Low-molecular weight heparin protamine complex augmented the potential of adipose-derived stromal cells to ameliorate limb ischemia.
Heparin/protamine micro/nanoparticles (LH/P-MPs) were recently developed as low-molecular weight, biodegradable carriers for adipose-derived stromal cells (ADSCs). These particles can be used for a locally delivered stem cell therapy that promotes angiogenesis. LH/P-MPs bind to the cell surface of ADSCs and promote cell-to-cell interaction and aggregation of ADSCs. Cultured ADSC/LH/P-MP aggregates remain viable. Here, we examined the ability of these aggregates to rescue limb loss in a mouse model of hindlimb ischemia.



Unilateral hindlimb ischemia was induced in adult male BALB/c mice by ligation of the iliac artery and hindlimb vein. For allotransplantation of ADSCs from the same inbred strain, we injected ADSC alone or ADSC/LH/P-MP aggregates or control medium (sham-treated) directly into the ischemic muscles. Ischemic limb blood perfusion, vessel density, and vessel area were recorded. The extent of ischemic limb necrosis or limb loss was assessed on postoperative days 2, 7, and 14.



Compared with the sham-treatment control, treatment with ADSCs alone showed modest effects on blood perfusion recovery and increased the number of α-SMA-positive vessels. Response to ADSC/LH/P-MP aggregates was significantly greater than ADSCs alone for every endpoint. ADSC/LH/P-MP aggregates more effectively prevented the loss of ischemic hindlimbs than ADSCs alone or the sham-treatment.



The LH/P-MPs augmented the effects of ADSCs on angiogenesis and reversal of limb ischemia. Use of ADSC/LH/P-MP aggregates offers a novel and convenient treatment method and potentially represents a promising new therapeutic approach to inducing angiogenesis in ischemic diseases.
Research Support Center, Dokkyo Medical University, Mibu, Tochigi, Japan; Center for Regenerative Medicine, Dokkyo Medical University, Mibu, Tochigi, Japan. Electronic address: skishi@dokkyomed.ac.jp.
Satoko
S
Kishimoto
Research Support Center, Dokkyo Medical University, Mibu, Tochigi, Japan; Center for Regenerative Medicine, Dokkyo Medical University, Mibu, Tochigi, Japan.
Ken-Ichi
K
Inoue
Division of Biomedical Engineering, Research Institute, National Defense Medical College, Saitama, Japan.
Shingo
S
Nakamura
Division of Biomedical Engineering, Research Institute, National Defense Medical College, Saitama, Japan.
Hidemi
H
Hattori
Division of Biomedical Engineering, Research Institute, National Defense Medical College, Saitama, Japan.
Masayuki
M
Ishihara
Center for Regenerative Medicine, Dokkyo Medical University, Mibu, Tochigi, Japan; Department of Cardiovascular Medicine, Dokkyo Medical University, Mibu, Tochigi, Japan.
Masashi
M
Sakuma
Center for Regenerative Medicine, Dokkyo Medical University, Mibu, Tochigi, Japan; Department of Cardiovascular Medicine, Dokkyo Medical University, Mibu, Tochigi, Japan.
Shigeru
S
Toyoda
Center for Regenerative Medicine, Dokkyo Medical University, Mibu, Tochigi, Japan; Department of Cardiovascular Medicine, Dokkyo Medical University, Mibu, Tochigi, Japan.
Hideki
H
Iwaguro
Center for Regenerative Medicine, Dokkyo Medical University, Mibu, Tochigi, Japan; Department of Cardiology, Koshigaya Hospital, Dokkyo Medical University, Koshigaya, Saitama, Japan.
Isao
I
Taguchi
Research Support Center, Dokkyo Medical University, Mibu, Tochigi, Japan; Center for Regenerative Medicine, Dokkyo Medical University, Mibu, Tochigi, Japan; Department of Cardiovascular Medicine, Dokkyo Medical University, Mibu, Tochigi, Japan.
Teruo
T
Inoue
Center for Regenerative Medicine, Dokkyo Medical University, Mibu, Tochigi, Japan.
Ken-Ichiro
K
Yoshida
D017667:Adipocytes / Q000166:cytology*; D000273:Adipose Tissue / Q000166:cytology; D000327:Adsorption; D000818:Animals; D002450:Cell Communication; D002470:Cell Survival; D006495:Heparin, Low-Molecular-Weight / Q000627:therapeutic use*; D006614:Hindlimb / Q000473:pathology; D007511:Ischemia / Q000473:pathology / Q000628:therapy*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D018919:Neovascularization, Physiologic / Q000187:drug effects; D011479:Protamines / Q000627:therapeutic use*; D033581:Stem Cell Transplantation; D017154:Stromal Cells / Q000166:cytology*
D006495:Heparin, Low-Molecular-Weight; D011479:Protamines
Adipose-derived stromal cells; Angiogenesis therapy; Limb ischemia; Low-molecular-weight heparin/protamine micro/nanoparticles; Neovascularization