RIPk3 adenovirus

The molecular features of necroptosis in cardiac ischemia-reperfusion (IR) injury have been extensively studied. However, the physiological regulatory mechanisms of melatonin on necroptosis in cardiac IR injury have not been investigated. Here, the results show that Ripk3 is mainly activated by IR injury and, as a result, exacerbates endothelial necroptosis, microvascular barrier dysfunction, capillary hyperpermeability, inflammatory responses, microcirculatory vasospasm, and microvascular perfusion defects. However, melatonin administration inhibits Ripk3 activation and provides a prosurvival advantage to the endothelial system in the context of cardiac IR injury, similar to the results obtained by genetic knockout of Ripk3. Functional studies clearly demonstrated that activated Ripk3 upregulated the expression of PGAM5, which in turn increased the phosphorylation of CypD, which led to necroptosis in endothelial cells by promoting the opening of the mPTP (mitochondrial permeability transition pore). Interestingly, melatonin supplementation suppressed mPTP opening and interrupted endothelial necroptosis via blocking the Ripk3-PGAM5-CypD signal pathways. Taken together, these studies identify the Ripk3-PGAM5-CypD-mPTP axis as a novel pathway that leads to reperfusion-mediated microvascular injury by initiating endothelial cell necroptosis.

The fluorescence intensity of vascular endothelial cadherin (VEcadherin), a junctional protein that maintains microvascular barrier function, decreased to approximately 75% of the baseline level after ischemia-reperfusion injury, but increased to approximately 97% of the baseline level after melatonin treatment or Ripk3 knockout (Figure 1A and C). However, Ad-Ripk3 transfection inhibited the melatonin-induced enhancement of VE-cadherin expression. Endothelial barrier dysfunction leads to capillary leakage and interstitial edema, which inevitably impairs tissue oxygen exchange and cardiomyocyte contraction. By immunohistochemical analysis of plasma albumin, the researchers found that ischemia-reperfusion injury caused more plasma albumin to leak from blood vessels into myocardial tissue, while Ripk3 knockout or melatonin treatment weakened the diffusion of plasma albumin to the outer surface of the vascular wall (Figure 1B-D). However, despite the use of melatonin treatment, Ad-Ripk3 treatment induced plasma albumin leakage again (Figure 1B-D).

Figure 1. Melatonin sustains microvascular barrier function via limiting Ripk3 expression. (Zhou H, et al., 2018)