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Abstract
Critical limb ischemia (CLI) is the most severe peripheral artery disease and caused by thrombus formation in blood vessel. The current strategies for treating CLI does not protect limb amputation and reduction in the risk of mortality. Recently, human bone marrow derived mesenchymal stem cells (BD-MSC) were reported to have a paracrine effects on angiogenesis in several ischemic diseases. So, we validate to determine whether BD-MSC protect against ferric chloride treated CLI and induce angiogenesis.
To characterized human bone marrow derived stem cell, BD-MSC differentiated to osteocytes and adipocytes and validated stemness using flow cytometry. Endothelial cell induced angiogenesis followed by mesenchymal stem cell cultured medium treatment in HUVEC in vitro. We also mimicked CLI patients condition using FeCl3 treated CLI mouse and injected one hundred thousand of BD-MSC along the femoral artery to leg muscle. We validated stem cell survival, blood vessel formation, leg muscle condition and fibrosis compared by saline injected mice 28 days later.
In this study, BD-MSC cultured medium treatment increased migration and tube formation of HUVEC and BD-MSC injection had an effective blood vessel formation in FeCl3 treated CLI. As well as blood vessel formation, limb salvage rate also improved and fibrosis area statistically decreased in BD-MSC injected mice.
In conclusion, bone marrow derived mesenchymal stem cell improved not only blood vessel formation but also reduction of fibrosis in FeCl3 treated CLI mice and finally protected limb amputation.
References
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Fig. 1.
Characterization of Bone marrow derived mesenchymal stem cell and validation of angiogenesis in vitro. A. Flow cytometry analysis showed BD-MSC of negative stem cell markers such as CD45 and CD14 expression. B. BD-MSC of left panel differentiated to os-teocytes (right, top) and adipocytes (right, bottom) respectively and the differentiated cells stained with alkaline phosphatase and Oil red O respectively. Magnification = × 40. C. HUVEC showed tube formation in growth factor reduced Matrigel assay after PBS treated (PBS) or BD-MSC incubated medium (BD-MSC). Magnification = × 40. D. HUVEC showed wound migration assay after PBS treated (PBS) or BD-MSC incubated medium (BD-MSC). Scale bar = 1,000 pixel, ∗p<0.05, ∗∗p<0.01 vs. PBS treated HUVEC. PBS: phosphate buffer saline, BD-MSC: bone marrow-derived mesenchymal stem cell.
Fig. 2.
Validation of injected BD-MSC survival to FeCl3 treated mouse model. A. In vivo images showed expression of injected BD-MSC in FeCl3 treated mouse leg (BD-MSC) after 3 and 7 days. B. Confocal microscopy images showed Hu-Nu intensity of saline injected (saline) or BD-MSC injected (BD-MSC) in FeCl3 treated mouse model after 3 and 7 days (green: Hu-Nu, blue: DAPI (nucleus)). Scale bar = 50 μm, Hu-Nu: human nuclei, BD-MSC: bone marrow-drived mesenchymal stem cell.
Fig. 3.
Validation of angiogenesis in FeCl3 treated mouse model. A. Confocal microscopy images showed vWF (endothelial cells) intensity of saline injected (saline) or BD-MSC injected (BD-MSC) in FeCl3 treated mouse model (green: vWF, blue: DAPI (nucleus)). Scale bar = 50 μm. B. Density for each group of blood vessels: under 10, 10–20, 20–30 and above 31 μm/field was obtained from vWF stained slides and measured by Zen software. ∗∗P<0.01 vs. saline treated mice. BD-MSC: bone marrow-derived mesenchymal stem cell, vWF: von Willebrand factor.
Fig. 4.
Validation of BD-MSC protection from FeCl3 induced damage. A. left side of leg appearance showed skin color and limb condition of saline injected (saline) or BD-MSC injected (BD-MSC) in FeCl3 treated mouse model (arrow) and graph indicated improved functional outcomes in BD-MSC injected group. B. Central nucleus of damaged skeletal muscles was indicated (green arrow) and confirmed by Hematoxylin and Eosin staining. C. Confocal fluorescence analysis showed apoptosis cell (red) in FeCl3 treated mouse model. D. Fibrous (blue) was validated by Masson's trichrome in FeCl3 induced muscle. Scale bar=100 μm, ∗P<0.01 vs. saline treated mice.
Table 1.
Ischemia scale used for functional test of limb ischemia leg
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