Repurposing an anti‐cancer agent for the treatment of hypertrophic heart disease

Figures

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  Figure 1. β3‐integrin expression is upregulated in human dilated and ischaemic cardiomyopathy. (A) Western blotting analysis of β3 integrin in protein lysates from human non‐failing heart, non‐failing heart with hypertrophy, and dilated and ischaemic myocardium. GAPDH was used as a loading control. Bar charts show mean densitometric readings ± SEM. N, 5 or 6 individual patient samples. (B) Immunofluorescence reveals β3‐integrin expression in sections of human non‐failing heart with an apparent increase in dilated and ischaemic hearts, but less in non‐failing heart with hypertrophy. The non‐specific primary antibody control showed minimal background level staining. N, four individual patient samples per group. Scale bar = 50 μm. Student's t‐test, *p < 0.05.

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  Figure 2. Low‐dose cilengitide treatment restores the effect of pressure overload induced by abdominal aortic constriction. (A) Wild‐type mice underwent either sham or abdominal aortic constriction (AAC) surgery and were treated with either cilengitide (Cil, 50 or 500 μg/kg) or vehicle alone immediately after surgery in a preventative mode trial. Representative endpoint echocardiographic images in vehicle and Cil‐treated sham‐surgery or AAC‐surgery mice (n, seven mice per group). Double‐headed arrows, LV internal diameter at diastole (LVIDd) and systole (LVIDs). (B) Treatment of mice with 50 μg/kg, but not 500 μg/kg, cilengitide corrected the fractional shortening effects after AAC surgery. Line graphs show summary fractional shortening (FS %) analysis over time, and the bar chart the endpoint FS % from the mice treated in A. (C) Three weeks after AAC surgery, both FS % and the ejection fraction (EF %) were reduced, whilst left ventricular internal diameter (during systole) (LVID;s) was enhanced compared with sham controls (n, five mice per group). (D) Treatment of mice with ldCil rescued the fractional shortening effects of AAC surgery in an intervention mode trial. Wild‐type mice…

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  Figure 3. Treatment with low‐dose cilengitide enhances cardiac blood vessel density in vivo and stimulates cardiac endothelial cell DNA‐replication, mitosis, and cell‐cycle transition transcriptional profiles. (A) Wild‐type mice underwent either sham or AAC surgery and were treated with ldCil or vehicle control for 3 weeks. Representative immunofluorescence images of myocardium stained for the endothelial cell marker CD31. Treatment with ldCil enhanced blood vessel density (BVD) in the myocardium of mice after AAC but not sham surgery. Mean ± SEM. One‐way ANOVA with Tukey's post hoc test; *p < 0.05. NS, not significant. (B) Duplicate preparations of mouse cardiac endothelial cells were treated for 0, 24 or 48 h with ldCil and RNA‐Seq was performed. Heatmap of differentially expressed (DE) up‐ and down‐regulated transcripts in cardiac endothelial cells treated with ldCil. Results are shown for duplicate samples. Fifty‐four DE genes were concordant at 24 and 48 h ldCil treatment compared with control. (C) Enrichment analysis of RNA‐Seq data demonstrates that treatment of cardiac endothelial cells significantly enriches for cell cycle, DNA replication, cell cycle, mitosis, and DNA strand…

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  Figure 4. Low‐dose cilengitide treatment reverses the transcriptomic profiles of AngII‐stimulated cardiomyocytes to control levels. (A) Mouse cardiomyocytes were isolated and either exposed to angiotensin II (AngII) to mimic the molecular stress of heart failure or exposed to AngII and also treated with ldCil. Heatmap of differentially expressed protein coding genes indicates that AngII stimulation resulted in 13 down‐ and nine up‐regulated transcripts. ldCil treatment of AngII‐stimulated cardiomyocytes rescues the differentially expressed (DE) transcript profiles. (B) Data from A represented in order of fold‐change. DE protein‐coding genes with **p < 0.01 in AngII versus control, also with high average expression across samples, became not DE (p > 0.05) in the AngII + ldCil group compared with control. Green bars show the levels of fold‐change. (C) Treatment with ldCil restores aberrant signalling pathways induced by AngII stimulation of cardiomyocytes. GSEA of KEGG and reactome signalling pathways reveal that dysregulated pathways with FDR q < 0.1 in AngII versus control became not significant (q > 0.1) in AngII + ldCil compared with control.

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  Figure 5. Low‐dose cilengitide treatment restores transcriptomic profiles in mouse cardiomyocytes similar to those found in non‐failing human heart. (A) Treatment of mouse cardiomyocytes with AngII + ldCil compared with AngII restores the transcriptomic signature similar to that found in human non‐failing versus failing heart. Heatmap illustrates concordant DE transcripts between human failing (ischaemic and dilated cardiomyopathies) versus non‐failing heart and mouse cardiomyocytes treated with AngII versus control or AngII versus AngII + ldCil. (B) Venn diagrams show the overlap of gene sequence expression analysis (GSEA) pathways (p < 0.05) in mouse cardiomyocytes stimulated with AngII versus control and human dilated cardiomyopathy (CMP) versus non‐failing heart. Scatterplots of normalised enrichment scores (NES) of the overlapping pathways illustrate how many of those change concordantly. Heatmap of NES for GSEA pathways that change concordantly in human dilated CMP versus non‐failing heart, mouse AngII‐stimulated versus control cardiomyocytes, and AngII‐stimulated cardiomyocytes treated with ldCil versus AngII‐stimulated cardiomyocytes. (C) Venn diagrams of the overlap of GSEA…