*4.7. Gene Ontology (GO) and Pathway Enrichment Analysis*

The biological function of each protein was annotated with Blastp (Blast2GO version 5, BioBam Bioinformatics, Spain) using the entire gene-expression database, and subsequent mapping was carried out with the GO database (www.geneontology.org/, Gene ontology resource, USA). To further understand the biological significance of differentially expressed exosome proteins, pathway analysis was carried out with the KEGG Orthology-Based Annotation System (http://kobas.cbi.pku.edu.cn, China). The association of proteins with different pathways was computed using the KEGG database (www.genome.jp/kegg, Japan). EuKaryotic Orthologous Groups (KOG) Analysis was based on the phylogenetic classification of proteins encoded in the complete genomes (www.ncbi.nlm.nih. gov/COG/, NCBI, MD, USA) project. IPA (QIAGEN, Germany) was used for additional functional annotation, including top canonical pathway, top disease and function and molecular and cellular functions, prediction of upstream, regulator effectors, and miRNA–mRNA relationship and interaction network analysis.

## *4.8. Statistical Analysis*

For the GO analysis, statistically significant alterations in functions of differentially expressed exosome proteins were assessed with Fisher's exact test in Blast2GO with an adjusted *p*-value (false discovery rate, FDR) of <0.05 and fold change > 1.5. The statistical significance of changes in pathways identified with IPA was assessed with the right-tailed Fisher's Exact test. A *p*-value of < 0.05 implies that the relationship of a set of targeted molecules and a process/pathway/transcription was randomly matched. A *Z* score of ≥ 2 or ≤ −2 indicated significant activation or significant inhibition, respectively. For all analysis, the difference was considered significant for *p* < 0.05.

**SupplementaryMaterials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/6/1908/s1.

**Author Contributions:** Formal analysis, H.Z.; Investigation, K.J.; Methodology, H.Z. Project administration, K.J.; Writing—original draft, H.Z.; Writing—review and editing, K.J. All authors have read and agreed to the published version of the manuscript.

**Funding:** American Heart Association: 18PRE34020126; National Institutes of Health: 5R21-NS094859-02.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


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International Journal of *Molecular Sciences*
