Circulatory Rejuvenated EPCs Derived from PAOD Patients Treated by CD34+ Cells and Hyperbaric Oxygen Therapy Salvaged the Nude Mouse Limb against Critical Ischemia.

Kuan-Hung Chen,Hon-Kan Yip,Pei-Lin Shao,Shun-Cheng Wu,Jiunn-Jye Sheu,Yi-Ling Chen,John Y Chiang,Pei-Hsun Sung,Yi-Chen Li,Yin-Chia Chen,Tien-Hung Huang

doi:10.3390/ijms21217887

Abstract

This study tested whether circulatory endothelial progenitor cells (EPCs) derived from peripheral arterial occlusive disease (PAOD) patients after receiving combined autologous CD34+ cell and hyperbaric oxygen (HBO) therapy (defined as rejuvenated EPCs) would salvage nude mouse limbs against critical limb ischemia (CLI). Adult-male nude mice (n = 40) were equally categorized into group 1 (sham-operated control), group 2 (CLI), group 3 (CLI-EPCs (6 × 105) derived from PAOD patient’s circulatory blood prior to CD34+ cell and HBO treatment (EPCPr-T) by intramuscular injection at 3 h after CLI induction) and group 4 (CLI-EPCs (6 × 105) derived from PAOD patient’s circulatory blood after CD34+ cell and HBO treatment (EPCAf-T) by the identical injection method). By 2, 7 and 14 days after the CLI procedure, the ischemic to normal blood flow (INBF) ratio was highest in group 1, lowest in group 2 and significantly lower in group 4 than in group 3 (p < 0.0001). The protein levels of endothelial functional integrity (CD31/von Willebrand factor (vWF)/endothelial nitric-oxide synthase (eNOS)) expressed a similar pattern to that of INBF. In contrast, apoptotic/mitochondrial-damaged (mitochondrial-Bax/caspase-3/PARP/cytosolic-cytochrome-C) biomarkers and fibrosis (Smad3/TGF-ß) exhibited an opposite pattern, whereas the protein expressions of anti-fibrosis (Smad1/5 and BMP-2) and mitochondrial integrity (mitochondrial-cytochrome-C) showed an identical pattern of INBF (all p < 0.0001). The protein expressions of angiogenesis biomarkers (VEGF/SDF-1α/HIF-1α) were progressively increased from groups 1 to 3 (all p < 0.0010). The number of small vessels and endothelial cell surface markers (CD31+/vWF+) in the CLI area displayed an identical pattern of INBF (all p < 0.0001). CLI automatic amputation was higher in group 2 than in other groups (all p < 0.001). In conclusion, EPCs from HBO-C34+ cell therapy significantly restored the blood flow and salvaged the CLI in nude mice.

Highlights

Peripheral arterial occlusive disease (PAOD), a high prevalence aging-associated chronic disease, incurs huge public healthcare costs and causes unacceptably high morbidity and mortality worldwide
Ischemic/Normal Blood Flow (INBF) Ratio Measured by Laser Doppler Scan at Days 2, 7 and 14 after Left Femoral Artery Ligated and Totally Removed and Automatic Amputation of Distal Critical Ischemic Limb
To elucidate whether intramuscular administration of endothelial progenitor cells (EPCs) which were derived from PAOD patients prior to and after receiving combined therapy of Hyperbaric oxygen (HBO) and CD34+ cells would salvage the critical limb ischemia (CLI) animals, a laser Doppler scan was utilized for determining the INBF ratio in each group of animals

Summary

Introduction

Peripheral arterial occlusive disease (PAOD), a high prevalence aging-associated chronic disease, incurs huge public healthcare costs and causes unacceptably high morbidity and mortality worldwide. Patients with PAOD may develop critical limb ischemia (CLI) at the late stage of the disease process [2,3]. The CLI commonly occurs when arterial blood flow is greatly restricted, resulting in perfusion in capillary beds and inadequately sustained microvasculature. Hypoxia and exhausted energy develop in the tissues and cells in the ischemic area [4,5]. Clinical studies have delineated that thousands of patients are asymptomatic prior to the development of CLI [6,7], which poses an obstacle to early diagnosis and early treatment for the purposes of slowing or abolishing disease progression and the development of unacceptable complications

Methods

Results

Conclusion