Serum C-peptide level correlates with the course of muscle tissue healing in the rabbit model of critical limb ischemia.

AIM: The therapeutic potential of adipose-derived stem cell conditioned medium (ASC-CM) was studied in the rabbit model of critical limb ischemia (CLI). METHODS: Rabbits received treatment with ASC-CM or placebo. Gastrocnemius muscle tissue was collected 35 days after ischemia induction. Ischemic changes were evaluated in hematoxylin-eosin stained tissues for early (necrotic lesions/granulation tissue) and late (fibrous scars) phases of tissue repair. The expression of proangiogenic miR-126 was also evaluated using in situ hybridization. The levels of cytokines, insulin, and C-peptide were measured in blood. RESULTS: Early repair phases were observed more often in placebo-treated samples (45.5%) than in ASC-CM-treated ones (22.2%). However, the difference was not statistically significant. We demonstrated a statistically significant positive correlation between the early healing phases in tissue samples and C-peptide levels in peripheral blood. The expression of proangiogenic miR-126 was also shown in a number of structures in all phases of ischemic tissue healing. CONCLUSION: Based on our results, we believe that treatment with ASC-CM has the potential to accelerate the healing process in ischemic tissues in the rabbit model of CLI. The whole healing process was accompanied by miR-126 tissue expression. C-peptide could be used to monitor the course of the tissue healing process.

The Role of miR-126 in Critical Limb Ischemia Treatment Using Adipose-Derived Stem Cell Therapeutic Factor Concentrate and Extracellular Matrix Microparticles.

BACKGROUND Paracrine factors secreted by adipose-derived stem cells can be captured, fractionated, and concentrated to produce therapeutic factor concentrate (TFC). The present study examined whether TFC effects could be enhanced by combining TFC with a biological matrix to provide sustained release of factors in the target region. MATERIAL AND METHODS Unilateral hind limb ischemia was induced in rabbits. Ischemic limbs were injected with either placebo control, TFC, micronized small intestinal submucosa tissue (SIS), or TFC absorbed to SIS. Blood flow in both limbs was assessed with laser Doppler perfusion imaging. Tissues harvested at Day 48 were assessed immunohistochemically for vessel density; in situ hybridization and quantitative real-time PCR were employed to determine miR-126 expression. RESULTS LDP ratios were significantly elevated, compared to placebo control, on day 28 in all treatment groups (p=0.0816, p=0.0543, p=0.0639, for groups 2-4, respectively) and on day 36 in the TFC group (p=0.0866). This effect correlated with capillary density in the SIS and TFC+SIS groups (p=0.0093 and p=0.0054, respectively, compared to placebo). A correlation was observed between miR-126 levels and LDP levels at 48 days in SIS and TFC+SIS groups. CONCLUSIONS A single bolus administration of TFC and SIS had early, transient effects on reperfusion and promotion of ischemia repair. The effects were not additive. We also discovered that TFC modulated miR-126 levels that were expressed in cell types other than endothelial cells. These data suggested that TFC, alone or in combination with SIS, may be a potent therapy for patients with CLI that are at risk of amputation.

Therapeutic Potential of Adipose-Derived Therapeutic Factor Concentrate for Treating Critical Limb Ischemia.

Transplantation of adipose-derived stem cells (ADSCs) is an emerging therapeutic option for addressing intractable diseases such as critical limb ischemia (CLI). Evidence suggests that therapeutic effects of ADSCs are primarily mediated through paracrine mechanisms rather than transdifferentiation. These secreted factors can be captured in conditioned medium (CM) and concentrated to prepare a therapeutic factor concentrate (TFC) composed of a cocktail of beneficial growth factors and cytokines that individually and in combination demonstrate disease-modifying effects. The ability of a TFC to promote reperfusion in a rabbit model of CLI was evaluated. A total of 27 adult female rabbits underwent surgery to induce ischemia in the left hindlimb. An additional five rabbits served as sham controls. One week after surgery, the ischemic limbs received intramuscular injections of either (1) placebo (control medium), (2) a low dose of TFC, or (3) a high dose of TFC. Limb perfusion was serially assessed with a Doppler probe. Blood samples were analyzed for growth factors and cytokines. Tissue was harvested postmortem on day 35 and assessed for capillary density by immunohistochemistry. At 1 month after treatment, tissue perfusion in ischemic limbs treated with a high dose of TFC was almost double (p < 0.05) that of the placebo group [58.8 ± 23 relative perfusion units (RPU) vs. 30.7 ± 13.6 RPU; mean ± SD]. This effect was correlated with greater capillary density in the affected tissues and with transiently higher serum levels of the angiogenic and prosurvival factors vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). The conclusions from this study are that a single bolus administration of TFC demonstrated robust effects for promoting tissue reperfusion in a rabbit model of CLI and that a possible mechanism of revascularization was promotion of angiogenesis by TFC. Results of this study demonstrate that TFC represents a potent therapeutic cocktail for patients with CLI, many of whom are at risk for amputation of the affected limb.