| Background:The prevalence of hypertension increases year by year in the world. The danger of hypertension is that it can cause organ damages, in which cardiac injury is very important. In the course of hypertension induced cardiac injury, inflammation plays a important role. Transcriptome study based on RNA-Seq technology, revealed the biological process and molecular mechanism of many diseases. RNA-Seq has been widely used in basic research, clinical diagnostics and drug development etc. However RNA-Seq technology has not yet been used in hypertension-induced cardiac damage. It is recently reported that the endoplasmic reticulum(ER) stress pathway is involved in the inflammatory response, whereby inflammation induces ER stress, and ER stress induces an inflammatory response. However, whether ER stress is involved in hypertension induced cardiac injury is unknown and the role of ER stress in this process remains unclear. Therefore, we investigated the molecular mechanisms and the role of ER stress in hypertension induced cardiac inflammation and cardiac injury using RNA-Seq technology. Aims:To conform the mechanisms of how hypertension caused the cardiac inflammation and cardiac injury at a molecular level. To clarify the role of ER Stress and CHOP in hypertension-induced cardiac injury and investigate the underlying mechanism in this process. Methods:1. Preparation of hypertension model: Angiotensin II(Ang II) induced hypertensive cardiac fibrosis model was made by micro pump(1500ng/kg * min) infusion. The infusion time was 1 day, 3 days or 7 days respectively. Sham groups were infused with saline as control groups. Hypertension model was successful made when blood pressure was greater than 140 / 90 mmHg after Ang II infusion.2. Blood pressure measurement: Systolic blood pressure was measured at the indicated day using a computerized mouse tail cuff system(BP98A, Softron, Tokyo, Japan).3. Cardiac function measurement: Cardiac echocardiography was performed using the Vevo 2100 high resolution micro imaging system(VisualSonic, Toronto, Canada).4. Inflammatory cells detection: Flow cytometry analysis was used to measure the type and number of inflammatory cells infiltrating in saline-infused and Ang II-infused hearts.5. Cardiac fibrosis determination: The deposition of collagen in the cardiac tissue was detected by Masson trichrome staining, Picrosirius Red staining, IHC(α-SMA、TGF-β1) and realtime-PCR(Collagen 1,Collagen 3 and Fibronectin).6. Transcriptome detection and analysis: RNA-Seq, the high-throughput sequencing technology was used to detect the transcriptome of hypertensive hearts after Ang II infusion. A comprehensive bioinformatics analysis was used to enrich the dataset for genes that were most likely to be associated with the development of Ang II-induced acute cardiac injury, including clustering analysis, Gene ontology(GO) analysis, pathway analysis, and dynamic gene network analysis.7. Inflammatory cytokines expression measurement: RNA-Seq and RT-PCR were used to detect the mRNA expression level of inflammation associate genes(S100a8, S100a9, CXCL1, CXCL2, CCL2, CCL9 etc.)8. Bone Marrow transplantation: 8-12 weeks recipient mice were explored to 10 Gy irradiation, then 5x106 donor bone marrow cells were reperfusion within 4-6 hours, mice were kept in the SPF environment 8 weeks for bone marrow reconstitution.9. Isolation of neutrophils: Density gradient centrifugation was used to isolate the bone marrow neutrophils.10. Apoptosis detection: TUNEL staining was used to detect the apoptosis in Ang II-infused hearts.11. Pathway of apoptosis measurement: Western Blot was used to detect apoptosis associated protein including Bcl-2, Bcl-XL.12. Statistical analysis: Data were expressed as the means ± standard error of the mean(SEM). Differences between groups were tested for statistical significance using Student’s t-tests or one-way analysis of variance(ANOVA) followed by Newman-Keuls multiple comparison tests using Graph Pad Prism 5.0. P < 0.05 denoted the statistically significant difference. Results: Part I:1. Ang II-induced hypertension, cardiac inflammation and fibrosis model was successfully made.2. Differentially expressed genes(DEGs) analysis showed 1,801 genes were significantly expressed at day 1, day 3, day 7 after Ang II infusion compared to Sham.3. Gene Ontology(GO) analysis of DEGs showed the high enrichment Go terms were respectively response to endogenous stimulus, response to stress, immune system process, extracellular structure organization and extracellular matrix organization et al. after Ang II infusion.4. KEGG pathway analysis of DEGs showed the significantly overrepresented pathways were respectively cytokine-cytokine receptor interaction, chemokine signaling pathway, ECM-receptor interaction, focal adhesion and TGF-β signaling pathway et al. after Ang II infusion.5. Cluster analysis of DEGs showed the 1801 genes could been classified into 16 profiles based on expression patterns, and each cluster has similar expression patterns after Ang II infusion.6. Protein-protein interaction network analysis of DEGs showed ubiquitin C connected with 118 genes and other genes, including Isg15, Cdk1, Rnf2, Runx1 and Crk interacted with more than 10 genes. Part II:7. RNA-Seq and RT-PCR showed hypertension induced endoplasmic reticulum(ER) stress after Ang II infusion.8. Knockout of CHOP, a key mediator of ER stress, increased expression of cytokines in Ang II- infused hypertensive hearts.9. CHOP deficiency increased the inflammatory cells infiltration in Ang II induced hypertensive heart damage.10. CHOP deficiency increased Ang II-induced cardiac fibrotic injury.11. CHOP deficiency in bone marrow-derived cells was responsible for Ang II induced hypertensive cardiac injury.12. CHOP deficiency decreased the apoptosis of neutrophils in Ang II-infusedhearts.13. CHOP deficiency decreased the apoptosis of neutrophils in vitro. Conclusion Part I:1. Totally 1,801 genes were differently expressed at 1, 3 and 7 days after Ang II infusion.2. GO terms including response to endogenous stimulus, response to stress, immune system process, extracellular structure organization and extracellular matrix organization et al. played critical roles in the pathobiology of hypertension induced cardiac injury.3. KEGG pathways associated with cytokine-cytokine receptor interaction, chemokine signaling pathway, ECM-receptor interaction, focal adhesion and TGF-β signaling pathway et al. played important roles in the pathobiology of hypertension induced cardiac injury.4. The 1801 genes could been classified into 16 profiles based on expression patterns, and these patterns were important for hypertension induced cardiac injury.5. Ubiquitin C, Rnf2, Eed, Npm1 and Myc may play a key role in regulating the processes of hypertension induced cardiac injury. Part II:6. ER stress was activated in Ang II induced hypertensive cardiac injury.7. Deficiency of CHOP, a key mediator of ER stress, increased Ang II-induced cardiac inflammation and injury.8. CHOP deficiency in bone marrow-derived cells was responsible for Ang II-induced hypertensive cardiac injury.9. CHOP deficiency increased Ang II induced cardiac inflammatory response, maybe partly by reducing neutrophil apoptosis and delaying of inflammation resolution.In summary, inflammatory response plays important role in Ang II infusion induced cardiac damage. Several pathways, including acute stress reaction, immune system response, extracellular structure organization and extracellular matrix organization et al. were activated in this process. ER stress was activated in the early stage of Ang II induced hypertensive cardiac injury. And the deficiency of ER stress or CHOP increased Ang II-induced cardiac inflammation and injury. |
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