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 5

Inhibition of the MEK1/-2/ERK1/-2 signaling induces the switch from human cardiac PCs into VSMC-like cells in vitro.

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Inhibition of the MEK1/-2/ERK1/-2 signaling induces the switch from huma...
(A) Schematic showing EGF and bFGF signaling in cardiac PCs and the MEK1/2 inhibitor employed. (B–I) PCs were cultured for 10 days with different media as indicated, in the presence of PD0325901 (PD, 250 nM) or DMSO (Veh), before using them for the functional assays. (B and C) Analyses of protein expression using Western blotting. Representative blots are from 1 patient, and graphs show blot densitometry for n = 5 patients’ PCs. (D) Representative immunofluorescence images of PCs from 1 patient show contractile VSMC proteins and cytoskeletal F-actin expression (green). Scale bars: 50 μm. n = 5 patients’ PCs. (E) 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. (F) Gap closure migration assay. Migration time was 24 hours. Bar graphs report the area of the final gap. n = 4 patients’ PCs (E and F). Representative images are from 1 patient. SDF-1α, stromal cell–derived factor 1α. (G) Secreted angiogenic factors. n = 6 patients’ PCs. (H) 2D-Matrigel assay with human coronary artery ECs (CAECs) and PCs. CAECs were used in monoculture or cocultures with either Veh-PC or PD0325901-treated PC (PD-PC). n = 3 or 4 patients’ PCs. n = 1 CAEC (assayed 5 times). (I) 2D-Matrigel assay with PCs alone. n = 5 patients’ PCs. All data are plotted as individual values and mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by ordinary 2-way ANOVA followed by Tukey’s multiple comparisons test (C); 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 Veh and All GFs PD) per treatment (E); unpaired Mann-Whitney U test (F, G, and I); or unpaired Kruskal-Wallis followed by Dunn’s multiple comparisons test (H).