Korean J Physiol Pharmacol.  2016 Nov;20(6):657-667. 10.4196/kjpp.2016.20.6.657.

Increasing injection frequency enhances the survival of injected bone marrow derived mesenchymal stem cells in a critical limb ischemia animal model

• Kang WC
• Oh PC
• Lee K
• Ahn T
• Byun K

• KMID: 2364270
• DOI: http://doi.org/10.4196/kjpp.2016.20.6.657

Abstract

Critical limb ischemia (CLI) is one of the most severe forms of peripheral artery diseases, but current treatment strategies do not guarantee complete recovery of vascular blood flow or reduce the risk of mortality. Recently, human bone marrow derived mesenchymal stem cells (MSCs) have been reported to have a paracrine influence on angiogenesis in several ischemic diseases. However, little evidence is available regarding optimal cell doses and injection frequencies. Thus, the authors undertook this study to investigate the effects of cell dose and injection frequency on cell survival and paracrine effects. MSCs were injected at 10⁶ or 10⁵ per injection (high and low doses) either once (single injection) or once in two consecutive weeks (double injection) into ischemic legs. Mice were sacrificed 4 weeks after first injection. Angiogenic effects were confirmed in vitro and in vivo, and M2 macrophage infiltration into ischemic tissues and rates of limb salvage were documented. MSCs were found to induce angiogenesis through a paracrine effect in vitro, and were found to survive in ischemic muscle for up to 4 weeks dependent on cell dose and injection frequency. In addition, double high dose and low dose of MSC injections increased vessel formation, and decreased fibrosis volumes and apoptotic cell numbers, whereas a single high dose did not. Our results showed MSCs protect against ischemic injury in a paracrine manner, and suggest that increasing injection frequency is more important than MSC dosage for the treatment CLI.

Keyword

Bone marrow derived mesenchymal stem cells; Critical limb ischemia; High frequency injection; High MSCs dose injection

MeSH Terms

Animals*
Bone Marrow*
Cell Count
Cell Survival
Extremities*
Fibrosis
Humans
In Vitro Techniques
Ischemia*
Leg
Limb Salvage
Macrophages
Mesenchymal Stromal Cells*
Mice
Models, Animal*
Mortality
Peripheral Arterial Disease

Figure

• 

  Fig. 1 Increased MSCs survival after double injections of MSCs in the limb ischemia animal model.(A) Double stained confocal microscopic images showing that distribution of CD44 positive MSCs (green) in ischemic muscle at 4 weeks after first injections depended on injection frequency and MSCs dose. Below graph came from representative pictures (above) and arrows indicated existed MSCs in the ischemic muscles. (B) CD29 and CD44 mRNA levels were assessed by qRT-PCR in the ischemic muscles of mice treated single or double with high or low dose of MSCs injection. Scale bar=100 µm, original magnification=200×, None; no signal detectable, *Different (p<0.05) versus a single injection of low dose, §Different (p<0.05) versus double low dose of MSCs injections, †Different (p<0.05) versus double high dose of MSCs injections.

• 

  Fig. 2 Paracrine induction of angiogenesis by MSCs secretions.HUVECs were treated with MSCs conditioned medium (MSC-CM) of PBS (control) to determine the ability of MSCs of promote angiogenesis, (A) HUVECs migration, (B) tube-like structure formation, and (C) HUVECs proliferation. Scale bar=1000 pixels, original magnification=40× *p<0.05 vs. PBS treated HUVECs (D) Levels of ANG-1, HGF, and VEGF in MSC-CM were measured by ELISA. The ELISA plot shows fold levels of proteins in MSC-CM versus basal medium (Basal-M). (E) Immunoblotting results showing that the expressions of pAKT and MAPK pathway-related molecules in Basal-M and MSC-CM treated HUVECs in vitro. *p<0.05 vs. Basal-M treated HUVECs (F) Confocal microscopic images showing the effects of MSCs dose and injection frequency on vWF (green) positive blood vessels and nuclei (DAPI; blue) in ischemic muscle. Vessel sizes were calculated by Zen 2009 software and described in graph. Scale bar=100 µm, original magnification=200 ×, *Different (p<0.05) versus a single injection of low dose, §Different (p<0.05) versus double low dose of MSCs injections, †Different (p<0.05) versus double high dose of MSCs injection.

• 

  Fig. 3 High dose of MSCs injection enhanced M2 macrophage polarization in the limb ischemia animal model.(A) Triple stained microscopic images showing total activated macrophages (Iba1), M2 macrophage marker (Arg 1) and nuclei (DAPI; blue). (B) Graphs were derived from representative microscopic images. The graph in the left panel shows numbers of activated macrophages (Iba1) and the right panel shows the ratio of Arg1 positive cells to Iba1 cells. Scale bar=100 µm, original magnification=200×, *Different (p<0.05) versus a single injection of low dose, §Different (p<0.05) versus double low dose of MSCs injections, †Different (p<0.05) versus double high dose of MSCs injection.

• 

  Fig. 4 Protective effects of double injections of MSCs in the limb ischemia animal model.(A) Fibrotic fibers (blue) in ischemic muscle were confirmed by Masson's trichrome staining and (B) apoptotic cells (arrows) were TUNEL stained. Scale bar=100 µm (C) Skin color immediately changed after ferric chloride had been applied to femoral arteries followed by MSCs injection visibly ameliorated limb ischemia. Limb salvage, necrosis and amputation ratios were described by white, gray and black color, respectively. *Different (p<0.05) versus a single injection of low dose, §Different (p<0.05) versus double low dose of MSCs injections, †Different (p<0.05) versus double high dose of MSCs injection.

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