Cardiac pericyte reprogramming by MEK inhibition promotes arteriologenesis and angiogenesis of the ischemic heart
Elisa Avolio, Rajesh Katare, Anita C. Thomas, Andrea Caporali, Daryl Schwenke, Michele Carrabba, Marco Meloni, Massimo Caputo, Paolo Madeddu
Elisa Avolio, Rajesh Katare, Anita C. Thomas, Andrea Caporali, Daryl Schwenke, Michele Carrabba, Marco Meloni, Massimo Caputo, Paolo Madeddu
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Research Article Angiogenesis Vascular biology

Cardiac pericyte reprogramming by MEK inhibition promotes arteriologenesis and angiogenesis of the ischemic heart

Abstract

Pericytes (PCs) are abundant yet remain the most enigmatic and ill-defined cell population in the heart. Here, we investigated whether PCs can be reprogrammed to aid neovascularization. Primary PCs from human and mouse hearts acquired cytoskeletal proteins typical of vascular smooth muscle cells (VSMCs) upon exclusion of EGF/bFGF, which signal through ERK1/2, or upon exposure to the MEK inhibitor PD0325901. Differentiated PCs became more proangiogenic, more responsive to vasoactive agents, and insensitive to chemoattractants. RNA sequencing revealed transcripts marking the PD0325901-induced transition into proangiogenic, stationary VSMC-like cells, including the unique expression of 2 angiogenesis-related markers, aquaporin 1 (AQP1) and cellular retinoic acid–binding protein 2 (CRABP2), which were further verified at the protein level. This enabled us to trace PCs during in vivo studies. In mice, implantation of Matrigel plugs containing human PCs plus PD0325901 promoted the formation of αSMA+ neovessels compared with PC only. Two-week oral administration of PD0325901 to mice increased the heart arteriolar density, total vascular area, arteriole coverage by PDGFRβ+AQP1+CRABP2+ PCs, and myocardial perfusion. Short-duration PD0325901 treatment of mice after myocardial infarction enhanced the peri-infarct vascularization, reduced the scar, and improved systolic function. In conclusion, myocardial PCs have intrinsic plasticity that can be pharmacologically modulated to promote reparative vascularization of the ischemic heart.

Authors

Elisa Avolio, Rajesh Katare, Anita C. Thomas, Andrea Caporali, Daryl Schwenke, Michele Carrabba, Marco Meloni, Massimo Caputo, Paolo Madeddu

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Figure 3

Human cardiac PCs differentiated without GFs display functional properties of contractile VSMCs.

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Human cardiac PCs differentiated without GFs display functional properti...
PCs were cultured with different GF combinations for 10 days and then used for functional assays. All GFs: VEGF, IGF-1, EGF, bFGF. No GFs: depletion of all GFs. – EGF/bFGF: only VEGF and IGF1. + EGF/bFGF: only EGF and bFGF. (A) Contraction assay. Cells were embedded in collagen gels, treated with a contraction inhibitor (inhib), and stimulated with endothelin-1 (ET-1). Bar graphs indicate the percentage of gel contraction after 24 hours. n = 4 patients’ PCs. Representative images are from 1 patient. (B) Fluo-4 calcium assay. Cells were loaded with the Fluo-4 dye and stimulated with ET-1 or vehicle. The intracellular calcium flux was measured as relative fluorescence units (RFU, green). Scale bars: 50 μm. Curves summarize n = 4 patients’ PCs (mean ± SEM are reported for each time point). Bar graphs show the quantification of the area under the curve and the peak fluorescence intensity. Representative images are from 1 patient. (C) Gap closure migration assay. Migration time was 24 hours. The absence of stimuli served as control (CTRL). Bar graphs show the final area of the gap. n = 3 patients’ PCs. Representative images are from 1 patient. (D and E) Expression of extracellular matrix proteins and transcripts. mRNA data are expressed as a fold change versus coronary artery SMCs (CASMCs) used as reference population (dashed line at y = 1). n = 3 to 5 patients’ PCs. All data are individual values and mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by unpaired Kruskal-Wallis followed by Dunn’s multiple comparisons test to compare the 3 treatment groups (CTRL, ET-1, ET-1 + inhib) per experimental condition, and unpaired Mann-Whitney U test to compare the 2 experimental groups (All GFs and No GFs) per treatment (A); ordinary 2-way ANOVA followed by Tukey’s multiple comparisons test (B, D, and E); or unpaired Mann-Whitney U test (C).