Circulating Total Extracellular Vesicles Cargo Are Associated with Age-Related Oxidative Stress and Susceptibility to Cardiovascular Diseases: Exploratory Results from Microarray Data

Abstract

Aging is a risk factor for many non-communicable diseases such as cardiovascular and neurodegenerative diseases. Extracellular vesicles and particles (EVP) carry microRNAs that may play a role in age-related diseases and may induce oxidative stress. We hypothesized that aging could impact EVP miRNA and impair redox homeostasis, contributing to chronic age-related diseases. Our aims were to investigate the microRNA profiles of circulating total EVPs from aged and young adult animals and to evaluate the pro- and antioxidant machinery in circulating total EVPs. Plasma from 3- and 21-month-old male Wistar rats were collected, and total EVPs were isolated. MicroRNA isolation and microarray expression analysis were performed on EVPs to determine the predicted regulation of targeted mRNAs. Thirty-one mature microRNAs in circulating EVPs were impacted by age and were predicted to target molecules in canonical pathways directly related to cardiovascular diseases and oxidative status. Circulating total EVPs from aged rats had significantly higher NADPH oxidase levels and myeloperoxidase activity, whereas catalase activity was significantly reduced in EVPs from aged animals. Our data shows that circulating total EVP cargo-specifically microRNAs and oxidative enzymes-are involved in redox imbalance in the aging process and can potentially drive cardiovascular aging and, consequently, cardiac disease.

Keywords: NADPH oxidase; aging; cardiac aging; extracellular vesicles and particles; redox homeostasis.

Figure 1

Differentially expressed mature miRNA. (A). The 31 mature miRNAs with targets, based on current available IPA target annotations. The miRNA fold-change between aged and young adult rats is summarized. (B) Hierarchical clustering of the expression of differently expressed miRNAs from aged and young adult rats. The color bar indicates the standardized expression (z-score) of each miRNA to a mean of 0. Upregulated microRNAs have positive values and are displayed in red. Downregulated microRNAs have negative values and are displayed in blue.

Figure 2

Cardiovascular signaling canonical pathways. The top cardiovascular signaling canonical pathways with predicted targets for differentially expressed total EVP microRNAs between aged and young animals were identified by Ingenuity pathway analysis (IPA). Canonical pathways are displayed along the y-axis and the −log (p-value), calculated by Fisher’s exact test right-tailed, on the x-axis. The threshold line indicates the minimum significance level −log (p-value). The length of the bars represents significance, with longer bars representing more significant associations, and the orange color indicates that those pathways are predicted to be upregulated (z-score >0), while blue indicates downregulation (z-score < 0).

Figure 3

The Cardiac Hypertrophy Signaling pathway is predicted to be upregulated in EVPs from aged animals. Predicted up- and downregulation of mRNA targets in the cardiac hypertrophy signaling pathway. The red color indicates predicted upregulation while the green indicates predicted downregulation. The complete list of pathway targets and their predicted fold-change can be found in Supplementary Table S3.

Figure 4

Production of Nitric Oxide and Reactive Oxygen Species in Macrophages signaling pathways is predicted to be upregulated in EVPs from aged animals. Predicted up- and downregulation of mRNA targets in the cardiac hypertrophy signaling pathway. The red color indicates predicted upregulation while the green indicates predicted downregulation. The complete list of pathway targets and their predicted fold-change can be found in Supplementary Table S5.

Figure 5

Effect of the aging process on pro- and antioxidant markers in circulating extracellular vesicles and particles (EVPs) from Wistar rats, of 3- and 21-month-old. Columns represent the mean ± SD; representative images from a gel of Western Blot experiments, showing 3 bands for each experimental group. Student’s t-test; * significantly different from young adults, p < 0.05. (A) NADPH oxidase content (n = 6; p = 0.043); (B) MPO activity (n = 9; p = 0.008); (C) xanthine oxidase content (n = 6; p = 0.040); and (D) xanthine oxidase activity (n = 9; p = 0.024); (E) catalase content (n = 6; p = 0.34) and (F) catalase activity (n = 9; 0.0002).