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Biomedical subjects

Yang Shi

Publications and source records attributed to Yang Shi.

At least 19 recordsLinked to original sources
PubMed2026

Comprehensive transcriptomics and proteomics analysis of neointima formation in human saphenous vein: implications for bypass graft disease.

Human saphenous veins (SVs) are widely used as grafts in coronary artery bypass (CABG) surgery but often fail due to neointima formation. Little is known, however, regarding the cellular, transcriptomic, and proteomic dynamics of neointima formation in human veins. Here, we performed transcriptomics and proteomics analysis in an ex vivo tissue culture model of neointima formation in human SVs procured for CABG surgery. Histological examination demonstrated significant elastin degradation and neointima formation (indicated by increased neointima area and neointima-to-media ratio) in SVs subjected to tissue culture. Analysis of data from 72 patients suggests that the progression of SV remodeling and neointima formation differs according to sex and body mass index, which is negatively associated with neointima formation in males only. RNA sequencing demonstrated upregulation of proinflammatory and proliferation-related genes during neointima formation and identified novel processes, including increased cellular stress and DNA damage responses, reflecting tissue trauma associated with vein harvesting. Proteomic analysis identified upregulated extracellular matrix-related and coagulation/thrombosis proteins and downregulated metabolic proteins. Spatial transcriptomics, used to infer regionally enriched gene expression, suggested dynamic alterations in fibroblast and vascular smooth muscle cell (VSMC) states during neointima formation. Specifically, we identified the emergence of HES1+ and matrix metalloproteinase 2- and 14-positive (MMP2+/MMP14+) expression in VSMCs and fibroblasts, respectively, during neointima formation. Furthermore, our data suggest that MIR647, identified through screening, maintains VSMC contractile gene expression. Our findings suggest dynamic transcriptomic and proteomic changes during neointima formation in human veins and provide useful mechanistic information for the pathogenesis of SV graft disease.NEW & NOTEWORTHY Using multiomics and spatial transcriptomics, we uncover dynamic molecular and cellular changes driving neointima proliferation in human saphenous veins, the most common conduit for bypass surgery. Our study highlights sex- and body mass index-associated differences, novel fibroblast and smooth muscle cell states, and a role for microRNA-647 in preserving vascular contractile phenotype. These findings provide new insight into the mechanisms of vein graft failure and may guide future strategies to improve coronary bypass outcomes.

Humans↗
PubMed2006

Integrins control dendritic spine plasticity in hippocampal neurons through NMDA receptor and Ca2+/calmodulin-dependent protein kinase II-mediated actin reorganization.

The formation of dendritic spines during development and their structural plasticity in the adult brain are critical aspects of synaptogenesis and synaptic plasticity. Many different factors and proteins have been shown to control dendritic spine development and remodeling (Ethell and Pasquale, 2005). The extracellular matrix (ECM) components and their cell surface receptors, integrins, have been found in the vicinity of synapses and shown to regulate synaptic efficacy and play an important role in long-term potentiation (Bahr et al., 1997; Chavis and Westbrook, 2001; Chan et al., 2003; Lin et al., 2003; Bernard-Trifilo et al., 2005). Although molecular mechanisms by which integrins affect synaptic efficacy have begun to emerge, their role in structural plasticity is poorly understood. Here, we show that integrins are involved in spine remodeling in cultured hippocampal neurons. The treatment of 14 d in vitro hippocampal neurons with arginine-glycine-aspartate (RGD)-containing peptide, an established integrin ligand, induced elongation of existing dendritic spines and promoted formation of new filopodia. These effects were also accompanied by integrin-dependent actin reorganization and synapse remodeling, which were partially inhibited by function-blocking antibodies against beta1 and beta3 integrins. This actin reorganization was blocked with the NMDA receptor (NMDAR) antagonist MK801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate]. The Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 (N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulfonamide) also suppressed RGD-induced actin reorganization and synapse remodeling. Our findings show that integrins control ECM-mediated spine remodeling in hippocampal neurons through NMDAR/CaMKII-dependent actin reorganization.

Actins↗
PubMed2006

Vaccine with beta-defensin 2-transduced leukemic cells activates innate and adaptive immunity to elicit potent antileukemia responses.

Murine beta-defensin 2 (MBD2) is a small antimicrobial peptide of the innate immune system. Recent study showed that MBD2 could not only recruit immature dendritic cells but also activate them by Toll-like receptor 4 and thus may provide a critical link between the innate immune system and the adaptive immune response. In this report, we examined the antileukemia activity of MBD2 in a murine model of acute lymphoid leukemia (ALL) L1210. L1210 cells were engineered to secrete biologically functional MBD2. MBD2-modified L1210 (L1210-MBD2) showed significantly reduced leukemogenecity, resulting in a 80% rate of complete leukemia rejection. Inoculation of mice with L1210-MBD2 induced enhanced CTL and natural killer (NK) activity and augmented interleukin-12 and IFN-gamma production. All the recovered mice from the inoculation showed a protective immunity to the following challenge with parental L1210 cells and generate leukemia-specific memory CTL. Vaccines with irradiated L1210-MBD2 cells could cure 50% leukemia-bearing mice. Depletion of CD8+ T cells but not CD4+ T cells completely abrogated the antileukemia activity of MBD2. Interestingly, NK cells were also required for the MBD2-mediated antileukemia response, although ALL generally display a high degree of resistance to NK-mediated lysis. Our results suggest that MBD2 can activate both innate and adaptive immunity to generate potent antileukemia response, and MBD2 immunotherapy warrants further evaluation as a potential treatment for ALL.

Acute Disease↗
PubMed2005

Characterization of human cardiac mitochondrial ATP-sensitive potassium channel and its regulation by phorbol ester in vitro.

Activation of the mitochondrial ATP-sensitive K+ channel (mitoKATP) and its regulation by PKC are critical events in preconditioning induced by ischemia or pharmaceutical agents in animals and humans. The properties of the human cardiac mitoKATP channel are unknown. Furthermore, there is no evidence that cytosolic PKC can directly regulate the mitoKATP channel located in the inner mitochondrial membrane (IMM) due to the physical barrier of the outer mitochondrial membrane. In the present study, we characterized the human cardiac mitoKATP channel and its potential regulation by PKC associated with the IMM. IMM fractions isolated from human left ventricles were fused into lipid bilayers in symmetrical potassium glutamate (150 mM). The conductance of native mitoKATP channels was usually below 80 pS ( approximately 70%), which was reduced by ATP and 5-hydroxydecanoic acid (5-HD) in a dose- and time-dependent manner. The native mitoKATP channel is activated by diazoxide and inhibited by ATP and 5-HD. The PKC activator phorbol 12-myristate 13-acetate (2 microM) increased the cumulative open probability of the mitoKATP channel previously inhibited by ATP (P < 0.05), but its inactive analog 4alpha-phorbol 12,13-didecanoate had no effect. Western blot analysis detected an inward rectifying K+ channel (Kir6.2) immunoreactive protein at 56 kDa and PKC-delta in the IMM. These data provide the first characterization of the human cardiac mitoKATP channel and its regulation by PKC(s) in IMM. This local PKC control mechanism may represent an alternative pathway to that proposed previously for cytosolic PKC during ischemic/pharmacological preconditioning.

Adenosine Triphosphate↗
PubMed2005

EphB receptors regulate dendritic spine morphogenesis through the recruitment/phosphorylation of focal adhesion kinase and RhoA activation.

Dendritic filopodia are small protrusions on the surface of neuronal dendrites that transform into dendritic spines upon synaptic contact with axon terminals. The formation of dendritic spines is a critical aspect of synaptic development. Dendritic spine morphogenesis is characterized by filopodia shortening followed by the formation of mature mushroom-shaped spines. Here we show that activation of the EphB receptor tyrosine kinases in cultured hippocampal neurons by their ephrinB ligands induces morphogenesis of dendritic filopodia into dendritic spines. This appears to occur through assembly of an EphB-associated protein complex that includes focal adhesion kinase (FAK), Src, Grb2, and paxillin and the subsequent activations of FAK, Src, paxillin, and RhoA. Furthermore, Cre-mediated knock-out of loxP-flanked fak or RhoA inhibition blocks EphB-mediated morphogenesis of dendritic filopodia. Finally, EphB-mediated RhoA activation is disrupted by FAK knock-down. These data suggest that EphB receptors are upstream regulators of FAK in dendritic filopodia and that FAK-mediated RhoA activation contributes to assembly of actin filaments in dendritic spines.

Animals↗
PubMed2005

Regulation of LSD1 histone demethylase activity by its associated factors.

LSD1 is a recently identified human lysine (K)-specific histone demethylase. LSD1 is associated with HDAC1/2; CoREST, a SANT domain-containing corepressor; and BHC80, a PHD domain-containing protein, among others. We show that CoREST endows LSD1 with the ability to demethylate nucleosomal substrates and that it protects LSD1 from proteasomal degradation in vivo. We find hyperacetylated nucleosomes less susceptible to CoREST/LSD1-mediated demethylation, suggesting that hypoacetylated nucleosomes may be the preferred physiological substrates. This raises the possibility that histone deacetylases and LSD1 may collaborate to generate a repressive chromatin environment. Consistent with this model, TSA treatment results in derepression of LSD1 target genes. While CoREST positively regulates LSD1 function, BHC80 inhibits CoREST/LSD1-mediated demethylation in vitro and may therefore confer negative regulation. Taken together, these findings suggest that LSD1-mediated histone demethylation is regulated dynamically in vivo. This is expected to have profound effects on gene expression under both physiological and pathological conditions.

Enzyme Stability↗
PubMed2005

Peroxynitrite reduces the endothelium-derived hyperpolarizing factor component of coronary flow-mediated dilation in PECAM-1-knockout mice.

Platelet endothelial cell adhesion molecule 1 (PECAM-1) is capable of transducing signals in endothelial cells exposed to shear; however, the biological consequences of this signal transduction are unknown. Because shear stress elicits flow-mediated dilation (FMD), we examined whether steady-state FMD in mouse coronary arteries (MCAs) is affected in the PECAM-1 knockout (KO) mouse. MCAs were isolated from wild-type (WT) or KO mice and prepared for videomicroscopy, histofluorescence, Western blotting, and immunohistochemistry. FMD was examined in the absence and presence of N(omega)-nitro-l-arginine methyl ester (l-NAME) and l-NAME+indomethacin (INDO). FMD was reduced in KO relative to WT MCAs, but the l-NAME-inhibitable portion of FMD was similar between the two. The INDO-sensitive component of FMD was diminished in KO MCAs. In contrast, the residual component of dilation, presumably because of endothelium-derived hyperpolarizing factor (EDHF), was abolished in KO MCAs. Histofluorescence showed relatively more superoxide (O2-.; oxy-ethidium fluorescence) and peroxide production (dihydrochlorofluorescene fluoresecence) in KO MCAs at rest. Flow augmented O2-. and peroxide production in WT MCAs but had little effect on KO MCAs. Enhanced nitric oxide generation was observed in arteries from KO mice, accompanied with increased eNOS S1177 phosphorylation. In vessels from KO mice, treatment with ebselen decreased peroxynitrite (ONOO-) formation and improved the reduced FMD, largely due to restoration of the presumed EDHF component. These results suggest that PECAM-1 is necessary for normal FMD in the mouse coronary circulation. In the absence of this adhesion and signaling molecule, ONOO- production is increased concomitant with a reduction in both the EDHF and INDO-sensitive components of FMD.

Animals↗
PubMed2005

Par-4 links dopamine signaling and depression.

Prostate apoptosis response 4 (Par-4) is a leucine zipper containing protein that plays a role in apoptosis. Although Par-4 is expressed in neurons, its physiological role in the nervous system is unknown. Here we identify Par-4 as a regulatory component in dopamine signaling. Par-4 directly interacts with the dopamine D2 receptor (D2DR) via the calmodulin binding motif in the third cytoplasmic loop. Calmodulin can effectively compete with Par-4 binding in a Ca2+-dependent manner, providing a route for Ca2+-mediated downregulation of D2DR efficacy. To examine the importance of the Par-4/D2DR interaction in dopamine signaling in vivo, we used a mutant mouse lacking the D2DR interaction domain of Par-4, Par-4DeltaLZ. Primary neurons from Par-4DeltaLZ embryos exhibit an enhanced dopamine-cAMP-CREB signaling pathway, indicating an impairment in dopamine signaling in these cells. Remarkably, Par-4DeltaLZ mice display significantly increased depression-like behaviors. Collectively, these results provide evidence that Par-4 constitutes a molecular link between impaired dopamine signaling and depression.

Amino Acid Motifs↗
PubMed2005

Regulation of tissue-specific and extracellular matrix-related genes by a class I histone deacetylase.

Class I histone deacetylases (HDACs) repress transcription by deacetylating histones and have been shown to play crucial roles in mouse, Xenopus, zebrafish, and C. elegans development. To identify the molecular networks regulated by a class I HDAC in a multicellular organism, we carried out a global gene expression profiling study using C. elegans embryos, and identified tissue-specific and extracellular matrix (ECM)-related genes as major HDA-1 targets. Ectopic expression of HDA-1 or C. elegans cystatin, an HDA-1 target identified from the microarray, significantly perturbed mammalian cell invasion. Similarly, RNAi depletion or overexpression of human HDAC-1 also affected cell migration. These findings suggest that HDA-1/HDAC-1 may play a critical, evolutionarily conserved role in regulating the extracellular microenvironment. Because human HDACs are targets for cancer therapy, these findings have significant implications in cancer treatment.

Animals↗
PubMed2005

Design and synthesis of a solid-phase fluorescent mass tag.

In this study, we demonstrate the design of a new solid-phase fluorescent mass tag (FMT) that contains the following features: (1) the FMT is synthesized using Fmoc chemistry which is simple, rapid, and cost-effective; (2) lysine is used as a uniformly labeled amino acid (using stable isotopes) to allow 8 Da difference between "heavy" and "light" tags; (3) a fluorescent molecule is coupled to the isotope tag that allows a tagged peptide to be detected by online fluorescence; and (4) an iodoacetyl reactive group provides cysteine reactivity. Using MALDI-TOF MS and HPLC, we show that the FMT reagent can be used to label standard cysteine-containing peptides as well as cysteine-containing peptides from a BSA tryptic digest.

Amino Acid Sequence↗
PubMed2005

Increased resistance to myocardial ischemia in the Brown Norway vs. Dahl S rat: role of nitric oxide synthase and Hsp90.

Hearts from Brown Norway (BN/Mcw) rats are more resistant to ischemia than hearts from Dahl S (SS/Mcw) rats. We determined whether nitric oxide (.NO) is responsible for increased cardioprotection in BN/Mcw vs. SS/Mcw hearts. Hearts from the two strains were treated with N(G)-monomethyl-L-arginine (L-NMA) or S-nitrosoglutathione (GSNO) before ischemia and reperfusion. Infarct size in untreated BN/Mcw hearts was approximately 63% less than in SS/Mcw hearts. Inhibiting NOS with L-NMA increased infarct size in BN/Mcw hearts to that observed in untreated SS/Mcw hearts but did not further increase injury in SS/Mcw hearts. The .NO donor GSNO decreased infarct size in SS/Mcw rats but had no effect on BN/Mcw hearts. Plasma and heart tissue from BN/Mcw rats contained 80% and 130% more nitrite + nitrate than that from SS/Mcw rats. These data suggest that increased .NO production protects BN/Mcw hearts from ischemic injury. Real time PCR showed no differences in NOS1, NOS2 or NOS3 isozyme transcripts in the hearts from the two strains. NOS3 was the only isozyme detected by western analysis. Both strains exhibited the same level of NOS3 and hsp90 protein expression. However, hsp90 association with NOS3 in BN/Mcw hearts was increased twofold compared with SS/Mcw hearts. Inhibiting hsp90-NOS3 interaction with geldanamycin decreased the resistance to ischemia in BN/Mcw hearts but not in SS/Mcw hearts. SS/Mcw hearts also generated three times more N(omega)-nitro-L-arginine-methylester inhibitable superoxide than BN/Mcw hearts. These findings indicate that hsp90 with NOS3 increases .NO production and decreases uncoupled NOS3 activity. We conclude increased association of hsp90 with NOS3 is a major mechanism by which BN/Mcw hearts are more resistant to ischemia than SS/Mcw hearts.

Animals↗
PubMed2005

Endoplasmic reticulum stress induction of the Grp78/BiP promoter: activating mechanisms mediated by YY1 and its interactive chromatin modifiers.

The unfolded protein response is an evolutionarily conserved mechanism whereby cells respond to stress conditions that target the endoplasmic reticulum (ER). The transcriptional activation of the promoter of GRP78/BiP, a prosurvival ER chaperone, has been used extensively as an indicator of the onset of the UPR. YY1, a constitutively expressed multifunctional transcription factor, activates the Grp78 promoter only under ER stress conditions. Previously, in vivo footprinting analysis revealed that the YY1 binding site of the ER stress response element of the Grp78 promoter exhibits ER stress-induced changes in occupancy. Toward understanding the underlying mechanisms of these unique phenomena, we performed chromatin immunoprecipitation analyses, revealing that YY1 only occupies the Grp78 promoter upon ER stress and is mediated in part by the nuclear form of ATF6. We show that YY1 is an essential coactivator of ATF6 and uncover their specific interactive domains. Using small interfering RNA against YY1 and insertional mutation of the gene encoding ATF6alpha, we provide direct evidence that YY1 and ATF6 are required for optimal stress induction of Grp78. We also discovered enhancement of the ER-stressed induction of the Grp78 promoter through the interaction of YY1 with the arginine methyltransferase PRMT1 and evidence of its action through methylation of the arginine 3 residue on histone H4. Furthermore, we detected ER stress-induced binding of the histone acetyltransferase p300 to the Grp78 promoter and histone H4 acetylation. A model for the ER stress-mediated transcription factor binding and chromatin modifications at the Grp78 promoter leading to its activation is proposed.

Acetyltransferases↗
PubMed2005

[Changes in intrahepatic portal systemic shunt flow in a rat model of acute intrahepatic presinusoidal portal hypertension].

OBJECTIVES: To investigate the changes in intrahepatic portal systemic shunt flow (IHSF) and their relationship with microspheres induced acute portal hypertension. METHODS: Following acute intrahepatic presinusoidal obstruction by intraportal injection of 15 microm diameter microspheres in male Wistar rats, functional hepatic blood flow (FHBF) and IHSF were determined by hepatic sorbitol uptake methods. The percentage of large shunts of diameter > 15 microm were estimated by intraportal injection of 51Cr labeled 15 mum diameter microspheres. RESULTS: In normal control rats, hepatic sorbitol uptake was 97.9%+/-0.5% and IHSF was negligible, with FHBF equaling total hepatic blood flow [(2.52 +/- 0.23) ml/min x 100 g body weight-1]. Microsphere injection decreased sorbitol uptake to 12.8% +/- 4.3% and further to 4.1% +/- 0.7% when followed by hepatic arterial ligation. In the latter two groups, IHSF (1.46 +/- 0.15 and 1.16 +/- 0.19 ml/min x 100 g body weight-1, respectively) was not significantly different from portal venous flow [(1.36 +/- 0.20) and (1.20 +/- 0.20) ml/min x 100 g body weight-1, respectively; t = 2.013 and t = 2.116]. Portal venous flow remained at 70% of basal values and portal venous pressure only increased by 50% from baseline. 51Cr labeled microsphere shunt fraction through large shunts (> 15 microm) was less than 1.0%. CONCLUSION: Intrahepatic portasystemic shunts in the normal rat liver predominantly have diameters less than 15 microm and, when activated by intraportal injection of microspheres, they divert up to 70% of portal venous blood flow away from hepatic sinusoids and thereby they reduce acute increases in portal venous pressure.

Animals↗
PubMed2005

[Synthesis of derivatives of (9S)-12-methylene erythromycin and their antibacterial activity in vitro].

AIM: To synthesizs of derivatives of (9S)-12-methylene erythromycin possessed potent antibacterial activity. METHODS: Using erythromycin A as a starting material, via two intermediate compounds protected 12,21-dehydroerythromycin A and 6,7: 12,21-didehydro erythromycin A, several 9-O, 11-O-ethylidene compounds were obtained. During this process, benzyl and isopropyl have been selected as the protecting group. The structures of compounds obtained were confirmed with 13C NMR and MS-FAB. Their antibacterial activity in vitro was tested. RESULTS: Eleven derivatives of erythromycin were synthesized. Five of them were unknown compounds. CONCLUSION: The preliminary biological test showed that two target compounds exhibited less potent antibacterial activity in vitro.

Anti-Bacterial Agents↗
PubMed2005

Multistep mass tagging coupled with 2D LC-MS--an approach to increasing the number of identified proteins.

Identification of large numbers of proteins from complex biological samples is a continuing challenge in the area of quantitative proteomics. We introduce here a simple and reliable multistep mass tagging technique using our recently developed solid phase mass tagging reagents. When coupled with two-dimensional liquid chromatography/nano-electrospray ionization ion trap mass spectrometry (2D-LC/nano-ESI-MS), this method allows enhanced protein identification when tested on samples from prokaryotic and eukaryotic sources. The proteome of Escherichia coli D21 grown to either mid-exponential or stationary phase, and the membrane proteome from established breast cancer cell lines BT474 and MCF7 were used as model systems in these experiments. In both experiments, the numbers of total identified proteins are at least twice the numbers identified from a single tagging cycle. The sample complexity can be effectively reduced with corresponding increases in protein identification using the multistep method. The strategy described here represents a potentially powerful technique for large-scale qualitative and quantitative proteome research.

Cell Line, Tumor↗
PubMed2004

Histone demethylation mediated by the nuclear amine oxidase homolog LSD1.

Posttranslational modifications of histone N-terminal tails impact chromatin structure and gene transcription. While the extent of histone acetylation is determined by both acetyltransferases and deacetylases, it has been unclear whether histone methylation is also regulated by enzymes with opposing activities. Here, we provide evidence that LSD1 (KIAA0601), a nuclear homolog of amine oxidases, functions as a histone demethylase and transcriptional corepressor. LSD1 specifically demethylates histone H3 lysine 4, which is linked to active transcription. Lysine demethylation occurs via an oxidation reaction that generates formaldehyde. Importantly, RNAi inhibition of LSD1 causes an increase in H3 lysine 4 methylation and concomitant derepression of target genes, suggesting that LSD1 represses transcription via histone demethylation. The results thus identify a histone demethylase conserved from S. pombe to human and reveal dynamic regulation of histone methylation by both histone methylases and demethylases.

Conserved Sequence↗