Abstract
Introduction: Whilst blood flow restoration is critical following myocardial infarction (MI), ischaemia-reperfusion injury (IRI) accounts for ~50% of the final infarct size. The newly discovered cytokine, interleukin-36 (IL-36), could potentially mediate these disturbances. However, its role in myocardial IRI is not known. Although several anti-inflammatory therapies have been successful in pre-clinical models of MI, they have failed in the clinical setting. This translational failure may be linked to a lack of inclusion of comorbidities and/or risk factors, as well as an early benefit at the level of the coronary microcirculation. We firstly investigated if IL-36 cytokines and its receptor (IL-36R) were present in the heart, and whether their expression varied in an injury-, age-, and sex-related manner. We then determined whether coronary microcirculatory disturbances and infarct size post-IRI were modified by age and sex, and whether IL-36Ra could confer vasculoprotection.
Methods: Myocardial IRI was induced in adult (3-months) and aged (>18-months) female mice, with sex differences assessed in adult male and female mice. Myocardial IL-36R/α/β, VCAM-1 expression, and oxidative stress were investigated by immunostaining, flow cytometry, and/or using Western blot. Expression on human heart tissue samples of varying ages was also determined. IL-36R/α/β expression was also determined on stimulated vena cava endothelial cells (VCECs). The beating heart coronary microcirculation was imaged in vivo intravitally for neutrophils, platelets, and functional capillary density, and also ex vivo using multiphoton microscopy. Laser speckle contrast imaging was used to determine overall left ventricular perfusion. The effects of topical IL-36 cytokine application was also observed intravitally. In some studies, recombinant mouse IL-36Ra (15μg/mouse) was injected intra-arterially at 5 minutes pre-reperfusion and 60 minutes post-reperfusion. Infarct size was measured using dual TTC/Evans Blue staining.
Results: Expression of IL-36 cytokine and its receptor was predominantly on the vasculature and cardiomyocytes of both the murine and human hearts, and their expression increased with age and injury. Expression was significantly higher in healthy and injured adult female hearts compared to male hearts. Basal VCEC surface expression of IL-36R increased after cytokine stimulation in a concentration-dependent manner. Intravital imaging of the beating heart demonstrated heightened basal and injury-induced neutrophil recruitment and poorer blood flow in the aged coronary microcirculation when compared with adult hearts. These events were mirrored in deeper myocardial layers when imaged using multiphoton microscopy. A greater burden of thrombotic disease was noted in adult injured male coronary microvessels, whilst a greater neutrophil presence was identified in adult injured female coronary microvessels. Infarct size was significantly larger in injured adult female hearts when compared to males. All IL-36 cytokines were able to induce an inflammatory response when topically applied to the adult and aged hearts. An IL-36R antagonist (IL-36Ra) decreased neutrophil recruitment, improved blood flow and ventricular perfusion, and reduced infarct size in both adult and aged mice. This may be mechanistically explained by attenuated endothelial oxidative damage and VCAM-1 expression in IL-36Ra–treated mice.
Conclusion: These novel results are the first to demonstrate myocardial presence of IL-36 and its receptor and how expression changes with age and sex. Our findings of enhanced coronary microcirculatory perturbations associated with age may explain the poorer outcomes in elderly MI patients. The cellular nature of the thromboinflammatory response may explain the sex-related differences in outcomes after MI. Importantly, we are the first to demonstrate that targeting IL-36/IL-36R pathway may be a potential novel therapy for treatment of myocardial IRI.
Methods: Myocardial IRI was induced in adult (3-months) and aged (>18-months) female mice, with sex differences assessed in adult male and female mice. Myocardial IL-36R/α/β, VCAM-1 expression, and oxidative stress were investigated by immunostaining, flow cytometry, and/or using Western blot. Expression on human heart tissue samples of varying ages was also determined. IL-36R/α/β expression was also determined on stimulated vena cava endothelial cells (VCECs). The beating heart coronary microcirculation was imaged in vivo intravitally for neutrophils, platelets, and functional capillary density, and also ex vivo using multiphoton microscopy. Laser speckle contrast imaging was used to determine overall left ventricular perfusion. The effects of topical IL-36 cytokine application was also observed intravitally. In some studies, recombinant mouse IL-36Ra (15μg/mouse) was injected intra-arterially at 5 minutes pre-reperfusion and 60 minutes post-reperfusion. Infarct size was measured using dual TTC/Evans Blue staining.
Results: Expression of IL-36 cytokine and its receptor was predominantly on the vasculature and cardiomyocytes of both the murine and human hearts, and their expression increased with age and injury. Expression was significantly higher in healthy and injured adult female hearts compared to male hearts. Basal VCEC surface expression of IL-36R increased after cytokine stimulation in a concentration-dependent manner. Intravital imaging of the beating heart demonstrated heightened basal and injury-induced neutrophil recruitment and poorer blood flow in the aged coronary microcirculation when compared with adult hearts. These events were mirrored in deeper myocardial layers when imaged using multiphoton microscopy. A greater burden of thrombotic disease was noted in adult injured male coronary microvessels, whilst a greater neutrophil presence was identified in adult injured female coronary microvessels. Infarct size was significantly larger in injured adult female hearts when compared to males. All IL-36 cytokines were able to induce an inflammatory response when topically applied to the adult and aged hearts. An IL-36R antagonist (IL-36Ra) decreased neutrophil recruitment, improved blood flow and ventricular perfusion, and reduced infarct size in both adult and aged mice. This may be mechanistically explained by attenuated endothelial oxidative damage and VCAM-1 expression in IL-36Ra–treated mice.
Conclusion: These novel results are the first to demonstrate myocardial presence of IL-36 and its receptor and how expression changes with age and sex. Our findings of enhanced coronary microcirculatory perturbations associated with age may explain the poorer outcomes in elderly MI patients. The cellular nature of the thromboinflammatory response may explain the sex-related differences in outcomes after MI. Importantly, we are the first to demonstrate that targeting IL-36/IL-36R pathway may be a potential novel therapy for treatment of myocardial IRI.
| Original language | English |
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| Qualification | ???thesis.qualification.phd??? |
| Publisher | |
| Publication status | Published - Jul 2022 |
