Negative Regulation of miR-375 by Interleukin-10 Enhances Bone Marrow-Derived Progenitor Cell-Mediated Myocardial Repair and Function After Myocardial Infarction - PubMed (original) (raw)

. 2015 Dec;33(12):3519-29.

doi: 10.1002/stem.2121. Epub 2015 Aug 25.

Prasanna Krishnamurthy 2, Suresh Kumar Verma 1, Mohsin Khan 1, Tatiana Abramova 3, Alexander R Mackie 3, Gangjian Qin 3, Cynthia Benedict 1, Emily Nickoloff 1, Jennifer Johnson 1, Ehre Gao 1, Douglas W Losordo 3, Steven R Houser 4, Walter J Koch 1 5, Raj Kishore 1 5 3

Affiliations

Negative Regulation of miR-375 by Interleukin-10 Enhances Bone Marrow-Derived Progenitor Cell-Mediated Myocardial Repair and Function After Myocardial Infarction

Venkata Naga Srikanth Garikipati et al. Stem Cells. 2015 Dec.

Abstract

Poor survival and function of transplanted cells in ischemic and inflamed myocardium likely compromises the functional benefit of stem cell-based therapies. We have earlier reported that co-administration of interleukin (IL)-10 and BMPAC enhances cell survival and improves left ventricular (LV) functions after acute myocardial infarction (MI) in mice. We hypothesized that IL-10 regulates microRNA-375 (miR-375) signaling in BMPACs to enhance their survival and function in ischemic myocardium after MI and attenuates left ventricular dysfunction after MI. miR-375 expression is significantly upregulated in BMPACs upon exposure to inflammatory/hypoxic stimulus and also after MI. IL-10 knockout mice display significantly elevated miR-375 levels. We report that ex vivo miR-375 knockdown in BMPAC before transplantation in the ischemic myocardium after MI significantly improve the survival and retention of transplanted BMPACs and also BMPAC-mediated post-infarct repair, neovascularization, and LV functions. Our in vitro studies revealed that knockdown of miR-375-enhanced BMPAC proliferation and tube formation and inhibited apoptosis; over expression of miR-375 in BMPAC had opposite effects. Mechanistically, miR-375 negatively regulated 3-phosphoinositide-dependent protein kinase-1 (PDK-1) expression and PDK-1-mediated activation of PI3kinase/AKT signaling. Interestingly, BMPAC isolated from IL-10-deficient mice showed elevated basal levels of miR-375 and exhibited functional deficiencies, which were partly rescued by miR-375 knockdown, enhancing BMPAC function in vitro and in vivo. Taken together, our studies suggest that miR-375 is negatively associated with BMPAC function and survival and IL-10-mediated repression of miR-375 enhances BMPAC survival and function.

Keywords: Angiogenesis; Bone marrow progenitor angiogenic cells; Inflammation; Left ventricular remodeling; miRNA.

© 2015 AlphaMed Press.

PubMed Disclaimer

Figures

Fig. 1

Fig. 1. IL-10 regulates miR-375 expression in BMPAC

(A) IL-10 inhibits MI-induced myocardial miR-375 expression. ***p<0.001Vs sham injured hearts; ##p<0.01Vs MI injured hearts (n=4) **(B)** WT-BMPAC/IL-10 KO BMPAC were stimulated with LPS, with LPS + IL-10 or IL-10 alone. Expression of miR-375 was measured by RT-PCR. **(C)** BMPAC were subjected to hypoxia, hypoxia +IL-10 or IL-10 alone and miR-375 expression was measured by RT-PCR normalized to U6 with or without IL-10. _n_ = 3. ***_P_< 0.001 Vs WT-Ctrl BMPAC; $.p<0.001 Vs WT/IL-10 KO BMPAC +LPS. (D) BMPACs were treated with LPS or IL-10 or both before the addition of 5μg actinomycin D (Act-D). BMPACs were harvested 30, 60, and 120 min after the addition of actinomycin D (time 0), RT-qPCR was performed for miR-375. Expression data are expressed as percent of miR-375 remaining at each time point vs. miR-375 levels at time 0. miR expression was normalized to U6 snRNA. **P< 0.01 ***P< 0.001 Vs LPS alone; ##. P<0.01###.p<0.001 Vs LPS+ actinomycin-D.

Fig.2

Fig.2. Knockdown of miR-375 enhances WT BMPAC functions and rescues IL-10 KO BMPAC phenotype

(A) The representative photomicrographs (10X, 20uM scale bar) of tube formation by matrigel angiogenesis assays in WT- BMPAC/IL-10 KO BMPAC transfected with scrambled or anti-miR-375. (B) Relative quantification of branch points, (C) Quantification of percentage of Tunel+ cells were measured by fluorescence microscopy after apoptotic stimuli of 100μmol/L H202 after WT- BMPAC/IL-10 KO BMPAC transfected with scrambled or anti-miR-375. (D) Quantification of apoptosis by caspase 3/7 assay. Quantification of BMPAC proliferation measured by (D) ciQuant assay after WT- BMPAC/IL-10 KO BMPAC transfected with scrambled or anti-miR-375. Results are presented as s.e.m for three independent experiments. ***P < 0.001, **P < 0.01, *P<0.05 Vs WT Scrambled BMPAC; ###p<0.001, ##p<0.01, #p<0.05 Vs IL-KO scrambled BMPAC.

Fig.3

Fig.3. miR-375 directly targets PDK-1

(A) Relative mRNA expression of PDK-1 normalized to 18S (B) Representative immune-blot of PDK-1. (C) Relative quantification of PDK-1 protein. (D) Study of the interaction between miR-375 and 3′UTR of PDK-1 mRNA by luciferase assay (E) Overexpression of miR-375 attenuates AKT phosphorylation. Results are presented as s.e.m for three independent experiments. n = 3. ***P< 0.00, **P< 0.01, *P< 0.05 Vs WT Scrambled BMPACs.

Fig.4

Fig.4. Down-regulation of PDK-1 inhibits the effects of anti-miR-375 on HUVECs apoptosis and tube formation

HUVECs were transfected with NC-siRNA or PDK-1 for 24h. (A) Representative PDK-1 protein levels (B) Quantification of PDK-1 levels normalized to β-actin. HUVECs transfected with NC-siRNA, anti-miR-375, PDK-1 siRNA, PDK-1 siRNA+ anti miR-375. (C) Quantification of apoptosis by tunel assay. (D) Relative quantification of branch points, Results are presented as s.e.m for three independent experiments. n = 3. ***p<0.001Vs Scrambled ctrl HUVECs. ### P<.001 Vs anti miR-375 treated HUVECs.

Fig.5

Fig.5. Increased survival of miR-375 Knockdown BMPACs in situ in the heart following myocardial infarction

BMPAC retention and survival in the myocardium at 5 days after MI in anti-miR-375 BMPAC or scrambled BMPAC treated mice. Tunel staining for detecting apoptosis (red) of BMPAC (GFP-positive, green florescence) and DAPI (blue) for nuclear staining. Inset is higher magnification of yellow-boxed area. Arrows indicate GFP+TUNEL+cells (40x, Scale bar 100μm). (A–C) Quantification of GFP+(BMPAC) at 5 days post MI. (D) Quantitative analysis of GFP/TUNEL double positive cells at 5 days after MI. (E–F) Increased GFP+BrdU+ cells (40x, Scale bar 100μm) within hearts treated with anti-miR-375 BMPAC compared scrambled BMPAC-treated hearts stained with green fluorescent protein (GFP; green), BrdU (red), DAPI (blue) for nuclei staining. Inset is higher magnification of yellow-boxed area. Arrows indicate GFP+BrdU+cells. (G). Quantification of BrdU+/ GFP+ cells in mice treated with anti-miR-375 BMPAC and Scrambled BMPAC (n=5). (H–I) Representative TUNEL staining image for cardiomyocyte apoptosis (green nuclei), alpha actinin (red), DAPI (blue) in border zone of LV infarct at 5 d post-MI. (J) Quantitative analysis of TUNEL+ cardiomyocytes at 5 d post-MI. n = 5/group. ***P<0.001, **P<0.05 vs scrambled WT-BMPAC treated groups.

Fig.6

Fig.6. PDK-1 is up-regulated in the anti-miR-375 BMPACs transplanted hearts after MI

Representative western blot and its quantification for PDK-1, pAKT and total AKT protein expression in LV at 5 d post-MI normalized to β-actin. n = 5/group. ##P < 0.01 vs saline group and $ P<0.05 vs Scrambled BMPAC group.

Fig.7

Fig.7. Transplantation of miR-375 Knockdown BMPACs reduces fibrosis and enhances neovascularization and LV functional recovery 4 weeks after MI

(A–C) Representative masons trichome stained heart (1X, 100μm scale bar) treated with saline or WT-BMPAC treated with scrambled or anti-miR-375. (D) Quantitative analysis of infarct size (%LV area). (E–G) Representative immunofluorescence (20x, Scale bar 100μm) capillaries images taken within the infarct border zone of mice treated with saline or WT-BMPAC treated with scrambled or anti-miR-375. Capillaries were stained with BS-lectin-Alexa-555 (red) and nuclei were counterstained with DAPI (blue). (H) Quantification of border zone capillary number across treatments presented as the number of isolectin B4–positive capillaries and DAPI-stained nuclei per high power field (LPF). Representative echocardiography analysis shown in bars, in the hearts treated with saline or WT-BMPAC treated with scrambled or anti-miR-375. Mice receiving miR-375 knockdown WT-BMPAC: (I) %EF, (J) FS %. n = 6/group*P< 0.05, **P< 0.01, ***P< 0.001 vs saline group; #P< 0.05, ##P< 0.01, ###P< 0.001 vs scrambled BMPAC group. (n=6).

Fig.8

Fig.8. Proposed mechanisms demonstrating the role of miR-375 in BMPAC mediated cardiac regeneration

BMPACs inhibits IL-10 regulated miR-375 leading to activation of PDK-1/AKT signaling, PDK-1 (potential target of miR-375), thereby enhancing the neovascularization and also BMPAC survival post transplantation in MI mice. BMPAC indicates bone marrow progenitor angiogenic cell; IL-10, interleukin -10; PDK-1, 3-phosphoinositide-dependent protein kinase 1.

References

    1. Assmus B, Schachinger V, Teupe C, et al. Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI) Circulation. 2002;106:3009–3017. - PubMed
    1. Losordo DW, Henry TD, Davidson C, et al. Intramyocardial, autologous CD34+ cell therapy for refractory angina. Circulation research. 2011;109:428–436. - PMC - PubMed
    1. Strauer BE, Brehm M, Zeus T, et al. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation. 2002;106:1913–1918. - PubMed
    1. Losordo DW, Schatz RA, White CJ, et al. Intramyocardial transplantation of autologous CD34+ stem cells for intractable angina: a phase I/IIa double-blind, randomized controlled trial. Circulation. 2007;115:3165–3172. - PubMed
    1. Kandala J, Upadhyay GA, Pokushalov E, et al. Meta-analysis of stem cell therapy in chronic ischemic cardiomyopathy. The American journal of cardiology. 2013;112:217–225. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources