Interleukin-10 deficiency impairs bone marrow-derived endothelial progenitor cell survival and function in ischemic myocardium - PubMed (original) (raw)

Interleukin-10 deficiency impairs bone marrow-derived endothelial progenitor cell survival and function in ischemic myocardium

Prasanna Krishnamurthy et al. Circ Res. 2011.

Abstract

Rationale: Endothelial progenitor cell (EPC) survival and function in the injured myocardium is adversely influenced by hostile microenvironment such as ischemia, hypoxia, and inflammatory response, thereby compromising full benefits of EPC-mediated myocardial repair.

Objective: We hypothesized that interleukin-10 (IL-10) modulates EPC biology leading to enhanced survival and function after transplantation in the ischemic myocardium.

Methods and results: Myocardial infarction (MI)-induced mobilization of bone marrow EPC (Sca-1+Flk1+cells) into the circulation was significantly impaired in IL-10 knockout (KO) mice. Bone marrow transplantation to replace IL-10 KO marrow with wild-type (WT) marrow attenuated these effects. Impaired mobilization was associated with lower stromal cell-derived factor (SDF)-1 expression levels in the myocardium of KO mice. Interestingly, SDF-1 administration reversed mobilization defect in KO mice. In vitro, hypoxia-mediated increases in CXCR4 expression and cell survival were lower in IL-10-deficient EPCs. Furthermore, SDF-1-induced migration of WT EPCs was inhibited by AMD3100, an inhibitor of CXCR4. To further study the effect of IL-10 on in vivo EPC survival and engraftment into vascular structures, GFP-labeled EPC were injected intramyocardially after induction of MI, and the mice were treated with either saline or recombinant IL-10. The IL-10-treated group showed increased retention of transplanted EPCs in the myocardium and was associated with significantly reduced EPC apoptosis after MI. Interestingly, increased EPC retention and their association with the vascular structures was observed in IL-10-treated mice. Increased EPC survival and angiogenesis in the myocardium of IL-10-treated mice corroborated with improved left ventricular function, reduced infarct size, and fibrosis in the myocardium. In vitro, IL-10-induced increase in VEGF expression in WT EPC was abrogated by STAT3 inhibitor, suggesting IL-10 signals through STAT3 activation.

Conclusions: Taken together, our studies demonstrate that MI-induced EPC mobilization was impaired in IL-10 KO mice and that IL-10 increases EPC survival and function possibly through activation of STAT3/VEGF signaling cascades, leading to attenuation of MI-induced left ventricular dysfunction and remodeling.

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Figures

Figure 1

Figure 1

FACS analysis on peripheral blood mononuclear cells for MI-induced EPC mobilization [Sca1+/Flk1+] in WT (A), IL-10 KO-mice (B) and following bone marrow transplantation (D) or SDF-1 administration (E). C. Bar graph shows that Flk1+/Sca1+ cell mobilization was impaired in KO-mice as compared to WT-mice (#p<0.05). and BMT or SDF-1 administration attenuated mobilization in KO-mice (*$p<0.05 vs KO-mice). F. SDF-1 mRNA expression (RT-PCR) in border zone of myocardial tissue at 3 days after MI. mRNA expression normalized to 18S and depicted as fold change vs control (WT-C). SDF-1 expression was lower in KO mice as compared to WT mice (*P<0.05 WT-MI versus KO-MI).

Figure 2

Figure 2

A. Immunofluroscence staining for CXCR4 protein expression (green) in EPCs from WT (WT-EPC) and IL-10 KO-mice (KO-EPC) and DAPI (blue) for nuclear staining. Inset is higher magnification of the yellow-boxed area. Also, IL-10 deficient EPCs showed increased cell death (arrows; rounding and no clear nuclear staining). B. Bar graph depicting semi-quantitative analysis of CXCR4 fluorescence signal expressed as % Arbitrary Fluorescence Units (%AFU). C. Real time-PCR data for CXCR4 mRNA expression in EPCs in response to LPS. mRNA expression normalized to 18S and depicted as fold change vs control untreated cells. CXCR4 expression (mRNA and protein) was lower in KO-EPCs as compared to WT-EPCs (*P<0.05 versus KO-EPC).

Figure 3

Figure 3

SDF-1 induced migration EPC from WT and IL-10-deficient mice. Migratory response of Ex vivo expanded EPCs toward 20ng/ml SDF-1 gradient was measured by modified Boyden chamber migration assay. a,d. Untreated control EPCs, SDF-1 stimulated migration (b,e) and EPCs incubated with AMD3100 (10μg/ml; CXCR4 inhibitor) (c,f). g. Higher magnification of cells attached to the membrane after migration towards SDF-1 gradient. h. Bar graph of migrated cell number after 18 hours of incubation. EPCs demonstrated a potent migratory activity toward SDF-1. SDF-1 induced migration was impaired in IL-10 deficient EPCs (KO-EPCs) as compared to WT-EPCs. *P<0.05, Control vs SDF-1; #P<0.05, SDF-1 induced, WT-EPC vs KO-EPC; $P<0.05, SDF-1 vs AMD3100.

Figure 4

Figure 4

A. Inflammatory stimuli (LPS)-induced apoptosis (TUNEL+, red fluorescence) in EPC's isolated from WT and IL-10 KO-mice. DAPI (blue) was used for nuclear staining. Inset is higher magnification of the yellow-boxed area. B. LPS-induced EPC apoptosis was lower in WT-EPC as compared to EPC from KO-mice (#P<0.01).

Figure 5

Figure 5

A. EPC retention and survival in the myocardium at 3 days post-MI in IL-10/saline treated mice. TUNEL staining for detecting apoptosis (Red) of EPC (GFP-positive, green fluorescence) and DAPI (blue) for nuclear staining. Inset is higher magnification of the yellow-boxed area. Arrows indicate GFP+TUNEL+ cells. B. Quantification of GFP+ (EPC) cells at 3 days post-MI. C. Quantitative analysis of GFP/TUNEL double-positive cells at 3 days post-MI. IL-10 increased GFP+ EPC retention and survival in the heart following transplantation, *P<0.01 vs EPC+saline group. hvf, high-power visual field.

Figure 6

Figure 6

A,B. EPC-mediated neovascularization in border zone of LV infarct at 28 days post-MI. Engraftment of EPC (GFP+, green fluorescence) into vascular structures (CD31 staining for capillaries, red fluorescence) is seen as yellow structures. However, some cells are not incorporated (green). Inset is higher magnification of the yellow-boxed area. Bar graph shows quantitative analysis of CD31+ capillaries per high-power visual field (hvf) (C) and number of GFP+ cells associated with CD31+ vasculature (D). Capillary density and EPC engraftment into vascular structures was higher in IL-10 treated mice (#P<0.05). E. Effect of STAT3 on VEGF-A mRNA expression in EPC cells. Curcurbitacin I (Cur, STAT3 inhibitor) treated cells inhibited IL-10 induced VEGF-A. mRNA expression normalized to 18S and depicted as fold change vs control (c) untreated cells. *P<0.01 vs control cells; #P<0.01 vs IL-10 treated cells.

Figure 7

Figure 7

A. M-mode echocardiographic tracings at baseline and 7, 14 and 28 days of MI in EPC+saline and EPC+IL-10 groups. Analysis of LV diameter in systole (B) and %EF (C) and %FS (D) calculations. IL-10 administration significantly improved LV function with decreased LVESD and increased %EF and %FS, as compared to EPC+saline group. LVESD, LV end-systolic diameter; %EF, percent ejection fraction; %FS, percent fractional shortening; #P<0.05 vs MI; *P<0.05 vs EPC+saline; #P<0.05 vs MI alone group.

Figure 8

Figure 8

A. Trichrome stained heart sections (28 days post-MI). B. Quantitative analysis of fibrosis area at 28 days post-MI. Mice that received EPC+IL-10 showed lower fibrosis area when compared to EPC+saline group. #P<0.01 vs MI; *P<0.01 vs EPC+saline.

Comment in

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