Simon X. Liang

Differentiation and migration of Sca1+/CD31- cardiac side population cells in a murine myocardial ischemic model.

BACKGROUND Side population cells are a rare subset of cells found in the adult heart that are highly enriched for stem and progenitor cell activity. Recent studies have suggested that Sca1+/CD31- cardiac side population cells are capable of differentiation into cardiomyocytes in vitro. However, the response of these cells to myocardial injury remains unknown in vivo. METHODS Sca1+/CD31- cardiac side population cells were isolated from mouse (C57BL6/J) hearts by FACS. These cells were labeled and delivered via an intramyocardial injection into an infracted mouse heart. The differentiation potential of these cells was determined by immunohistochemistry two weeks later. We further tested the migration potential and the relationship of SDF-1alpha/CXCR4 to these cells. RESULTS The transplanted cells were found to express cardiomyocyte or endothelial cell specific markers. Furthermore, when these cells were transplanted into non-infarct myocardium after myocardial infarction, they were found in the damaged myocardium. Consistent with their homing property, we found that SDF-1alpha and CXCR4 were up-regulated in the damaged myocardium and on Sca1+/CD31- cardiac side population cells respectively following myocardial infarction. We also show that SDF-1alpha induced migration of Sca1+/CD31- cardiac side population cells in vitro. CONCLUSIONS Our results have suggested that Sca1+/CD31- cardiac side population cells are able to migrate into damaged myocardium from non-ischemic area of the heart and differentiate into both cardiomyocyte- and endothelial-like cells following acute ischemic injury. The SDF-1alpha/CXCR4 system might play an important role in the migration of these cells.

Expression and localisation of dynamin and syntaxin during neural development and neuromuscular synapse formation.

The expression and subcellular localisation of dynamin and syntaxin were examined during the periods of motor neuron development and neuromuscular synaptogenesis in the mouse embryo. Both dynamin and syntaxin could be detected by immunoblotting in the spinal cord at embryonic day 10 (E10; 2 days before axon outgrowth) and at all subsequent ages examined. Reverse transcription and polymerase chain reaction (RT‐PCR) identified low levels of all three carboxy‐terminal splicing forms of dynamin I in spinal cord from as early as E10. During the period of maturation of spinal neurons, from E10 to the first postnatal day (P0), the short carboxy‐terminal splicing form of dynamin I (dynamin I*b) was up‐regulated, as was dynamin III, relative to dynamin II mRNA. Syntaxin immunostaining became colocalized with the synaptic vesicle protein, SV2, at neuromuscular synapses within 12 hours of the commencement of synapse formation and throughout subsequent development. In contrast, dynamin, which is important for activity‐dependent synaptic vesicle recycling and, thus, sustained neurotransmission, could not be detected at most newly formed synapses until several days after synapse formation. The delayed appearance of dynamin at the synapse, thus, heralds the neonatal development of robust synaptic transmission at the neuromuscular junction. J. Comp. Neurol. 410:531–540, 1999.

Development of fast purinergic transmission in the mouse vas deferens

ATP released by sympathetic varicosities of the mouse vas deferens binds to P2X receptors which activate fast, ligand‐gated channels, resulting in depolarisation of smooth muscle cells. We examined the development of fast neuromuscular transmission at surface longitudinal smooth muscle fibres of the mouse vas deferens. Sympathetic varicosities were visualised using DiOC2(5)‐fluorescence to aid in positioning loose patch electrodes over small sets of sympathetic varicosities to record the nerve terminal impulse (NTI) and excitatory junction currents (EJCs) evoked during nerve stimulation. At the earliest age at which EJCs could be detected, 21 days postnatal (PN), most recording sites rarely showed a detectable EJC over 100 trials, even though NTIs were recorded without failure. The extent of such intermittence in transmitter release progressively declined between 21 and 42 days PN. In addition, the mean amplitude of spontaneous EJCs (SEJCs) and EJCs increased by 2‐ and 2.4‐fold, respectively, between 21 and 42 days PN. The rise time of EJCs varied widely at each age but declined with development (e.g., 7–14 ms at 28 days PN, 3–12 ms at 42 days PN). All EJCs were abolished by suramin (100 μM). Fast rise time EJCs were rapidly abolished by α,β‐methylene ATP (20 μM) while some (34%) of the slower rise time EJCs were resistant to rapid desensitisation of this kind. P2X1 and P2X2 mRNAs were detected by reverse transcription and polymerase chain reaction (RT‐PCR) to determine whether levels of expression of the receptor subunits might explain the increased EJC amplitude. Between 10 and 42 days PN no marked change was observed in the P2X2 receptor mRNA or β‐actin mRNA (control). In contrast, the intensity of the RT‐PCR band for P2X1 receptor showed a progressive ∼4.3‐fold developmental increase relative to the P2X2 band. These observations suggest that both prejunctional and postjunctional mechanisms cause the maturation of fast purinergic junctional transmission at the longitudinal muscle of the mouse vas deferens between 21 and 42 days PN. Synapse 37:283–291, 2000.

In vitro and in vivo proliferation, differentiation and migration of cardiac endothelial progenitor cells (SCA1+/CD31+ side-population cells).

To cite this article: Liang SX, Khachigian LM, Ahmadi Z, Yang M, Liu S, Chong BH. In vitro and in vivo proliferation, differentiation and migration of cardiac endothelial progenitor cells (SCA1+/CD31+ side‐population cells). J Thromb Haemost 2011; 9: 1628–37.

Muscle-specific kinase (MuSK) autoantibodies suppress the MuSK pathway and ACh receptor retention at the mouse neuromuscular junction.

Myasthenic anti‐muscle‐specific‐kinase (MuSK) IgG was injected into mice to study its effect upon the MuSK signalling pathway and the homeostasis of postsynaptic acetylcholine receptor packing at the neuromuscular junction. Densities of MuSK, activated Src kinase, phosphorylated ACh receptors and rapsyn were all reduced at motor endplates while β‐dystroglycan was unaffected. Pulse‐labelling showed that the slow decline in junctional ACh receptor density could be explained largely by diminished retention of ACh receptors within the postsynaptic membrane scaffold. The results suggest that anti‐MuSK IgG reduces the density of MuSK, associated tyrosine phosphorylation and retention of junctional ACh receptors within the postsynaptic membrane.

Migration of Resident Cardiac Stem Cells in Myocardial Infarction

Ischemic heart disease is a major cause of morbidity and mortality worldwide. Stem cell‐based therapy, which aims to restore cardiac structure and function by regeneration of functional myocardium, has recently been proposed as a novel alternative treatment modality. Resident cardiac stem cells (CSCs) in adult hearts are a key cell type under investigation. CSCs have been shown to be able to repair damaged myocardium and improve myocardial function in both human and animal studies. This approach relies not only on the proliferation of the CSCs, but also upon their migration to the site of injury within the heart. Here, we briefly review reported CSC populations and discuss signaling factors and pathways required for the migration of CSCs. Anat Rec, 2013.

Drug-induced thrombocytopenia: development of a novel NOD/SCID mouse model to evaluate clearance of circulating platelets by drug-dependent antibodies and the efficacy of IVIG

Drug-induced immune thrombocytopenia (DITP) is an adverse drug effect mediated by drug-dependent antibodies. Intravenous immunoglobulin (IVIG) is frequently used to treat DITP and primary immune thrombocytopenia (ITP). Despite IVIGs proven beneficial effects in ITP, its efficacy in DITP is unclear. We have established a nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model of DITP in which human platelets survive for more than 24 hours, allowing platelet clearance by DITP/ITP antibodies to be studied. Rapid human platelet clearance was uniformly observed with all quinine-induced thrombocytopenia (QITP) patient sera studied (mean platelet lifespans: QITP 1.5 ± 0.3 hours vs controls 16.5 ± 4.3 hours), consistent with the clinical presentation of DITP. In contrast, clearance rates with ITP antibodies were more variable. IVIG treatment partially prevented platelet clearance by DITP and ITP antibodies. Our results suggest that the NOD/SCID mouse model is useful for investigating the efficacy of current and future DITP therapies, an area in which there is little experimental evidence to guide treatment.

Structure and chromosome location of the mouse P2X1 purinoceptor gene (P2rx1)

P2X1 receptors are ATP-gated cation channels that mediate the fast, purinergic component of sympathetic nerve–smooth muscle neurotransmission in the mouse vas deferens and may serve comparable functions in the urinary bladder and the arteries. The gene for mouse P2X1 (P2rx1) was cloned and its genomic structure defined by sequencing. The gene spans about 10 kb and consists of 12 exons. All splice sites conformed to the GT-AG motif and the exon-intron boundaries were largely conserved with other members of the P2X gene family so far cloned. A single transcription-starting site was identified by 5′ RACE analysis, 233 bp upstream of the translation start site. The P2X1 gene maps to the central region of mouse chromosome 11.

Forced expression of muscle specific kinase slows postsynaptic acetylcholine receptor loss in a mouse model of MuSK myasthenia gravis

We investigated the influence of postsynaptic tyrosine kinase signaling in a mouse model of muscle‐specific kinase (MuSK) myasthenia gravis (MG). Mice administered repeated daily injections of IgG from MuSK MG patients developed impaired neuromuscular transmission due to progressive loss of acetylcholine receptor (AChR) from the postsynaptic membrane of the neuromuscular junction. In this model, anti‐MuSK‐positive IgG caused a reduction in motor endplate immunolabeling for phosphorylated Src‐Y418 and AChR β‐subunit‐Y390 before any detectable loss of MuSK or AChR from the endplate. Adeno‐associated viral vector (rAAV) encoding MuSK fused to enhanced green fluorescent protein (MuSK‐EGFP) was injected into the tibialis anterior muscle to increase MuSK synthesis. When mice were subsequently challenged with 11 daily injections of IgG from MuSK MG patients, endplates expressing MuSK‐EGFP retained more MuSK and AChR than endplates of contralateral muscles administered empty vector. Recordings of compound muscle action potentials from myasthenic mice revealed less impairment of neuromuscular transmission in muscles that had been injected with rAAV‐MuSK‐EGFP than contralateral muscles (empty rAAV controls). In contrast to the effects of MuSK‐EGFP, forced expression of rapsyn‐EGFP provided no such protection to endplate AChR when mice were subsequently challenged with MuSK MG IgG. In summary, the immediate in vivo effect of MuSK autoantibodies was to suppress MuSK‐dependent tyrosine phosphorylation of proteins in the postsynaptic membrane, while increased MuSK synthesis protected endplates against AChR loss. These results support the hypothesis that reduced MuSK kinase signaling initiates the progressive disassembly of the postsynaptic membrane scaffold in this mouse model of MuSK MG.

The mouse passive‐transfer model of MuSK myasthenia gravis: disrupted MuSK signaling causes synapse failure

While the majority of myasthenia gravis patients express antibodies targeting the acetylcholine receptor, the second most common cohort instead displays autoantibodies against muscle‐specific kinase (MuSK). MuSK is a transmembrane tyrosine kinase found in the postsynaptic membrane of the neuromuscular junction. During development, MuSK serves as a signaling hub, coordinating the alignment of the pre‐ and postsynaptic components of the synapse. Adult mice that received repeated daily injections of IgG from anti‐MuSK+ myasthenia gravis patients developed muscle weakness, associated with neuromuscular transmission failure. MuSK autoantibodies are predominantly of the IgG4 type. They suppress the kinase activity of MuSK and the phosphorylation of target proteins in the postsynaptic membrane. Loss of postsynaptic acetylcholine receptors is the primary cause of neuromuscular transmission failure. MuSK autoantibodies also disrupt the capacity of the motor nerve terminal to adaptively increase acetylcholine release in response to the reduced postsynaptic responsiveness to acetylcholine. The passive IgG transfer model of MuSK myasthenia gravis has been used to test candidate treatments. Pyridostigmine, a first‐line cholinesterase inhibitor drug, exacerbated the disease process, while 3,4‐diaminopyridine and albuterol were found to be beneficial in this mouse model.