Endothelial nitric oxide synthase-engineered mesenchymal stromal cells induce anti-inflammation in experimental immune models.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) remain an area of interest in the field of regenerative medicine. Although there is clear evidence of safety, a lack of substantial efficacy has led to many MSC-based clinical trials to stall in phase 1. Therefore, potentiating MSCs with biologically relevant messenger RNA (mRNA) transcripts presents a relatively safe and efficient way to increase functionality. METHODS: In this study, human bone marrow-derived MSCs were transfected with endothelial nitric oxide synthase (eNOS) mRNA and evaluated for transfection efficiency and immunosuppressive ability. To assess MSC-eNOS functionality, T-cell proliferation assays and mouse models of experimental autoimmune encephalomyelitis and graft-versus-host disease were used. RESULTS: The authors found that MSC-eNOS retained MSC characteristics and exhibited significantly enhanced immunosuppressive effects compared with naive MSCs in both in vitro and in vivo models. CONCLUSIONS: It is feasible to pursue eNOS mRNA transfection to potentiate the immunomodulatory capacity of MSCs for clinical applications in the future.

Novel LCAT (Lecithin:Cholesterol Acyltransferase) Activator DS-8190a Prevents the Progression of Plaque Accumulation in Atherosclerosis Models.

OBJECTIVE: Enhancement of LCAT (lecithin:cholesterol acyltransferase) activity has possibility to be beneficial for atherosclerosis. To evaluate this concept, we characterized our novel, orally administered, small-molecule LCAT activator DS-8190a, which was created from high-throughput screening and subsequent derivatization. We also focused on its mechanism of LCAT activation and the therapeutic activity with improvement of HDL (high-density lipoprotein) functionality. Approach and Results: DS-8190a activated human and cynomolgus monkey but not mouse LCAT enzymes in vitro. DS-8190a was orally administered to cynomolgus monkeys and dose dependently increased LCAT activity (2.0-fold in 3 mg/kg group on day 7), resulting in HDL cholesterol elevation without drastic changes of non-HDL cholesterol. Atheroprotective effects were then evaluated using Ldl-r KO×hLcat Tg mice fed a Western diet for 8 weeks. DS-8190a treatment achieved significant reduction of atherosclerotic lesion area (48.3% reduction in 10 mg/kg treatment group). Furthermore, we conducted reverse cholesterol transport study using Ldl-r KO×hLcat Tg mice intraperitoneally injected with J774A.1 cells loaded with [3H]-cholesterol and confirmed significant increases of [3H] count in plasma (1.4-fold) and feces (1.4-fold on day 2 and 1.5-fold on day3) in the DS-8190a-treated group. With regard to the molecular mechanism involved, direct binding of DS-8190a to human LCAT protein was confirmed by 2 different approaches: affinity purification by DS-8190a-immobilized beads and thermal shift assay. In addition, the candidate binding site of DS-8190a in human LCAT protein was identified by photoaffinity labeling. CONCLUSIONS: This study demonstrates the potential of DS-8190a as a novel therapeutic for atherosclerosis. In addition, this compound proves that a small-molecule direct LCAT activator can achieve HDL-C elevation in monkey and reduction of atherosclerotic lesion area with enhanced HDL function in rodent.

Development and evaluation of IL-6 overexpressing mesenchymal stem cells (MSCs).

Mesenchymal stem/stromal cell (MSC) therapy has been investigated in multiple diseases and conditions. Although the mechanisms of MSC-based therapies are not fully understood, we and others have shown interleukin 6 (IL-6) to be an important factor in MSC function. IL-6 contributes to many biological events, such as immune response, neurogenesis, and bone remodeling. In our study, we tested the feasibility of engineering MSCs by IL-6 mRNA transfection (eMSCs-IL6) and evaluated the optimal time to harvest them after transfection. We then assessed the functional characteristics of eMSCs-IL6. Quantitative real-time PCR and ELISA results have shown that mature IL-6 mRNA was efficiently transfected into MSCs using a lipofectamine based method. The IL-6 mRNA and protein overexpression peaked after 1 day of transfection and the secreted IL-6 protein was sustained for at least 6 days. A short time course experiment demonstrated that 4 h after transfection was the best time point to harvest and freeze eMSCs-IL6 for future studies. In addition, eMSCs-IL6 maintained their characteristics as defined by International Society for Cell & Gene Therapy. The immunosuppressive capacity of conditioned culture medium (CCM) from eMSCs-IL6 (CCM-IL6) was significantly enhanced compared to naïve MSCs conditioned culture medium (CCM-control). Our studies established for the first time the feasibility of efficiently generating IL-6 overexpressing MSCs which have enhanced immunosuppressive capacity. This is providing a novel approach to improve the efficacy of MSCs for potential application in regenerative medicine.

IL-10 mRNA Engineered MSCs Demonstrate Enhanced Anti-Inflammation in an Acute GvHD Model.

Mesenchymal stem cells (MSCs) are used in various studies to induce immunomodulatory effects in clinical conditions associated with immune dysregulation such as graft versus host disease (GvHD). However, most of these clinical trials failed to go beyond early phase 2 studies because of limited efficacy. Various methods have been assessed to increase the potency of MSCs. IL-10 is an anti-inflammatory cytokine that is known to modulate immune responses in GvHD. In this study, we evaluated the feasibility of transfecting IL-10 mRNA to enhance MSC therapeutic potential. IL-10 mRNA engineered MSCs (eMSCs-IL10) maintained high levels of IL-10 expression even after freezing and thawing. IL-10 mRNA transfection did not appear to alter MSC intrinsic characteristics. eMSCs-IL10 significantly suppressed T cell proliferation relative to naïve MSCs in vitro. In a mouse model for GvHD, eMSCs-IL10 induced a decrease in plasma level of potent pro-inflammatory cytokines and inhibited CD4+ and CD8+ T cell proliferation in the spleen. In summary, our studies demonstrate the feasibility of potentiating MSCs to enhance their immunomodulatory effects by IL-10 mRNA transfection. The use of non-viral transfection may generate a safe and potent MSC product for treatment of clinical conditions associated with immune dysregulation such as GvHD.

Whole organism high content screening identifies stimulators of pancreatic beta-cell proliferation.

Inducing beta-cell mass expansion in diabetic patients with the aim to restore glucose homeostasis is a promising therapeutic strategy. Although several in vitro studies have been carried out to identify modulators of beta-cell mass expansion, restoring endogenous beta-cell mass in vivo has yet to be achieved. To identify potential stimulators of beta-cell replication in vivo, we established transgenic zebrafish lines that monitor and allow the quantification of cell proliferation by using the fluorescent ubiquitylation-based cell cycle indicator (FUCCI) technology. Using these new reagents, we performed an unbiased chemical screen, and identified 20 small molecules that markedly increased beta-cell proliferation in vivo. Importantly, these structurally distinct molecules, which include clinically-approved drugs, modulate three specific signaling pathways: serotonin, retinoic acid and glucocorticoids, showing the high sensitivity and robustness of our screen. Notably, two drug classes, retinoic acid and glucocorticoids, also promoted beta-cell regeneration after beta-cell ablation. Thus, this study establishes a proof of principle for a high-throughput small molecule-screen for beta-cell proliferation in vivo, and identified compounds that stimulate beta-cell proliferation and regeneration.

NMDAR2B tyrosine phosphorylation is involved in thermal nociception.

Previous studies found that the NMDA receptor-mediated signaling regulates thermal nociception, though the underlying molecular mechanism remains unclear. The GluN2B subunit of the NMDA receptor is tyrosine-phosphorylated, Tyr-1472 being the major phosphorylation site. In this study, we have found that homozygous knock-in mice that express a Tyr-1472-Phe mutant of GluN2B display defects in the nociceptive response in the hot plate test. Expression of the neurotensin receptor subtype 2 (NTSR2), which is relevant to the regulation of thermal nociception, is decreased in the amygdala of GluN2B Tyr-1472-Phe knock-in mice. In addition, NTSR2-mediated c-fos induction is impaired in the amygdala of these mice. These data suggest that Tyr-1472 phosphorylation on GluN2B is involved in thermal nociception through regulating the NTSR2 mRNA expression in the amygdala.

NMDAR2B tyrosine phosphorylation regulates anxiety-like behavior and CRF expression in the amygdala.

BACKGROUND: Anxiety disorders are a highly prevalent and disabling class of psychiatric disorders. There is growing evidence implicating the glutamate system in the pathophysiology and treatment of anxiety disorders, though the molecular mechanism by which the glutamate system regulates anxiety-like behavior remains unclear. RESULTS: In this study, we provide evidence suggesting that tyrosine phosphorylation of the NMDA receptor, an ionotropic glutamate receptor, contributes to anxiety-like behavior. The GluN2B subunit of the NMDA receptor is tyrosine-phosphorylated: Tyr-1472 is the major phosphorylation site. Homozygous knock-in mice that express a Tyr-1472-Phe mutant of GluN2B, which prevents phosphorylation of this site, show enhanced anxiety-like behavior in the elevated plus-maze test. Expression of corticotropin-releasing factor (CRF), which is important for the regulation of anxiety-like behavior, is increased in the amygdala of the knock-in mice. Furthermore, injection of CRF receptor antagonist attenuated the enhanced anxiety-like behavior of the knock-in mice. We also show that elevated plus-maze exposure simultaneously induced de-phosphorylation of Tyr-1472 and increased CRF expression. CONCLUSIONS: These data suggest that Tyr-1472 phosphorylation on GluN2B is important for anxiety-like behavior by negative regulation of CRF expression in the amygdala.

Molecular characterization of a novel RhoGAP, RRC-1 of the nematode Caenorhabditis elegans.

The GTPase-activating proteins for Rho family GTPases (RhoGAP) transduce diverse intracellular signals by negatively regulating Rho family GTPase-mediated pathways. In this study, we have cloned and characterized a novel RhoGAP for Rac1 and Cdc42, termed RRC-1, from Caenorhabditis elegans. RRC-1 was highly homologous to mammalian p250GAP and promoted GTP hydrolysis of Rac1 and Cdc42 in cells. The rrc-1 mRNA was expressed in all life stages. Using an RRC-1::GFP fusion protein, we found that RRC-1 was localized to the coelomocytes, excretory cell, GLR cells, and uterine-seam cell in adult worms. These data contribute toward understanding the roles of Rho family GTPases in C. elegans.

Neurotensin type 2 receptor is involved in fear memory in mice.

Neurotensin receptor subtype 2 (Ntsr2) is a levocabastine-sensitive neurotensin receptor expressed diffusely throughout the mouse brain. Previously, we found that Ntsr2-deficient mice have an abnormality in the processing of thermal nociception. In this study, to examine the involvement of Ntsr2 in mouse behavior, we performed a fear-conditioning test in Ntsr2-deficient mice. In the contextual fear-conditioning test, the freezing response was significantly reduced in Ntsr2-deficient mice compared with that of wild-type mice. This reduction was observed from 1 h to 3 weeks after conditioning, and neither shock sensitivity nor locomotor activity was altered in Ntsr2-deficient mice. In addition, we found that Ntsr2 mRNA was predominantly expressed in cultured astrocytes and weakly expressed in cultured neurons derived from mouse brain. The combination of in situ hybridization and immunohistochemistry showed that Ntsr2 mRNA was dominantly expressed in glial fibrillary acidic protein positive cells in many brain regions including the hypothalamus, while Ntsr2 gene was co-expressed with neuron-specific microtubule associated protein-2 in limited numbers of cells. These results suggest that Ntsr2 in astrocytes and neurons may have unique function like a modulation of fear memory in the mouse brain.

NR2B tyrosine phosphorylation modulates fear learning as well as amygdaloid synaptic plasticity.

Phosphorylation of neural proteins in response to a diverse array of external stimuli is one of the main mechanisms underlying dynamic changes in neural circuitry. The NR2B subunit of the NMDA receptor is tyrosine-phosphorylated in the brain, with Tyr-1472 its major phosphorylation site. Here, we generate mice with a knockin mutation of the Tyr-1472 site to phenylalanine (Y1472F) and show that Tyr-1472 phosphorylation is essential for fear learning and amygdaloid synaptic plasticity. The knockin mice show impaired fear-related learning and reduced amygdaloid long-term potentiation. NMDA receptor-mediated CaMKII signaling is impaired in YF/YF mice. Electron microscopic analyses reveal that the Y1472F mutant of the NR2B subunit shows improper localization at synapses in the amygdala. We thus identify Tyr-1472 phosphorylation as a key mediator of fear learning and amygdaloid synaptic plasticity.

The dual functions of biphenyl-degrading ability of Pseudomonas sp. KKS102: energy acquisition and substrate detoxification.

The bph operon of Pseudomonas sp. KKS102 is constituted of 11 bph genes which encode enzymes for biphenyl assimilation. Growth of a mutant in which a large part of the bph operon was deleted was inhibited by biphenyl in a concentration-dependent manner. We constructed a series of bph operon deletion mutants and tested for their biphenyl sensitivity. Growth inhibition by biphenyl was more prominent with the mutants defective in bphA1, bphB, bphC, and bphD, which were clustered in the bph operon and working in the early stage of the biphenyl degradation. The mutant defective in bphE, which was working at the late stage and forming a different cluster from the early stage genes, was not much inhibited by biphenyl. These indicate that biphenyl is detoxified by enzymes which function in the early stage of biphenyl assimilation and thus detoxification of substrates as well as energy acquisition could have played an important role in the evolution of the KKS102 bph operon.

Novel approach to the improvement of biphenyl and polychlorinated biphenyl degradation activity: promoter implantation by homologous recombination.

To improve the capabilities of microorganisms relevant for biodegradation, we developed a new genetic approach and applied it to the bph operon (bphEGF[orf4]A1A2A3CD[orf1]A4R) of Pseudomonas sp. strain KKS102 to enhance its biphenyl- and polychlorinated biphenyl (PCB)-degrading activity. A native promoter of the bph operon, which was under control, was replaced through homologous recombination by a series of promoters that had constitutive activity. By testing a series of promoters with various strengths, we were able to obtain strains that have enhanced degradation activity for biphenyl and PCBs. This strategy removes the rate-limiting factor associated with transcription and has the potential to improve the degradation activity of a wide variety of microorganisms involved in biodegradation.