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

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

doi:10.3390/ijms21217887

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

Int J Mol Sci. 2020 Oct 23;21(21):7887. doi: 10.3390/ijms21217887.

Authors

Yin-Chia Chen¹, Jiunn-Jye Sheu^{1

2

3}, John Y Chiang^{4

5}, Pei-Lin Shao⁶, Shun-Cheng Wu^{7

8

9}, Pei-Hsun Sung^{2

3

10}, Yi-Chen Li¹⁰, Yi-Ling Chen^{2

3

10}, Tien-Hung Huang^{2

3

6

10}, Kuan-Hung Chen¹¹, Hon-Kan Yip^{2

3

6

10

12

13}

Affiliations

¹ Division of Thoracic and Cardiovascular Surgery, Department of Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung 83301, Taiwan.
² Institute for Translational Research in Biomedicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung 83301, Taiwan.
³ Center for Shockwave Medicine and Tissue Engineering, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung 83301, Taiwan.
⁴ Department of Computer Science and Engineering, National Sun Yat-Sen University, Kaohsiung 80424, Taiwan.
⁵ Department of Healthcare Administration and Medical Informatics, Kaohsiung Medical University, Kaohsiung 80756, Taiwan.
⁶ Department of Nursing, Asia University, Taichung 41354, Taiwan.
⁷ Regenerative Medicine and Cell Therapy Research Center, Kaohsiung Medical University, Kaohsiung 80756, Taiwan.
⁸ Orthopaedic Research Center, Kaohsiung Medical University, Kaohsiung 80756, Taiwan.
⁹ Post-Baccalaureate Program in Nursing, Asia University, Taichung 41354, Taiwan.
¹⁰ Department of Cardiology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung 83301, Taiwan.
¹¹ Department of Anesthesiology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung 83301, Taiwan.
¹² Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 40402, Taiwan.
¹³ Division of Cardiology, Department of Internal Medicine, Xiamen Chang Gung Hospital, Xiamen 361028, China.

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 (EPC^Pr-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 (EPC^Af-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.

Keywords: angiogenesis; critical limb ischemia; endothelial progenitor cells; hyperbaric oxygen therapy; nude mice.

MeSH terms

Animals
Antigens, CD34 / metabolism*
Arterial Occlusive Diseases / blood
Arterial Occlusive Diseases / therapy*
Disease Models, Animal
Endothelial Progenitor Cells / transplantation*
Hindlimb / blood supply
Humans
Hyperbaric Oxygenation / methods*
Injections, Intramuscular
Ischemia / therapy*
Male
Mice
Mice, Nude
Neovascularization, Physiologic
Peripheral Arterial Disease / blood
Peripheral Arterial Disease / therapy*
Regional Blood Flow
Stem Cell Transplantation
Transplantation, Autologous
Treatment Outcome

Substances

Antigens, CD34

Grants and funding

MOST 106-2314-B-182A-108 -MY3/National Science Council