Therefore, the senolytic strategy with ABT263-PLGA in the early stage of myocardial IR injury may be an effective therapeutic option for myocardial infarction. STATEMENT OF SIGNIFICANCE: This study describes a local injection of senolytic drug-loaded nanoparticles that selectively kills stress-induced senescent cells (SISCs) in infarcted heart. Removal of SISCs decreases inflammatory cytokines and normal cell death. We firstly revealed that further efferocytosis of apoptotic senescent cells by macrophages restores cardiac function after myocardial ischemia-reperfusion injury. Importantly, a local injection of senolytic drug did not exhibit systemic toxicity, but a systemic injection did. Our findings not only spotlight the basic understanding of therapeutic potential of senolysis in infarcted myocardium, but also pave the way for the further application of senolytic drug for non-aging related diseases.

Multilayered Cell Sheets of Cardiac Reprogrammed Cells for the Evaluation of Drug Cytotoxicity.

BACKGROUND: Various cell-culture systems have been used to evaluate drug toxicity in vitro. However, factors that affect cytotoxicity outcomes in drug toxicity evaluation systems remain elusive. In this study, we used multilayered sheets of cardiac-mimetic cells, which were reprogrammed from human fibroblasts, to investigate the effects of the layer number on drug cytotoxicity outcomes. METHODS: Cell sheets of cardiac-mimetic cells were fabricated by reprogramming of human fibroblasts into cardiac-mimetic cells via coculture with cardiac cells and electric stimulation, as previously described. Double-layered cell sheets were prepared by stacking the cell sheets. The mono- and double-layered cell sheets were treated with 5-fluorouracil (5-FU), an anticancer drug, in vitro. Subsequently, apoptosis and lipid peroxidation were analyzed. Furthermore, effects of cardiac-mimetic cell density on cytotoxicity outcomes were evaluated by culturing cells in monolayer at various cell densities. RESULTS: The double-layered cell sheets exhibited lower cytotoxicity in terms of apoptosis and lipid peroxidation than the mono-layered sheets at the same 5-FU dose. In addition, the double-layered cell sheets showed better preservation of mitochondrial function and plasma membrane integrity than the monolayer sheets. The lower cytotoxicity outcomes in the double-layered cell sheets may be due to the higher intercellular interactions, as the cytotoxicity of 5-FU decreased with cell density in monolayer cultures of cardiac-mimetic cells. CONCLUSION: The layer number of cardiac-mimetic cell sheets affects drug cytotoxicity outcomes in drug toxicity tests. The in vitro cellular configuration that more closely mimics the in vivo configuration in the evaluation systems seems to exhibit lower cytotoxicity in response to drug.

Interleukin-4 Gene Transfection and Spheroid Formation Potentiate Therapeutic Efficacy of Mesenchymal Stem Cells for Osteoarthritis.

Osteoarthritis (OA) is a painful intractable disease that significantly affects patients' quality of life. However, current therapies, such as pain killers and joint replacement surgery, do not lead to cartilage protection. Mesenchymal stem cells (MSCs) have been proposed as an alternative strategy for OA therapy because MSCs can secrete chondroprotective and anti-inflammatory factors. However, interleukin-4 (IL-4), a potent anti-inflammatory cytokine, is barely produced by MSCs, and MSC therapy suffers from rapid MSC death following intra-articular implantation. MSCs in spheroids survive better than naïve MSCs in vitro and in vivo. IL-4-transfected MSCs in spheroids (IL-4 MSC spheroid) show increased chondroprotective and anti-inflammatory effects in an OA chondrocyte model in vitro. Following intra-articular implantation in OA rats, IL-4 MSC spheroids show better cartilage protection and pain relief than naïve MSCs. Thus, IL-4 MSC spheroid may potentiate the therapeutic efficacy of MSCs for OA.

T-Cell-Mimicking Nanoparticles for Cancer Immunotherapy.

Cancer immunotherapies, including adoptive T cell transfer and immune checkpoint blockades, have recently shown considerable success in cancer treatment. Nevertheless, transferred T cells often become exhausted because of the immunosuppressive tumor microenvironment. Immune checkpoint blockades, in contrast, can reinvigorate the exhausted T cells; however, the therapeutic efficacy is modest in 70-80% of patients. To address some of the challenges faced by the current cancer treatments, here T-cell-membrane-coated nanoparticles (TCMNPs) are developed for cancer immunotherapy. Similar to cytotoxic T cells, TCMNPs can be targeted at tumors via T-cell-membrane-originated proteins and kill cancer cells by releasing anticancer molecules and inducing Fas-ligand-mediated apoptosis. Unlike cytotoxic T cells, TCMNPs are resistant to immunosuppressive molecules (e.g., transforming growth factor-ß1 (TGF-ß1)) and programmed death-ligand 1 (PD-L1) of cancer cells by scavenging TGF-ß1 and PD-L1. Indeed, TCMNPs exhibit higher therapeutic efficacy than an immune checkpoint blockade in melanoma treatment. Furthermore, the anti-tumoral actions of TCMNPs are also demonstrated in the treatment of lung cancer in an antigen-nonspecific manner. Taken together, TCMNPs have a potential to improve the current cancer immunotherapy.