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Vascular smooth muscle cell (VSMC) accumulation and hypertrophy are common in vascular disorders, and inflammation has a crucial role in the development of these diseases. To investigate the effect of inflammation on the neurotransmission of VSMC, bioinformatic analysis was performed, following next generation sequencing. Genes of lipopolysaccharide (LPS)-treated A7r5 cells and phosphate-buffered saline (PBS)-treated A7r5 cells were sequenced via next generation sequencing, and each assay was repeated three times. Differentially expressed genes (DEGs) were obtained using the NOISeq package in R. Subsequently, their potential functions were predicted by functional and pathway enrichment analyses using the Database for Annotation, Visualization and Integrated Discovery online tool. Interaction relationships of the proteins enriched in pathways associated with neurological diseases, the proteins which had interaction relationships with adrenoceptor α 1D (ADRA1D) or calcium voltage-gated channel subunit α1 S (CACNA1S), separately, were obtained from STRING, and protein-protein interaction (PPI) networks were constructed using Cytoscape software. A total of 2,038 DEGs, including 1,094 upregulated and 944 downregulated genes in the LPS treatment group were identified when compared with the control group. Enrichment analyses showed that NADH:Ubiquinone Oxidoreductase Core Subunit V2 (NDUFV2) was involved in several neurological diseases, including oxidative phosphorylation, Alzheimer's disease, Parkinson's disease and Huntington's disease. Furthermore, NDUFV2 (degree, 20) had a higher degree in the PPI network for DEGs enriched in pathways associated with neurological diseases. In the PPI network for ADRA1D, CACNA1S and the DEGs interacting with them, prohibitin (PHB), oxytocin receptor (OXTR), collapsin response mediator protein 1 (CRMP1) and dihydropyrimidinase like 2 (DPYSL2) had interaction relationships with both ADRA1D and CACNA1S. To conclude, the present study revealed that NDUFV2, PHB, OXTR, CRMP1 and DPYSL2 may have key roles in the effect of inflammation on neurotransmission of VSMC.
As a highly specialized cell in mature animals, the vascular smooth muscle cell (VSMC) has a principal function of contraction; however, production of matrix components of the blood vessel wall and proliferation becomes the primary function of VSMCs during vasculogenesis (1). Abnormal contraction of SMC is a major incentive of vasospasm of the cerebral and coronary arteries, as well as hypertension (2). VSMC accumulation and hypertrophy are common in vascular disorders, such as atherosclerosis, hypertension, restenosis (3,4) and inflammation, which can be induced by hypoxia and has crucial roles in the development of these diseases (5,6). Thus, there is an urgency to elucidate the effect of inflammation on the neurotransmission of VSMCs.
Several pharmacological agents are capable of inducing inflammation. In human aortic smooth muscle cells, lipopolysaccharide (LPS) promotes the production of nitric oxide (NO) and Toll-like receptor 4 (TLR4) expression, inducing inflammatory responses (4). A previous study demonstrated that propranolol has a negative chronotropic effect on the expression levels of pro-inflammatory cytokines after myocardial infarction (MI) in rats (7). In rat aorta, β-adrenoceptors were overstimulated by the agonist, isoproterenol, which resulted in an increase of vascular inflammatory mediators, such as interleukin (IL)-1β, IL-6 and nuclear factor κB (NF-κB) (8). At a concentration of 20 µg/ml, the non-selective β-adrenergic receptor agonist, propranolol, was revealed to suppress cell growth of infantile hemangioma endothelial cells (IHECs) in vitro once the proliferation stage of IHECs has been affected for between 72 and 96 h, whereas isoproterenol yielded the opposite results (9).
Previous reports have been conducted to survey the effect of inflammation on VSMC. As an adipocytokine, extracellular pre-B cell colony-enhancing factor/nicotinamide phosphoribosyltransferase/visfatin (ePBEF/NAMPT/visfatin) functions as a direct contributor to vascular inflammation via its NAMPT activity (10). Through regulating vascular cell activation and inflammatory cell recruitment, the adhesion protein, cluster differentiation (CD) 44, has an important role in the development of atherosclerotic diseases (11). By inhibiting the activation of hypoxia-inducible factor-1α, transcription factors, NF-κB and activator protein-1 (AP-1), and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors have anti-proliferative and anti-inflammatory effects on human endothelial and vascular smooth muscle cells; thus, statins can be used to treat atherosclerosis (12).
In the present study, next generation sequencing was conducted to obtain sequence data. Differentially expressed genes (DEGs) between the LPS treatment group and the control group were screened and their functions were predicted by enrichment analyses. Moreover, a protein-protein interaction (PPI) network was constructed to investigate the interaction relationships between these DEGs.
Rat VSMC cell line A7r5, which was purchased from Shanghai enzyme research Biotechnology Co., Ltd. (Shanghai, China), was cultivated in Dulbecco's modified Eagle medium (DMEM) (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and maintained at 37°C using an incubator (Thermo Fisher Scientific, Inc). DMEM was discarded, and A7r5 cells were digested with pancreatin (Gibco; Thermo Fisher Scientific, Inc) for 5 min. Subsequently, 6-fold DMEM was added to terminate digestion and cells were centrifuged at room temperature with 157 × g for 5 min, and the supernatant and resuspension was discarded. A7r5 cells were cultured in new culture flasks in a 5% CO2 incubator at 37°C (Thermo Fisher Scientific, Inc.). Using the frozen stock solution made of 10% dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) and 90% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.), A7r5 cells were resuspended at a density of 5×106 cells/ml. Following stewing at 4°C for 10 min, cells were cryopreserved at −20°C for 2 h and stored at −80°C overnight.
A7r5 cells were inoculated into confocal plates (Wohong Biotechnology Co., Ltd., Shanghai, China) (2×104 cells/plate) and cultivated overnight. Cells were washed with phosphate-buffered saline (PBS) twice with 100 µl PBS containing 5 µmol/l Fluo-4/AM and lucifugally incubated at 37°C in a 5% CO2 incubator for 45 min. Subsequently, cells were washed with PBS twice again and induced by LPS (100 µg/ml) for 30 min. In the control groups, PBS was used instead of LPS. Under confocal laser scanning microscope, A7r5 cells were observed and images were captured before and after treatment with isoprenaline (10 µmol/l, Melone pharmaceutical, Co., Ltd., Dalian, China) or propranolol (10 µmol/l, Melone pharmaceutical, Co., Ltd.).
Cells were inoculated with 0.5% FBS in confocal plates (4×105 cells/plate). After being cultivated for 24 h, the 0.5% FBS medium was replaced with 10% FBS and cells were treated with cobalt dichloride (200 µmol/l) for 24 h. Subsequently, cells were induced by LPS (100 µg/ml) for 30 min; however, cells in the control group received PBS. After cell digestion by pancreatin (Gibco; Thermo Fisher Scientific, Inc.) for 5 min, the mixture was centrifuged at room temperature at 157 × g for 5 min. Then, cells were washed three times with DMEM at room temperature and further centrifuged at room temperature at 157 × g for 5 min. The supernatant was discarded and A7r5 cells were preserved in TRIzol (Invitrogen; Thermo fisher Scientific, Inc.) at −80°C. Each assay was performed in triplicate.
Total RNA of the LPS treatment group and the control group were extracted using a TRIzol total RNA extraction kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The integrity and purity of total RNA was detected using 2% agarose gel electrophoresis and a spectrophotometer (Merinton Instrument, Ltd., Beijing, China), respectively. The RNA-sequence library was constructed using methods described in a previous study (13). Subsequently, DNA cluster amplification was performed and high-throughput sequencing was conducted for the library, using Illumina Hiseq 2000 100PE (Illumina Inc., San Diego, CA, USA). After the raw data was obtained, sequences containing an adaptor, >50% low quality bases and >3% unknown bases were filtered out.
Following filtering, the sequences were mapped to the rat genome (rn5), using bowtie1 in TopHat software (version 2.1.0, accessible at http://ccb.jhu.edu/software/tophat/index.shtml) (14). The maximum read mismatch number was set at 2, and the parameter ‘max-multihits’ was set at 1 (15). And the other parameters were set to defaults. Combining with annotation information of rn5 in ensemble, expression of each sample was annotated using Cufflinks software (version 2.2.1, accessible at http://cole-trapnell-lab.github.io/cufflinks/) (16) under the default parameter values. The NOISeq package (version 2.18.0, accessible at http://www.bioconductor.org/packages/release/bioc/html/NOISeq.html) (17) in R was used to screen the DEGs between the LPS treatment group and the control group. The probability of a gene being DEGs (q) was set to 0.99.
Gene Ontology (GO), which consists of three categories, including biological process (BP), molecular function (MF) and cellular component (CC), is used to generate the vocabulary that can be applied to all eukaryotes (18). As a database, Kyoto Encyclopedia of Genes and Genomes (KEGG) includes information of known genes and their biochemical functionalities (19). Using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) online tool (20), GO and KEGG pathway enrichment analyses were conducted for the upregulated and downregulated genes between the LPS treatment and control groups, respectively. P<0.01 was used as the cut-off criterion.
Using STRING online software (version 10.0, accessible at http://www.string-db.org/) (21), interaction relationships of the proteins encoded by the DEGs were searched, and the required confidence (combined score) >0.1 was used as the cut-off criterion. Subsequently, PPI network was visualized using Cytoscape (version 3.2.0, accessible at http://www.cytoscape.org/) (22). Proteins in the network were named as nodes and the degree of a node was equal to the number of nodes interacted with it. Moreover, the nodes with degrees higher than 20 were defined as hub nodes.
In the PBS control group, A7r5 cells prior to treatment with isoprenaline or propranolol are indicated in Fig. 1A. In A7r5 cells treated with isoprenaline, the concentration of calcium decreased (Fig. 1B); however, the level of calcium in propranolol-treated A7r5 cells increased (Fig. 1C).
The LPS-treated group (Fig. 1D-F) exhibited a reduced calcium signal prior to treatment with isoprenaline or propranolol when compared with the PBS control group (Fig. 1D). In the A7r5 cells that were treated with isoprenaline, the concentration of calcium decreased (Fig. 1E). However, the level of calcium in the A7r5 cells treated with propranolol had no change when compared with the LPS group cells treated with isoprenaline (Fig. 1F).
Compared with the control PBS group, a total of 2,038 DEGs, including 1,094 upregulated and 944 downregulated genes were identified in the LPS-treated group. These findings indicated that the number of upregulated genes was markedly higher in comparison to the number of downregulated genes.
The enriched GO functions for upregulated genes are listed in Table I. The enriched functions in the BP category included chemical homeostasis (P=4.81E-04), mitogen-activated protein kinase kinase kinase cascade (P=7.85E-04) and nucleoside monophosphate metabolic process (P=8.39E-04). The enriched functions in the CC category included condensed chromosome kinetochore (P=1.33E-04), chromosome, centromeric region (P=1.63E-04) and plasma membrane (P=2.96E-04). Enriched functions in the MF category included hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds in linear amidines (P=1.40E-04), quaternary ammonium group binding (P=8.28E-04) and phosphatidylcholine binding (P=9.81E-04).
The enriched KEGG pathways for upregulated genes are also listed in Table I, including purine metabolism (P=3.13E-04), neuroactive ligand-receptor interaction (P=0.003996) and pyrimidine metabolism (P=0.007245).
The enriched GO functions for downregulated genes are presented Table II. The enriched functions in the BP category included oxidation reduction (P=1.19E-05), cofactor metabolic process (P=0.001298) and erythrocyte homeostasis (P=0.0013). Enriched functions in the CC category included mitochondrion (P=6.87E-07), organelle membrane (P=6.03E-06) and mitochondrial part (P=1.67E-05). The enriched functions in the MF category included nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide hydride binding (P=3.63E-04), protein homodimerization activity (P=4.08E-04) and iron ion binding (P=4.97E-04).
The enriched KEGG pathways for downregulated genes are also listed in Table II, including oxidative phosphorylation (P=2.49E-04), Alzheimer's disease (P=0.00105), Parkinson's disease (P=0.00299), Huntington's disease (P=0.00397) and proteasome (P=0.008983). Furthermore, NADH dehydrogenase ubiquinone flavoprotein 2 (NDUFV2) was revealed to be involved in several neurological diseases, including oxidative phosphorylation, Alzheimer's disease, Parkinson's disease and Huntington's disease.
Pyrimidine metabolism, oxidative phosphorylation, Alzheimer's disease and Parkinson's disease were all metabolic pathways associated with neurological diseases. DEGs enriched in these pathways and adrenergic receptor genes α1d (ADRA1D) were used to construct a PPI network. The PPI network consisted of 39 nodes and 183 interactions (Fig. 2). ATP synthase, mitochondrial F1 complex, O subunit (ATP5O; degree, 22) and NDUFV2 (degree, 20) were hub nodes in the PPI network.
With the required confidence threshold of >0, the DEGs which had interaction relationships with ADRA1D and voltage-dependent L-type calcium channel subunit α-1S (ACNA1S) were indicated in Fig. 3A and B, respectively. DEGs which had interaction relationships with ADRA1D and CACNA1S were merged in Fig. 4. In the PPI network, prohibitin (PHB), oxytocin receptor (OXTR), collapsin response mediator protein 1 (CRMP1) and dihydropyrimidinase-like 2b (DPYSL2) exhibited an interaction relationship with both ADRA1D and CACNA1S.
The present study indicated that calcium signals in A7r5 cells treated with LPS were weaker when compared with PBS-treated cells (the control group). It has been reported that calcium oxalate crystals are able to trigger inflammation through regulating IL-1β (23). Calcium pyrophosphate has been revealed to induce a novel acute inflammation, pleurisy (23). Thus, LPS induced inflammation in A7r5 cells. In the present study, a total of 2,038 DEGs, including 1,094 upregulated and 944 downregulated genes, were identified in the LPS-treated and control groups. Enrichment analyses indicated that NDUFV2 was involved in several neurological diseases, including oxidative phosphorylation, Alzheimer's disease, Parkinson's disease and Huntington's disease. Furthermore, NDUFV2 (degree, 20) exhibited a higher degree in the PPI network for DEGs enriched in pathways associated with neurological diseases. NDUFV2, which is located on chromosome 18p11.31-p11.2, has been named as a causative gene in neurological diseases, such as schizophrenia, Parkinson's disease, and bipolar disorder (24,25). A human disease cell model has revealed that the injury of mitochondrial localization of NDUFV2 is related to the pathogenesis of early-onset hypertrophic cardiomyopathy and encephalopathy (26). Therefore, the expression of NDUFV2 may be involved in the effect of inflammation on neurotransmission of VSMC.
In the PPI network for ADRA1D, CACNA1S and the DEGs interacting with these components, PHB, OXTR, CRMP1 and DPYSL2 were revealed to have interaction relationships with both ADRA1D and CACNA1S. PHB, a member of the Band-7 family of proteins, has a neuro-damaging role following oxidative and excitotoxic stress, and may serve as target for designing agents to control neuronal death in brain injury, such as cerebral ischemia (27). In cardiomyocytes, overexpressed PHB contributes to the maintenance of the mitochondrial membrane potential and improves cell survival during hypoxia (28). It has been speculated that the function of PHB in protecting against oxidative and hypoxic stress may be correlated with its role in mediating the electron transport chain enzyme, cytochrome C oxidase (29). A previous report has implicated oxytocin (OXT) in inflammatory processes (30). As G-protein coupled receptors, oxytocin receptors (OXTRs) are regulated by G-proteins, which stimulate the phosphatidylinositol-calcium secondary messenger system (31). In addition, OXTRs are widely distributed in the central nervous system and mediate various behaviors (32), such as social memory and recognition, responses to stress and anxiety, sexual and maternal behaviors, and bonding (33). These may indicate that the expression levels of PHB and OXTR are correlated with the effect of inflammation on neurotransmission of VSMC.
CRMP1 is affiliated with a cytoplasmic family of proteins and is involved in the development of the central nervous system (34). CRMP1, which may be disturbed by Speedy A1 (Spy1) from interacting with actin, has a role in the collapse and regeneration of growth cones after sciatic nerve crush (35). Previous studies have identified that within hypertrophic cells of a brain that has suffered a stroke, overexpression of SPNA2 and DPYSL2 (also known as CRMP2) was revealed to be correlated with neurite outgrowth and plasticity, which suggests an early activation of neuronal regeneration, repair and development (36,37). Meanwhile, in the neonatal rat brain, the Akt/glycogen synthase kinase-3β/CRMP2 pathway modulates axonal injury following hypoxia-ischemia (38,39). Thus, the expression levels of CRMP1 and DPYSL2 may be associated with the effect of inflammation on neurotransmission of VSMC.
In conclusion, we screened 2,038 DEGs, including 1,094 upregulated and 944 downregulated genes in the LPS group compared with the control group. The present study identified that NDUFV2, PHB, OXTR, CRMP1 and DPYSL2 may have key roles in the effect of inflammation on the neurotransmission of VSMC. However, the results were speculations following bioinformatics analysis and require further experimental validation.
The present study was supported by Shanghai Science and Technology Commission Fund (grant no. 15495810202) and the Medical Engineering Cross Research Fund, Shanghai Jiaotong University (grant nos. YG2013MS08 and YG2014QN09).