Exosomes from adipose-derived stem cells promote angiogenesis and reduce necrotic grade in hindlimb ischemia mouse models

Abstract

Objectives: Acute hindlimb ischemia is a peripheral arterial disease that severely affects the patient's health. Injection of stem cells-derived exosomes that promote angiogenesis is a promising therapeutic strategy to increase perfusion and repair ischemic tissues. This study aimed to evaluate the efficacy of adipose stem cell-derived exosomes injection (ADSC-Exos) in treating acute mouse hindlimb ischemia.

Materials and methods: ADSC-Exos were collected via ultracentrifugation. Exosome-specific markers were analyzed via flow cytometry. The morphology of exosomes was detected by TEM. A dose of 100 ug exosomes/100 ul PBS was locally injected into acute mice ischemic hindlimb. The treatment efficacy was evaluated based on the oxygen saturation level, limb function, new blood vessel formation, muscle structure recovery, and limb necrosis grade.

Results: ADSC-exosomes expressed high positivity for markers CD9 (76.0%), CD63 (91.2%), and CD81 (99.6%), and have a cup shape. After being injected into the muscle, in the treatment group, many small and short blood vessels formed around the first ligation and grew down toward the second ligation. The SpO2 level, reperfusion, and recovery of the limb function are more positively improved in the treatment group. On day 28, the muscle's histological structure in the treatment group is similar to normal tissue. Approximately 33.33% of the mice had grade I and II lesions and there were no grade III and IV observed in the treatment group. Meanwhile, in the placebo group, 60% had grade I to IV lesions.

Conclusion: ADSC-Exos showed the ability to stimulate angiogenesis and significantly reduce the rate of limb necrosis.

Keywords: Acute; Adipose-derived stem cells; Angiogenesis; Exosome; Extracellular vesicles; Limb ischemia.

Figure 1

Human adipose-derived stem cells expressed the MSC phenotype proposed by ISCT

Figure 2

Flow cytometry characteristics of isolated exosomes from human adipose-derived stem cells. They expressed markers of CD63 (A), CD81 (B), and CD9 (C), and have a morphology like disk or cup (D)

Figure 3

Recovery of the peripheral capillary SpO2 level after ADSC-Exos injection

Figure 4

Recovery of the blood flow to the feet

Figure 5

Recovery of the limb function. The mobility of mice climbs sharply decreased immediately after induced ischemia in both groups. The treatment group completely recovered after 14 days, while the untreated group took up to 21 days

Figure 6

Formation of new blood vessels. Vascular structure in normal mice (A-C). After 7 days, in comparison to normal mice, the blood vessel under second ligation developed in both the placebo group (D-F) and the treatment group (G-H). After 28 days, the development of new blood vessels connected between the two ligation sites in both the untreated group (K-M) and treated mice (N-M) was stronger. However, the vascular development under second ligation and posterior thigh in treated mice were also stronger than in untreated mice groups. The arrows point to new blood vessels

Figure 7

Histological muscle tissue structure at 7 days and 28 days after injection

Figure 8

Ratio of hindlimb necrosis caused by acute ischemia in both groups. The degree of limb necrosis was assessed and categorized according to the recommendations of Goto et al. [28] (A). The damage process of untreated mice lasted longer than treated mice (B). The data revealed that injection of AD-EXOs dramatically reduced hindlimb necrosis compared with a placebo group (C)