**1. Introduction**

Aging is a highly complex biological process that is often accompanied by a general decline in tissue function and an increased risk for aging-related diseases, such as cardiovascular disease, stroke, cancer, and neurodegenerative diseases. Indeed, as average lifespan continues to increase, aging-related functional decline, such as cognitive impairment, will likely become a health care priority [1]. For example, the most common form of dementia is Alzheimer's disease (AD), but a large proportion of cognitive impairment cases in the aged population is not due to AD but rather to normal aging process. Thus, it is important to identify ways to maintain functional integrity during aging [2]. Many theories have been proposed to explain why we age [3]. Recently, we proposed a new theory positing that aging is the process of continuous impairment of microcirculation in the body [4]. Indeed, compelling evidence indicates that systemic factors in the blood profoundly

reverse aging-related impairments [5–7], which are influenced by specific rejuvenating or deteriorating factors, e.g., proteins, microRNAs (miRNAs), and mRNAs [8]. Thus, many circulating factors have been identified as attractive biomarkers for tissue-specific diseases and aging [9,10]. However, the mechanisms underlying the contributions of blood-derived factors to aging remain unclear.

Research over the last two decades has demonstrated that cells mainly communicate by releasing extracellular vesicles (EV) that can act on nearby cells (paracrine signaling) or end up in circulating body fluids, with possible effects at distant sites (endocrine signaling) [11]. Exosomes are small EVs (approximately 50–150 nm in diameter) of endosomal origin that initially form as intraluminal vesicles inside late endosomal compartments. Indeed, exosomes contain many specific proteins, mRNAs, miRNAs, and long noncoding RNAs [12] and play a vital role in cell communication by transferring their cargo between source and target cells, which is also important in aging and aging-related disease [13]. For example, injection of serum exosomes from young mice into old mice could alter the expression pattern of aging-associated molecules to mimic that of young mice [14]. In addition, studies have documented that exosomes from brain cells can cross the blood-brain barrier (BBB) and serve as peripheral circulating biomarkers of cognitive impairment in AD [15–17], and, blood exosomes can also cross the BBB to target brain cells and affect brain function [18–21]. Thus, peripheral circulating exosomes have diagnostic and therapeutic potential. However, most studies have focused on establishing exosomal protein or miRNA profiles for comparing disease states and matched controls, and few studies have focused on characterizing proteins and miRNAs in peripheral circulating exosomes during normal aging [22]. Therefore, it is critical to define the profiles for exosomal proteins and miRNAs that can be transferred from exosome to recipient cells. Importantly, it has been estimated that miRNAs regulate ~31% of all eukaryotic genes by promoting degradation of their mRNAs or inhibiting their translation [23,24]. Indeed, miRNA-mediated regulation governs metabolism, immunity, lifespan, cell proliferation, apoptosis, and development [25–27], as well as pathological processes such as cancer and cardiovascular and neurodegenerative disease [28–30]. Therefore, among the exosomal cargo that is transferred to recipient cells, miRNAs likely have the greatest downstream impact on cell functions. To explore the role of circulating exosomes in aging processes, exosomal miRNAs must be more broadly characterized. In addition, recent evidence suggests that numerous signaling pathways regulate normal aging processes. However, research is lacking concerning how aging affects co-expression profiles for exosomal miRNAs and mRNAs and how miRNA-mRNA regulatory networks systematically influence aging processes.

To address shortcomings in our knowledge of exosomal miRNA functions, we used next-generation sequencing to establish miRNA and mRNA profiles for circulating exosomes isolated from young and old rats. We also investigated the possible role of exosomal miRNAs in aging by analyzing the biological importance of the miRNA targets and in major signaling pathways associated with aging using bioinformatic tools including Gene Ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and pathways, eukaryotic orthologous groups (KOG) function classification, and Ingenuity Pathway Analysis (IPA). Our findings may provide a basis for understanding the physiological consequences of aging-related changes in the makeup of circulating miRNAs and could lead to potential interventions for aging-related diseases.

#### **2. Results**

#### *2.1. Characterization of Serum Exosomes*

We first characterized the protein content of serum exosomes isolated from young and old rats using Western blotting. Serum exosomes from each of young and old rats were positive for the exosome markers, CD63 and CD9 (Figure 1A). Nanoparticle tracking analysis (NTA) (Figure 1B) verified a strong enrichment of particles in the range 40–120 nm, with mean size of 82 +/− 0.8 nm, supporting a multimodal size distribution of exosomes with a peak diameter of 70–120 nm, consistent with previous reports [31,32]. In addition, transmission electron microscopy (TEM) was used to confirm that the purified particles were membrane bound, round and heterogeneous in size (40–120 nm) (Figure 1C).

**Figure 1.** Characterization of serum exosomes. (**A**) Western blotting for CD63 and CD9 in serum exosomes isolated from young and old rats. (**B**) Average overall size distribution of exosomes from serum of old rats using the Nanoparticle Tracking Analysis. (**C**) Representative transmission electron microscopy image showing the typical morphology and size range of exosomes from serum of old rat. Y, serum exosomes from young rats; O, serum exosomes from old rats.
