1. International Journal of Cardiology
Cyclophilin A modulates bone marrow-derived CD117+ cells and enhances ischemia- induced angiogenesis via the SDF-1/CXCR4 axis 
Gianluca L. Perrucci, Stefania Straino, Maria Corlianò, Alessandro Scopece, Monica Napolitano, Bradford C. Berk, Federico Lombardi, Giulio Pompilio, Maurizio C. Capogrossi, Patrizia Nigro 
International Journal of Cardiology 
Volume 212, Pages (June 2016)
DOI: /j.ijcard
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2. Fig. 1
Intracellular and extracellular levels of CyPA increase during HLI. CyPA gene (A) and protein (B, C) expression at different days after HLI (qRT-PCR data are fold change±SD, Western blot quantification data are mean±SD, n=5/group/time point). (D) Representative images of immunohistochemistry for CyPA in ischemic (right panel) and contralateral non-ischemic (left panel) adductor muscle sections 3days after HLI. (E) Serum levels of CyPA at different time points after HLI (data are mean±SD, n=5/time point). Student's t-test: *p<0.05. Magnification=20×. Scale bar=100μm.
International Journal of Cardiology  , DOI: ( /j.ijcard )
Copyright © 2016 The Authors Terms and Conditions

3. Fig. 2
CyPA increases limb perfusion and angiogenesis. (A) Representative contrast images (upper panels) and correspondent laser Doppler images (lower panels) of hind-limbs 3days after surgery and treatments. Colour scale in Doppler images represents a gradient from less perfused (blue) to highly perfused (red) areas. (B) Bar chart of perfusion rates on different days post-HLI. (C) CD31 immunofluorescence for capillary detection and relative quantification (D) 7days post-surgery. Magnification=40×. Scale bar=15μm. (E) α-smooth muscle actin (α-SMA) immunofluorescence and relative arteriole quantification (F) 7days post-surgery. Magnification=20×. Scale bar=40μm. All data are mean±SD, n=5/group/time point. Student's t-test: *p<0.05.
International Journal of Cardiology  , DOI: ( /j.ijcard )
Copyright © 2016 The Authors Terms and Conditions

4. Fig. 3
CyPA augments inflammatory cell number following HLI. Immunohistochemistry for CD45 (A), Mac-3 (C), and CD3 (E) on adductor muscle slices of ischemic mice treated with saline or with 10ng CyPA for 3days. Immunohistochemistry quantification (B, D, F) data are shown as mean±SD, n=5/group. Student's t-test: *p<0.05. Magnification=20×. Scale bar=40μm.
International Journal of Cardiology  , DOI: ( /j.ijcard )
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5. Fig. 4
CyPA increases the number of BM-derived CD117+ cells. Immunohistochemistry for CD117 on adductor muscle sections of ischemic mice treated with saline or with 10ng CyPA (A) and relative quantification (B). Ischemic samples were collected 7days after HLI. Data are shown as mean±SD, n=5/group. CD117 gene (C) and protein (D, E) expression in adductor muscle tissues (qRT-PCR data are fold change±SD, Western blot quantification data are mean±SD, n=5/group). Student's t-test: *p<0.05. Magnification=40×. Scale bar=50μm.
International Journal of Cardiology  , DOI: ( /j.ijcard )
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6. Fig. 5
CyPA enhances BM-derived CD117+ cell recruitment in ischemic mice. (A) Methodological approach for HLI surgery and BM-derived CD117+ cell isolation, labelling, and retro-orbital injection. WT C57Bl/6j mice underwent surgical femoral artery removal by cauterization and dissection (recipient mice). Ischemic mice were treated by intramuscular injection of saline solution (n=5) or 10ng CyPA (n=5). The day after surgery, WT C57Bl/6j mice (n=5; donor mice) were sacrificed to explant BM-derived selected CD117+ cells from hind-limb bones. Once isolated, cells underwent CFSE labelling and injection into recipient HLI mice. (B) Representative images of CFSE-labelled BM-derived CD117+ cells (green) in adductor muscle cryo-sections and relative signal quantification (C). Highlighted areas correspond to 40× magnification. Data are shown as mean±SD, n=5/group. Student's t-test: *p<0.05. Magnification=20×. Scale bar=200μm.
International Journal of Cardiology  , DOI: ( /j.ijcard )
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7. Fig. 6
CyPA is involved in BM-derived CD117+ cell survival, migration and adhesion. Assay graphs for cell number up to 72h (A), migration up to 4h (B), and adhesion after 24h (C) of treatment with increasing doses of CyPA. (D) FACS analysis reporting the percentage of AnnexinV negative and PI positive BM-derived CD117+ cells after serum deprivation and treatment with 100ng/ml CyPA for 48h. (E) FACS analysis and percentage of CD31 in BM-derived CD117+ cells after 7days of treatment with 100ng/ml CyPA. (F) Representative images of tube formation assay in BM-derived CD117+ cells±CyPA (100ng/ml). Results are means±SD of three experiments performed in triplicate. Student's t-test: *p<0.05. Magnification=20×. Scale bar=50μm.
International Journal of Cardiology  , DOI: ( /j.ijcard )
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8. Fig. 7
CyPA regulates CXCR4 expression and activity in vitro and in vivo. CXCR4 gene expression (A) in BM-derived CD117+ cells treated with CyPA. qRT-PCR was performed in triplicate and data are shown as fold change±SD. Western blot analysis (B) for active CXCR4 (phospho-CXCR4) and downstream mediators in BM-derived CD117+ cells treated for 5min with 100ng/ml CyPA and/or 25μg/ml AMD3100. Cells were pre-treated with AMD3100 for 30min before CyPA addition. Gene expression of CXCR4 (D) and SDF-1 (G) were evaluated in adductor muscles of ischemic mice treated with saline or 10ng CyPA (qRT-PCR data are fold change±SD *p<0.05; n=5/group/time point). CXCR4 protein expression (E) was analysed on adductor muscles by Western blot. Protein quantification (C, F) was performed by densitometric analysis (*p<0.05). SDF-1 levels (H) were measured by ELISA in the serum of mice, 3days after surgery and treatment with 10ng CyPA. Student's t-test: *p<0.05.
International Journal of Cardiology  , DOI: ( /j.ijcard )
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9. Fig. 8
Inhibition of CXCR4 signalling reverses CyPA-mediated neo-angiogenesis in vivo. Bar charts depict perfusion levels (A), number of capillaries (B), and arterioles (C) in mice treated with saline, 10ng CyPA, AMD3100 (360μg/die)+saline or AMD3100 (360μg/die)+10ng CyPA after 3days post-surgery. Immunohistochemistry (D) and its densitometric analysis (E) for CD117 in adductor muscle tissue sections from ischemic mice treated with AMD3100 or co-treated with 10ng CyPA 3days post-surgery. Data are means±SD, n=5. Student's t-test: *p<0.05. Magnification=20×. Scale bar=50μm.
International Journal of Cardiology  , DOI: ( /j.ijcard )
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10. Fig. 1S
Recruited BM-derived CD117+ cells in ischemic tissue express an endothelial marker.
Representative images of CD31 immunofluorescence on recruited BM-derived CD117+ 3days postsurgery.
Green=CFSE-labelled BM-derived CD117+, red=CD31, blue=Hoechst. Magnification=40×. Scale bar=25μm.
International Journal of Cardiology  , DOI: ( /j.ijcard )
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11. Fig. 2S
CyPA does not alter the cell cycle distribution of BM-derived CD117+ cells. FACS analysis of BrdU/PI assay was performed on BM-derived CD117+ cells in the presence or absence of different CyPA concentrations (10, 100 and 1000ng/ml).
International Journal of Cardiology  , DOI: ( /j.ijcard )
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12. Fig. 3S
CyPA does not alter BM-derived CD117+ leukocyte marker expression. FACS analysis and percentage of CD45 in BM-derived CD117+ cells after 7days of differentiation. Control cell peak and relative percentage are depicted in blue, cells treated with 100ng/ml CyPA are in red.
International Journal of Cardiology  , DOI: ( /j.ijcard )
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13. Fig. 4S
CyPA increases CXCR4 expression in ischemic tissues. Representative images of CXCR4 immunofluorescence on cryosections of control and ischemic tissue 7days post-surgery. Red=CXCR4, blue=Hoechst. Magnification=40×. Scale bar=25μm.
International Journal of Cardiology  , DOI: ( /j.ijcard )
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14. Fig. 5S
Proposed mechanism of CyPA-mediated neo-angiogenesis. In a HLI scenario, CyPA can play a crucial role in enhancing SDF-1/CXCR4 axis activation. This mechanism is important to increase the recruitment of BM-derived CD117+ cells into the ischemic site, leading to an improved neoangiogenic process.
International Journal of Cardiology  , DOI: ( /j.ijcard )
Copyright © 2016 The Authors Terms and Conditions