Prolyl-hydroxylase inhibition induces SDF-1 associated with increased CXCR4+/CD11b+ subpopulations and cardiac repair

Abstract SDF-1/CXCR4 activation facilitates myocardial repair. Therefore, we aimed to activate the HIF-1α target genes SDF-1 and CXCR4 by dimethyloxalylglycine (DMOG)-induced prolyl-hydroxylase (PH) inhibition to augment CXCR4+ cell recruitment and myocardial repair. SDF-1 and CXCR4 expression was analyzed under normoxia and ischemia ± DMOG utilizing SDF-1-EGFP and CXCR4-EGFP reporter mice. In bone marrow and heart, CXCR4-EGFP was predominantly expressed in CD45+/CD11b+ leukocytes which significantly increased after myocardial ischemia. PH inhibition with 500 μM DMOG induced upregulation of SDF-1 mRNA in human microvascular endothelial cells (HMEC-1) and aortic vascular smooth muscle cells (HAVSMC). CXCR4 was highly elevated in HMEC-1 but almost no detectable in HAVSMC. In vivo, systemic administration of the PH inhibitor DMOG without pretreatment upregulated nuclear HIF-1α and SDF-1 in the ischemic mouse heart associated with increased recruitment of CD45+/CXCR4-EGFP+/CD11b+ cell subsets. Enhanced PH inhibition significantly upregulated reparative M2 like CXCR4-EGFP+ CD11b+/CD206+ cells compared to inflammatory M2-like CXCR4-EGFP+ CD11b+/CD86+ cells associated with reduced apoptotic cell death, increased neovascularization, reduced scar size, and an improved heart function after MI. In summary, our data suggest increased PH inhibition as a promising tool for a customized upregulation of SDF-1 and CXCR4 expression to attract CXCR4+/CD11b+ cells to the ischemic heart associated with increased cardiac repair. Key messages DMOG-induced prolyl-hydroxylase inhibition upregulates SDF-1 and CXCR4 in human endothelial cells. Systemic application of DMOG upregulates nuclear HIF-1α and SDF-1 in vivo. Enhanced prolyl-hydroxylase inhibition increases mainly CXCR4+/CD11b+ cells. DMOG increased reparative M2-like CD11b+/CD206+ cells compared to M1-like cells after MI. Enhanced prolyl-hydroxylase inhibition improved cardiac repair and heart function. Electronic supplementary material The online version of this article (doi:10.1007/s00109-017-1543-3) contains supplementary material, which is available to authorized users.

intercostal space. The heart was exposed, the pericardial sac was opened and pulled apart, and the left anterior descending (LAD) artery was visualized. Ligation was proceeded with a 7-0 silk suture passed with a tapered needle underneath the LAD artery about 1-2 mm lower than the tip of the left auricle. Occlusion was confirmed by pallor of the anterior wall of the left ventricle. Lungs were overinflated, and the chest cavity, muscles, and skin were closed layer by layer with 4-0 silk sutures. After the operation, all mice received a single injection of analgesia with 10 mg/kg piritramid i.p. Afterwards the animals received further analgesia through drinking water (2.5 mg piritramid in 100 ml drinking water) for the next 7 days.
Euthanasia at the end of the experiment was performed by cervical dislocation. Animal care and all experimental procedures were performed in strict accordance to the Austrian (BMWF-66.011/0031-II/3b/2014) and Directive 2010/63/EU of the European Parliament guidelines.

Fluorescent Activated Cell Sorting (FACS) of BM and heart cells
10-12 week old CXCR4-EGFP BAC transgenic reporter mice with or without LAD ligation were either treated with saline or DMOG (80 mg/kg/d), respectively, daily for up to 7 days. BM mononuclear cells were separated by density-gradient centrifugation using 1.077 g/ml Histopaque solution (Sigma Chemicals), purified, and resuspended in PBS containing 1% BSA. Cells were incubated for 40 min in the dark at 4°C with the following fluoresceinisothiocyanate (FITC), phycoerythrin (PE), and peridininchlorophyll-protein

Gene expression of HIF-1 target genes, fibrosis and hypertrophy genes in the heart
Heart tissues were minced and homogenized in Trizol reagent (Invitrogen, USA) and total RNA was isolated according to the manufacturer's instructions. RNA was reversely transcribed to cDNA using the QuantiTect RT kit (Qiagen). Exon spanning primers for mouse HIF-1α target genes VEGFA, LDHA, PDK-1, fibrosis marker collagen-1a and the hypertrophy marker BNP were designed and are listed in Supplementary Table 1. Using 2 x SYBR green mastermix (Applied Biosystems, USA) quantitative gene expressions was calculated using the comparative Ct-method with ß-actin as a reference gene.

Immunohistochemistry of CXCR4+ cells in the heart
In order to characterize SDF-1+ and CXCR4+ expressing cells in the ischemic heart, adult non-infarcted and adult infarcted hearts (day 7, day 30 after MI) were harvested, washed in phosphate-buffered saline, and immersion fixed in 4% paraformaldehyde for 24 hours at 4°C, cryoprotected in 30% sucrose, embedded in OTC (Sakura Finetek, Torrance, CA) and sectioned at 10 µm. Afterwards, CXCR4+ expressing cells were identified by EGFP immune reactivity and further characterized for co-expression of PECAM (endothelial cells; Sigma clone# 8A9) and CD11b (Alexa Fluor® 594 anti-mouse/human CD11b, Biolegend).

Functional parameters
To evaluate functional parameters, mice were randomly assigned to the following groups: Sham operated control mice (n = 5), saline-treated infarcted mice (n = 10), and DMOG (80 mg/kg i.p.) treated infarcted mice (n = 10). Pharmacological treatment was administered for up to 7 days post-MI (Supp. Fig. 4). Pressure-volume relations in vivo were analysed 30 days after MI. Mice were anesthetized with a mixture of 100 mg/kg ketamine (Sigma Chemical Co., 9, St. Louis, MO) and 5 mg/kg xylazine (Sigma), intubated, and ventilated (MiniVent, HUGO SACHS, Freiburg, Germany). Via the right carotid artery, an impedance micromanometer catheter (Millar Instruments, Houston, Texas) was introduced into the left ventricle. Raw conductance volumes were corrected for parallel conductance by the hypertonic saline dilution method as described previously [1]. Euthanasia at the end of the experiment was performed by cervical dislocation. Haemodynamic measurements and data analyses were performed independently by a blinded person using LabChart 8 analysis software (ADInstruments Ltd, United Kingdom).

Histology, immunostaining and quantification of apoptosis and neovascularization
30 days after MI hearts (n=10) were excised and fixed in 4% phosphate-buffered formalin.
Thereafter, hearts were cut transversally into 2 mm thick slices and embedded in paraffin.
Sections 10 µm thick were cut and mounted on positively charged glass slides. Standard histological procedures (hematoxylin and eosin and Masson's trichrome) and immunostaining were performed. Capillaries were stained with antibodies against CD31 (goat anti-mouse, Santa Cruz -SC1506), AEC was used as chromogen. Apoptotic cells were detected using the TUNEL assay (DeadEnd™ Fluorometric TUNEL System, Promega). Sections were co-stained with DAPI to detect all cell nuclei. Digital photographs were taken at a magnification of 400x, and four random high-power fields (HPFs) from the infarct border zone of each heart sample (n = 6) were analysed utilizing NIH Image software.
For quantification, the apoptotic index (AI) was calculated as percentage of TUNEL+ nuclei (green) to total nuclei DAPI (blue). Scar size was calculated as the average of three transverse sections sampled at 2 mm intervals from the apex to the base using the following formula developed by Pfeffer et al. [2]: Scar size (%) = [transverse scar perimeter (epicardial plus endocardial)/total transverse perimeter (epicardial plus endocardial)] × 100. Infarct wall thickness was measured in Masson's trichrome stained sections by taking the average length of five segments along evenly spaced radii from the centre of the LV through the infarcted and non-infarcted LV wall [3].

Statistical analysis
Results were expressed as mean ± SD. Multiple group comparisons were performed by oneway analysis of variance (ANOVA) followed by the Bonferroni procedure for comparison of means. Comparisons between two groups were performed using the unpaired two sided Student's t test. Data were considered statistically significant at a value of p<0.05.   Immunofluorescence images 7 days after ischemia in the infarcted area revealed substantial upregulation of CXCR4-EGFP+ cells in the heart. Co-staining of CXCR4-EGFP+ cells with the endothelial cell marker CD31 and the monocyte marker CD11b revealed co-expression in the infarct region (2 nd row, 3 rd row). Scale bar representing 25µm.   FACS analyses of CD45 -/CXCR4-EGFP + cells showed no increase in numbers of cells in the ischemic heart after DMOG treatment. All data represent mean ± SD (n = 4); Control vs. MI.