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Objective. The aim of the present investigation was to study the expression of genes encoding IRS1 (insulin receptor substrate 1) and some other functionally active proteins in U87 glioma cells under silencing of polyfunctional chaperone HSPB8 for evaluation of the possible significance of this protein in intergenic interactions. Methods. Silencing of HSPB8 mRNA was introduced by HSPB8 specific siRNA. The expression level of HSPB8, IRS1, HK2, GLO1, HOMER3, MYL9, NAMPT, PER2, PERP, GADD45A, and DEK genes was studied in U87 glioma cells by quantitative polymerase chain reaction. Results. It was shown that silencing of HSPB8 mRNA by specific to HSPB8 siRNA led to a strong down-regulation of this mRNA and significant modification of the expression of IRS1 and many other genes in glioma cells: strong up-regulated of HOMER3, GLO1, and PERP and down-regulated of MYL9, NAMPT, PER2, GADD45A, and DEK gene expressions. At the same time, no significant changes were detected in the expression of HK2 gene in glioma cells treated by siRNA, specific to HSPB8. Moreover, the silencing of HSPB8 mRNA enhanced the glioma cells proliferation rate. Conclusions. Results of this investigation demonstrated that silencing of HSPB8 mRNA affected the expression of IRS1 gene as well as many other genes encoding tumor growth related proteins. It is possible that the dysregulation of most of the studied genes in glioma cells after silencing of HSPB8 is reflected by a complex of intergenic interactions and that this polyfunctional chaperone is an important factor for the stability of genome function and regulatory mechanisms contributing to the tumorigenesis control.

Long non‑coding RNAs (lncRNAs) play critical roles in the development and progression of cancers. The present study aimed to identify novel lncRNAs and associated microRNAs (miRNAs or miRs) and mRNAs in gastric cancer. Differentially expressed lncRNAs (DElncRNAs) and differentially expressed mRNAs (DEmRNAs) of 6 paired gastric cancer and normal tissues were identified using microarray. The DEmiRNAs between gastric cancer and the normal control tissues were identified using miRNA‑seq data from Cancer Genome Atlas. Common DElncRNAs from the Cancer RNA‑Seq Nexus database and circlncRNAnet database were analyzed. A DElncRNAs‑DEmiRNAs‑DEmRNAs network was constructed by target prediction. Functional enrichment analysis was employed to predict the function of DEmRNAs in the network. The correlation between the expression of DElncRNAS and DEmRNAs in the network was analyzed. The expression levels of several genes were validated by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). A total of 1,297 DElncRNAs, 2,037 DEmRNAs and 171 DEmiRNAs were identified. Among the 4 lncRNAs common to the 3 datasets, prostate androgen‑regulated transcript 1 (PART1) was selected for further analysis. The analysis identified 5 DEmiRNAs and 13 DEmRNAs in the PART1‑mediated ceRNA network. The DEmRNAs in the ceRNA network were markedly enriched in cancer‑related biological processes (response to hypoxia, positive regulation of angiogenesis and positive regulation of endothelial cell proliferation) and pathways (cGMP‑PKG signaling pathway, cAMP signaling pathway and proteoglycans in cancer). Out of the 13 DEmRNAs, 11 were positively associated with PART1. The downregulation of PART1, myosin light chain 9 (MYL9), potassium calcium‑activated channel subfamily M alpha 1 (KCNMA1), cholinergic receptor muscarinic 1 (CHRM1), solute carrier family 25 member 4 (SLC25A4) and ATPase Na+/K+ transporting subunit alpha 2 (ATP1A2) expression levels in gastric cancer was validated by RT‑qPCR. On the whole, the current study identified a novel lncRNA and associated miRNAs and mRNAs that are involved in the pathogenesis of gastric cancer that may serve as potential therapeutic targets for the treatment of gastric cancer.

CD69 is an activation marker on leukocytes. Early studies showed that the CD69+ cells were detected in the lung of patients with asthmatic and eosinophilic pneumonia, suggesting that CD69 might play crucial roles in the pathogenesis of such inflammatory diseases, rather than simply being an activation marker. Intensive studies using mouse models have since clarified that CD69 is a functional molecule regulating the immune responses. We discovered that Myosin light chain 9, 12a, 12b (Myl9/12) are ligands for CD69 and that platelet-derived Myl9 forms a net-like structure (Myl9 nets) that is strongly detected inside blood vessels in inflamed lung. CD69-expressing activated T cells attached to the Myl9 nets can thereby migrate into the inflamed tissues through a system known as the CD69-Myl9 system. In this review, we summarize the discovery of the CD69-Myl9 system and discuss how this system is important in inflammatory immune responses. In addition, we discuss our recent finding that CD69 controls the exhaustion status of tumor-infiltrating T cells and that the blockade of the CD69 function enhances anti-tumor immunity. Finally, we discuss the possibility of CD69 as a new therapeutic target for patients with intractable inflammatory disorders and tumors.

Background Extreme panting under heat stress threatens dairy cattle milk production. Previous research has revealed that the gas exchange-mediated respiratory drive in critically ill dairy cattle with low O2 saturation induces panting. Vascular endothelial growth factor (VEGF) signaling may play important roles in immunosuppression and oxidative stress during severe respiratory stress responses in heat-stressed cattle. The objectives of this study were to transcriptomically analyze mRNA expression mediating heat-induced respiratory stress-associated panting, evaluate gas exchange, screen hub genes, and verify the expression of proteins encoded by differentially expressed genes in lymphocyte pathways. Results Jersey cattle were naturally heat-exposed. Physiological data were collected for response evaluation, and blood was collected for gas exchange and gene expression assays at 06:00, 10:00 and 14:00 continuously for 1 week. Lymphocytes were isolated from whole-blood samples for mRNA-seq and expression analysis of key pathway genes/proteins. The cattle respiration rates differed with time, averaging 51 bpm at 06:00, 76 bpm at 10:00, and 121 bpm at 14:00 (p 2 differed with time: they were 7.41 and 41 mmHg at 06:00, 7.45 and 37.5 mmHg at 10:00, and 7.49 and 33 mmHg at 14:00, respectively (p Conclusion These findings help elucidate the underlying immune and oxygen regulation mechanisms associated with the VEGF signaling pathway in heat-stressed dairy cattle.

Tumor recurrence is one of the most important risk factors that can negatively affect the survival rate of colorectal cancer (CRC) patients. However, the key regulators dictating this process and their exact mechanisms are understudied. This study aimed to construct a gene co-expression network to predict the hub genes affecting CRC recurrence and to inspect the regulatory network of hub genes and transcription factors (TFs). A total of 177 cases from the GSE17536 dataset were analyzed via weighted gene co-expression network analysis to explore the modules related to CRC recurrence. Functional annotation of the key module genes was assessed through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. The protein and protein interaction network was then built to screen hub genes. Samples from the Cancer Genome Atlas (TCGA) were further used to validate the hub genes. Construction of a TFs-miRNAs–hub genes network was also conducted using StarBase and Cytoscape approaches. After identification and validation, a total of five genes (TIMP1, SPARCL1, MYL9, TPM2, and CNN1) were selected as hub genes. A regulatory network of TFs-miRNAs-targets with 29 TFs, 58 miRNAs, and five hub genes was instituted, including model GATA6-MIR106A-CNN1, SP4-MIR424-TPM2, SP4-MIR326-MYL9, ETS1-MIR22-TIMP1, and ETS1-MIR22-SPARCL1. In conclusion, the identification of these hub genes and the prediction of the Regulatory relationship of TFs-miRNAs-hub genes may provide a novel insight for understanding the underlying mechanism for CRC recurrence.

Protein interacting with C-kinase 1 (PICK1) is a 415-aa multidomain scaffold protein encoded by the PICK1 gene. Accumulating evidence suggests that PICK1 is involved in the progression of cancer. However, the role of PICK1 in gastric cancer (GC) remains largely unknown. Using integrated analysis of publicly available GC transcriptome data from the Gene Expression Omnibus (GEO) database and immunohistochemistry analysis of samples obtained from clinical GC patients, we found that PICK1 expression was significantly down-regulated in gastric tumor tissues in comparison with adjacent normal tissues. Our analyses also revealed that decreased expression of PICK1 conferred a disadvantage on overall survival time in GC patients. Additionally, PICK1 expression showed a strong association with the epithelial-mesenchymal transition (EMT) pathway, and PICK1 might represent a functional bridge for EMT. Moreover, PICK1 expression was significantly decreased in the EMT subtype of GC and was negatively correlated with the expression of fibronectin 1 (FN1) and myosin light chain 9 (MYL9) mRNAs. Thus, our study provides evidence that PICK1 is a promising biomarker for the molecular etiology of GC.

The numbers of patients with inflammatory bowel disease (IBD), such as ulcerative colitis (UC) and Crohn’s disease (CD), have been increasing over time, worldwide; however, the pathogenesis of IBD is multifactorial and has not been fully understood. Myosin light chain 9 and 12a and 12b (Myl9/12) are known as ligands of the CD69 molecule. They create “Myl9 nets” that are often detected in inflamed site, which play a crucial role in regulating the recruitment and retention of CD69-expressing effector cells in inflamed tissues. We demonstrated the strong expression of Myl9/12 in the inflamed gut of IBD patients and mice with DSS-induced colitis. The administration of anti-Myl9/12 Ab to mice with DSS-induced colitis ameliorated the inflammation and prolonged their survival. The plasma Myl9 levels in the patients with active UC and CD were significantly higher than those in patients with disease remission, and may depict the disease severity of IBD patients, especially those with UC. Thus, our results indicate that Myl9/12 are involved in the pathogenesis of IBD, and are likely to be a new therapeutic target for patients suffering from IBD.

The organ of Corti is an auditory organ located in the cochlea, comprising hair cells (HCs) and other supporting cells. Cellular shape changes of HCs are important for the development of auditory epithelia and hearing function. It was previously observed that HCs and inner sulcus cells (ISCs) demonstrate cellular shape changes similar to the apical constriction of the neural epithelia. Apical constriction is induced via actomyosin cable contraction in the apical junctional complex and necessary for the physiological function of the epithelium. Actomyosin cable contraction is mainly regulated by myosin regulatory light chain (MRLC) phosphorylation by myosin light chain kinase (MLCK). However, MRLC and MLCK isoforms expressed in HCs and ISCs are unknown. Hence, we investigated the expression patterns and roles of MRLCs and MLCKs in HCs. Droplet digital PCR revealed that HCs expressed MYL12A/B and MYL9, which are non-muscle MRLC and smooth muscle MLCK (smMLCK), respectively. Immunofluorescence staining throughout the organ of Corti demonstrated that only MYL12 was expressed in the apical portion of HCs, whereas MYL12 and MYL9 were expressed on ISCs. In addition, purified MYL12B was phosphorylated by smMLCK in vitro, and the harvested HCs contained phosphorylated MYL12. Furthermore, accompanied by the expansion of the cell area of outer HCs, MYL12 phosphorylation was reduced by ML-7, which is an inhibitor of smMLCK. In conclusion, MYL12 phosphorylation by smMLCK contributed to the apical constriction-like cellular shape change of HCs possibly relating to the development of auditory epithelia and hearing function.

Aims Acute aortic dissection (AAD) is a life-threatening disease with high morbidity and mortality. Previous studies have showed that vascular smooth muscle cell (VSMC) phenotype switching modulates vascular function and AAD progression. However, whether an endogenous signaling system that protects AAD progression exists, remains unknown. Our aim is to investigate the role of Anxa1 in VSMC phenotype switching and the pathogenesis of AAD. Methods and results We first assessed Anxa1 expression levels by immunohistochemical staining in control aorta and AAD tissue from mice. A strong increase of Anxa1 expression was seen in the mouse AAD tissues. In line with these findings, micro-CT scan results indicated that Anxa1 plays a role in the development of AAD in our murine model, with systemic deficiency of Anxa1 markedly progressing AAD. Conversely, administration of Anxa1 mimetic peptide, Ac2-26, rescued the AAD phenotype in Anxa1-/- mice. Transcriptomic studies revealed a novel role for Anxa1 in VSMC phenotype switching, with Anxa1 deficiency triggering the synthetic phenotype of VSMCs via down-regulation of the JunB/MYL9 pathway. The resultant VSMC synthetic phenotype rendered elevated inflammation and enhanced matrix metalloproteinases (MMPs) production, leading to augmented elastin degradation. VSMC-restricted deficiency of Anxa1 in mice phenocopied VSMC phenotype switching and the consequent exacerbation of AAD. Finally, our studies in human AAD aortic specimens recapitulated key findings in murine AAD, specifically that the decrease of Anxa1 is associated with VSMC phenotype switch, heightened inflammation, and enhanced MMP production in human aortas. Conclusions Our findings demonstrated that Anxa1 is a novel endogenous defender that prevents acute aortic dissection by inhibiting vascular smooth muscle cell phenotype switching, suggesting that Anxa1 signaling may be a potential target for AAD pharmacological therapy. Translational perspective Our studies herein may lead to a paradigm shift for pharmacologic therapy towards acute aortic dissection. Through careful examination of the pathological changes that occur during AAD onset in experimental animal models, we demonstrated that VSMC phenotype switching plays a critical role in the development of AAD. Inhibition of VSMC phenotype switching and its attendant impacts on aortic function may be a viable approach for future treatment. Toward that end, our studies highlighted the protective benefit of Anxa1 and its mimetic peptide Ac2-26 in AAD through prevention of the switching of VSMC to a synthetic phenotype.

To what extent stromal cells in the tumor microenvironment (TME) are transformed by colorectal cancer (CRC) cells is unexplored. To dissect alterations in these non-malignant cells, we performed single-cell multiomics sequencing of 21 patients with microsatellite-stable CRCs and 6 cancer-free, elderly individuals. Surprisingly, somatic copy number alterations (SCNAs) are prevalent in immune cells, fibroblasts, and endothelial cells in both the TME and the normal tissues of each individual. Moreover, the proportions of fibroblasts with SCNAs in tumors (11.1%-47.7%) are much higher than those in adjacent normal tissues (1.1%-10.6%), with gain of chromosome 7 strongly enriched in the TME, clearly indicating clonal expansion. Furthermore, five genes (BGN, RCN3, TAGLN, MYL9, and TPM2) are identified as fibroblast-specific biomarkers of poorer prognosis of CRC. Our study provides evidence and functional relevance of pervasive genomic alterations in the stromal cells of TME in CRC.

Epigenetic gene silencing of the tumor suppressor death-associated protein kinase 1 (DAPK1) is implicated in the progression of malignant gliomas. However, the mechanism underlying the repression of DAPK1 in gliomas remains elusive. In this study, we identified the existence of DAPK1-inositol 1,4,5-trisphosphate receptor (IP3R)-interacting protein (ITPRIP) -myosin regulatory light polypeptide 9 (MYL9) complex in malignant glioma cells. Lentivirus co-infection and coimmunoprecipitation showed that ITPRIP bound with the death domain (DD) of DAPK1 in vitro. Further, dissociating ITPRIP-DAPK1 interaction inhibited glioma tumor growth in vitro but not in vivo. Moreover, knockdown of ITPRIP or DAPK1 impaired the ternary complex formation, whereas MYL9 knockdown did not affect ITPRIP-DAPK1 association. We further found that ITPRIP recruited MYL9 to the kinase domain (KD) of DAPK1, and in turn impeded the phosphorylation of MYL9. Accordingly, interference of ITPRIP enhanced the suppressive effects of DAPK1-KD on glioma progression both in vitro and in vivo. Our results demonstrate that ITPRIP plays a crucial role in the inhibition of DAPK1 and enhancement of tumorigenic properties of malignant glioma cells.

Colorectal cancer (CRC) is ranked as one of the three most common cancers in the world. This research aims at exploring the molecular mechanisms of CRC. We used R language to analyze gene expression data from the Gene Expression Omnibus (GEO) database. By using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) database, the function and pathway enrichment of differentially expressed genes were analyzed. The Protein-protein interaction (PPI) network was constructed by STRING and Cytoscape. A total of 205 differentially expressed genes (DEGs) were screened out. These differentially expressed genes mainly enriched in the terms of regulation of leukocyte chemo taxis, chemokine receptor binding and chemokine-mediated signaling pathway. The PPI network of differentially expressed genes was constructed. Seven hub genes (CCL2, TPM1, DES, CXCL12, MYLK, MYL9 and TPM2) were identified. We conclude that these 7 genes may be involved in the occurrence of CRC.

AimsTumour recurrence is inevitable in glioblastoma (GBM) and mostly noted in the peritumoural brain zone (PT). In our previous microarray-based study, we identified Myosin Light Chain 9 (MYL9) as a highly expressed gene in the PT of GBM. Therefore, we aimed to study the expression pattern and clinical significance of MYL9 in GBM.MethodsPatient samples included three retrospective cohorts: 25 GBM cases with differential biopsies of tumour core and PT, 62 retrospective cases of newly diagnosed GBM with survival information and 20 paired samples (newly diagnosed and recurrent GBM). All tumour tissues, archived as formalin fixed paraffin embedded blocks were retrieved and immunohistochemistry for MYL9 and IDH1 R132H was performed. MYL9 expression was correlated with patient prognosis in our cohort and in The Cancer Genome Atlas (TCGA) and Rembrandt cohorts. It was further evaluated in the 20 paired samples of GBM.ResultsMYL9 showed a cytoplasmic membranous staining of tumour cells. The staining pattern was variable and patchy within tumours. Higher MYL9 expression was associated with poor overall and progression-free survival in our and in TCGA and Rembrandt cohorts. The expression of MYL9 was higher in IDH1 R132H immunonegative cases.ConclusionsWe show MYL9 as a novel biomarker, variably expressed in GBM. The association of high MYL9 expression with poor prognosis in newly diagnosed GBM patients and increased expression in recurrent GBM is indicative of its role in conferring tumour aggressiveness.

This study was designed to identify the potential key protein interaction networks, genes, and correlated pathways in early-onset colorectal cancer (CRC) via bioinformatics methods. We selected microarray data GSE4107 consisting 12 patient’s colonic mucosa and 10 healthy control mucosa; initially, the GSE4107 were downloaded and analyzed using limma package to identify differentially expressed genes (DEGs). A total of 131 DEGs consisting of 108 upregulated genes and 23 downregulated genes of patients in early-onset CRC were selected by the criteria of adjusted P values

Mechanical strain associated with the expanding uterus correlates with increased preterm birth rates. Mechanical signals result in a cascading network of protein phosphorylation events. These signals direct cellular activities and may lead to changes in contractile phenotype and calcium signaling. In this study, the complete phospho-proteome of uterine smooth muscle cells subjected to mechanical strain for 5 min was compared to un-strained controls. Statistically significant, differential phosphorylation events were annotated by Ingenuity Pathway Analysis to elucidate mechanically induced phosphorylation networks. Mechanical strain leads to the direct activation of ERK1/2, HSPB1, and MYL9, in addition to phosphorylation of PAK2, vimentin, DOCK1, PPP1R12A, and PTPN11 at previously unannotated sites. These results suggest a novel network reaction to mechanical strain and reveal proteins that participate in the activation of contractile mechanisms leading to preterm labor.

Objective Myosin light chain 9 (MYL9) is necessary for cytoskeletal dynamics and experimental metastasis, but its expression in esophageal squamous cell carcinoma (ESCC) has not been addressed. We investigated the expression pattern and clinical significance of MYL9 in patients with ESCC. Methods We examined MYL9 expression using quantitative real-time PCR and western blotting in NE1 immortalized esophageal epithelial cells, ESCC cell lines, and paired ESCC tissues. MYL9 protein in 136 primary ESCC tissues and other types of solid tumor was detected using immunohistochemistry. The association between MYL9 expression and clinical parameters and survival was evaluated by statistical analysis. Results MYL9 was significantly upregulated in the ESCC cell lines as compared with NE1 cells. In the paired ESCC samples, MYL9 mRNA and protein expression was not significantly different between lesion tissues and the matched adjacent noncancerous tissues. In ESCC tissue, both intratumoral and peritumoral stroma were positive for MYL9. In the 136 ESCC samples, high MYL9 expression in the tumor cells significantly correlated with histological differentiation (p = 0.028), recurrence (p = 0.01), and vital status (p < 0.01). Patients with high MYL9 expression in the tumor cells had poorer overall survival (OS) and recurrence-free survival. Multivariate analysis revealed that high MYL9 expression in tumor cells was an independent and significant risk factor affecting OS after curative treatment (hazard ratio = 2.254, 95% confidence interval = 1.347–3.771, p = 0.002). Conclusions MYL9 expression might be a promising prognostic marker and therapeutic target in ESCC.

Recent decades have witnessed a rapid worldwide increase in chronic inflammatory disorders such as asthma. CD4(+) T helper 2 cells play critical roles in the pathogenesis of allergic airway inflammation, and CD69 expression on activated CD4 T cells is required to induce allergic inflammation in tissues. However, how CD69 mechanistically controls allergic inflammation remains poorly defined. In lymphoid tissues, CD69 regulates cellular retention through inhibition of S1P1 expression and requires no specific ligands to function. In contrast, we show herein that myosin light chain (Myl) 9 and Myl12 are new functional ligands for CD69. Blockade of CD69-My19/12 interaction ameliorates allergic airway inflammation in ovalbumin-induced and house dust mite-induced mouse models of asthma. Within the inflamed mouse airways, we found that the expression of My19/12 was increased and that platelet-derived My19/12 localized to the luminal surface of blood vessels and formed intravascular netlike structures. Analysis of nasal polyps of eosinophilic chronic rhinosinusitis patients revealed that My19/12 expression was increased in inflammatory lesions and was distributed within net-like structures in the intravascular space. In addition, we detected My19/12 in perivascular spaces where many CD69(+) cells were positioned within My19/12 structures. Thus, CD69-My19/12 interaction is a key event in the recruitment of activated CD69(+) T cells to inflamed tissues and could be a therapeutic target for intractable airway inflammatory diseases. Refereed/Peer-reviewed

Gastric cancer (GC) is a prevalent cancer, which remains incurable, and therefore requires an alternative treatment method. Celecoxib is a nonsteroidal anti-inflammatory drug that targets cyclooxygenase-2, and exhibits anticancer effects. The present study aimed to investigate the anti-GC mechanism of celecoxib using bioinformatics methods. Gene expression datasets {"type":"entrez-geo","attrs":{"text":"GSE56807","term_id":"56807"}}GSE56807 (GC tissues and normal gastric tissues) and {"type":"entrez-geo","attrs":{"text":"GSE54657","term_id":"54657"}}GSE54657 (celecoxib-treated and non-treated human GC epithelial AGS cells) were downloaded from the Gene Expression Omnibus database. Two groups of differentially expressed genes (DEGs) were identified using limma package in R language. The criterion for {"type":"entrez-geo","attrs":{"text":"GSE56807","term_id":"56807"}}GSE56807 was a false discovery rate of

International audience; Efficient angiogenic sprouting is essential for embryonic, postnatal and tumor development. Serum response factor (SRF) is known to be important for embryonic vascular development. Here, we studied the effect of inducible endothelial-specific deletion of Srf in postnatal and adult mice. We find that endothelial SRF activity is vital for postnatal growth and survival, and is equally required for developmental and pathological angiogenesis, including during tumor growth. Our results demonstrate that SRF is selectively required for endothelial filopodia formation and cell contractility during sprouting angiogenesis, but seems dispensable for vascular remodeling. At the molecular level, we observe that vascular endothelial growth factor A induces nuclear accumulation of myocardin-related transcription factors (MRTFs) and regulates MRTF/SRF-dependent target genes including Myl9, which is important for endothelial cell migration in vitro. We conclude that SRF has a unique function in regulating migratory tip cell behavior during sprouting angiogenesis. We hypothesize that targeting the SRF pathway could provide an opportunity to selectively target tip cell filopodia-driven angiogenesis to restrict tumor growth.

Transcription factor (TF) mutations are increasingly recognized to play a major role in inherited platelet abnormalities. RUNX1, a major hematopoietic TF, acts in a combinatorial manner with other TFs to regulate numerous megakaryocyte (MK)/platelet genes. Human RUNX1 haplodeficiency is associated with thrombocytopenia, platelet function defects, and increased leukemia risk. We have described a patient with multiple abnormalities in platelet aggregation and secretion responses with a heterozygous RUNX1 nonsense mutation (Sun et al Blood 2004; 103; 948-54). Transcript expression profiling of patient platelets (Sun et al J Thromb Haemost 2007; 5:146-54)showed several genes were significantly downregulated, including myosin light chain (MYL9), platelet factor 4 (PF4), protein kinase C-θ (PRCKQ), and 12-lipoxygenase (ALOX12); these have been shown by us to be regulated by RUNX1. The profiling data also showed 10-fold downregulation of phosphatidylcholine transfer protein (PCTP) gene (fold change ratio 0.09, p=0.02) in the patient compared with normal controls. PCTP regulates the intermembrane transfer of phosphatidylcholine (PC), a major plasma membrane phospholipid. Platelet PCTP expression is associated with increased platelet aggregation and calcium mobilization upon activation of protease-activated receptor 4 (PAR4) thrombin receptors in black subjects as compared to white subjects (Edelstein et al Nat Med 2013; 19:1609-16). Pharmacologic inhibition of PCTP decreased platelet aggregation in response to PAR4 agonist and siRNA knockdown of PCTP in megakaryocytic cells blunted calcium mobilization induced by PAR4 (Edelstein et al Nat Med 2013; 19:1609-16). Little is known regarding the regulation of PCTP in MKs/platelets and its role in cardiovascular events. Based on the decreased platelet PCTP expression in our patient, we pursued the hypothesis that PCTP is regulated by RUNX1 and contributes to cardiovascular events. Corrected total cellular immunofluorescence with anti-PCTP antibody showed significantly reduced platelet PCTP expression by 58% in our patient compared to a normal control. In silico analysis revealed 5 RUNX1 consensus binding sites up to ~ 1 kb of the PCTP 5' upstream region from ATG. To assess for interaction of RUNX1 with the PCTP promoter, chromatin immunoprecipitation (ChIP) assay with anti-RUNX1 antibody was performed using human erythroid leukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 48 hours to induce megakaryocytic transformation. The ChIP studies showed RUNX1 binding to PCTP chromatin in the regions encompassing RUNX1 binding site 1 (-345/-340), site 3 (-632/-627), and encompassing sites 4 and 5 (-974/-969, -997/-992). Electrophoretic mobility shift assay (EMSA) using PMA-treated HEL cell nuclear extracts showed RUNX1 binding to DNA probes (28-37 bp) containing site 1 (-345/-340) and both sites 4 and 5 (-974/-969, -997/-992). PCTP mRNA and protein expression were increased with RUNX1 overexpression and reduced with RUNX1 knockdown in HEL cells, indicating that PCTP is regulated by RUNX1. To assess the clinical relevance of the findings, the relationship between RUNX1 and PCTP in peripheral blood RNA, and PCTP and death or myocardial infarction (MI) events were assessed in two separate patient cohorts (n = 587 total patients) with cardiovascular disease. RUNX1 is transcribed from two alternate promoters (P1 and P2) resulting in different isoforms. In both patient cohorts, there was strong correlation between RUNX1 and PCTP expression in a promoter specific manner. RUNX1 P1 probe sets were strongly and inversely correlated with PCTP expression (p < 0.0001), while the P2 probe sets were not. PCTP expression was associated with death or MI in both patient cohorts (odds ratio 2.1, 95% CI [1.61-2.95], P-value < 0.0001) independent of age, sex, race, platelet count, and cardiovascular risk factors. Conclusions: Our results provide evidence that PCTP is regulated by RUNX1 (potentially in a promoter specific manner), and that PCTP expression is associated with death or myocardial infarction in patients with cardiovascular disease. RUNX1 regulation of PCTP may play a role in the pathogenesis of platelet-mediated cardiovascular events. Disclosures No relevant conflicts of interest to declare.

Background Aging-associated changes in the cardiovascular system increase the risk for disease development and lead to profound alterations in vascular reactivity and stiffness. Elucidating the molecular response of arteries to injury and age will help understand the exaggerated remodeling of aging vessels. Methodology/Principal Findings We studied the gene expression profile in a model of mechanical vascular injury in the iliac artery of aging (22 months old) and young rats (4 months old). We investigated aging-related variations in gene expression at 30 min, 3 d and 7 d post injury. We found that the Myosin Light Chain gene (MYL9) was the only gene differentially expressed in the aged versus young injured arteries at all time points studied, peaking at day 3 after injury (4.6 fold upregulation (p