The Influence of Circulating Exosomes Derived From Younger and Older Donors on Hypoxia-Inducible Factor 1 Alpha Gene Expression and P21 Protein in Cord Blood Hematopoietic Stem Cells

Abstract

Background: Exosomes are a group of extracellular vesicles that are influential in intercellular signaling and can affect aging. Hypoxia-inducible factor 1α (HIF-1α) is the principal mediator in response to hypoxia and can regulate aging. Moreover, P21 is a part of the downstream signaling pathway of hypoxia and is elevated during aging. Therefore, this research was conducted to investigate the effect of plasma exosomes of younger and older individuals on the expression of HIF-1α gene and P21 protein in hematopoietic stem cells (HSCs).

Methods: Plasma exosomes were derived from older and younger men and were characterized. Then, HSCs were isolated from cord blood samples and treated with exosomes of older and younger men. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to evaluate cell viability. Next, the expression of HIF-1α gene and P21 protein were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively.

Results: HIF-1α gene expression was considerably increased in HSCs treated with 10 µg/mL of exosomes isolated from younger men (Y10-Exo) compared to the untreated group (P = 0.002). Moreover, HIF-1α gene expression was remarkably decreased in HSCs treated with 10 µg/mL of exosomes obtained from older men (O10-Exo) in comparison with the untreated group (P < 0.001). Additionally, the expression of P21 protein was significantly increased in HSCs treated with 5 µg/mL of exosomes derived from older individuals (O5-Exo) and O10-Exo compared to the untreated group (P = 0.000 and P = 0.002, respectively).

Conclusions: Our findings showed that exosomes isolated from younger participants cause elevation in HIF-1α and may lead to delayed aging in HSCs. In addition, exosomes isolated from older participants can probably lead to aging through the reduction in HIF-1α and elevation in P21.

Keywords: Aging; Exosomes; HIF-1α; Hematopoietic stem cells; Older individuals; P21.

Figure 1

Determining the characteristics of exosomes. Isolated exosomes were evaluated in size, zeta potential, morphology, and CD63 protein expression. (a) Determination of the characteristics of exosomes by DLS. (b) Evaluation of the size and morphology of exosomes by TEM. (c) Confirmation of the expression of the specific CD63 marker by Western blot. DLS: dynamic light scattering; TEM: transmission electron microscopy.

Figure 2

(a) MTT colorimetric assay for measuring the viability of hematopoietic stem cells (HSCs) after treatment with 5 µg/mL and 10 µg/mL concentration of exosomes isolated from young and old individuals. (b) The HIF-1α mRNA expression in the HSCs treated with the doses of 5 and 10 µg/mL Older-Exo (O5-Exo, O10-Exo) and Younger-Exo (Y5-Exo, Y10-Exo). O5-Exo: 5 µg/mL concentration of exosomes isolated from older individuals; O10-Exo: 10 µg/mL concentration of exosomes isolated from older individuals; Y5-Exo: 5 µg/mL concentration of exosomes isolated from younger individuals; Y10-Exo: 10 µg/mL concentration of exosomes isolated from younger individuals; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; HIF-1α: hypoxia-inducible factor 1α.

Figure 3

(a) The relative P21 protein level, based on P21/GAPDH ratio, in hematopoietic stem cells (HSCs) treated with the doses of 5 and 10 µg/mL Older-Exo (O5-Exo, O10-Exo) and Younger-Exo (Y5-Exo, Y10-Exo). (b) The levels of P21 protein and GAPDH evaluated by the Western blotting test. O5-Exo: 5 µg/mL concentration of exosomes isolated from older individuals; O10-Exo: 10 µg/mL concentration of exosomes isolated from older individuals; Y5-Exo: 5 µg/mL concentration of exosomes isolated from younger individuals; Y10-Exo: 10 µg/mL concentration of exosomes isolated from younger individuals; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.