Caspase-1 Inflammasome Activation Mediates Homocysteine-Induced Pyrop-Apoptosis in Endothelial Cells
Rationale: Endothelial injury is an initial mechanism mediating cardiovascular disease.
Objective: Here, we investigated the effect of hyperhomocysteinemia (HHcy) on programed cell death in endothelial cells (EC).
Methods and Results: We established a novel flow-cytometric gating method to define pyrotosis (Annexin V-/Propidium iodide+). In cultured human EC, we found that: 1). Hcy and Lipopolysaccharide (LPS) individually and synergistically induced inflammatory pyroptotic and non-inflammatory apoptotic cell death. 2). Hcy/LPS induced caspase-1 activation prior to caspase-8, -9, -3 activations. 3). Caspase-1/3 inhibitors rescued Hcy/LPS-induced pyroptosis/apoptosis, but caspase-8/9 inhibitors had differential rescue effect. 4). Hcy/LPS induced NLRP3 protein, caused NLRP3-containing inflammasome assembly, caspase-1 activation and IL-1β cleavage/activation. 5). Hcy/LPS elevated intracellular reactive oxidative species (ROS). 6). Intracellular oxidative gradient determined cell death destiny as intermediate intracellular ROS levels are associated with pyroptosis, whereas, high ROS corresponded to apoptosis. 7). Hcy/LPS induced mitochondrial membrane potential collapse and cytochrome-c release, and increased Bax/Bcl-2 ratio which were attenuated by antioxidants and caspase-1 inhibitor. 8). Antioxidants extracellular superoxide dismutase and catalase prevented Hcy/LPS-induced caspase-1 activation, mitochondrial dysfunction and pyroptosis/apoptosis. In cystathionine β-synthase deficient (Cbs-/-) mice, severe HHcy induced caspase-1 activation in isolated lung EC and caspase-1 expression in aortic endothelium, and elevated aortic caspase-1,9 protein/activity and Bax/Bcl-2 ratio in Cbs-/- aorta and HUVEC. Finally, Hcy-induced DNA fragmentation was reversed in caspase-1-/- EC. HHcy-induced aortic endothelial dysfunction was rescued in caspase-1-/- and NLRP3-/- mice.
Conclusions: HHcy preferentially induces EC pyroptosis via caspase-1-dependent inflammasome activation leading to endothelial dysfunction. We termed caspase-1 responsive pyroptosis and apoptosis as pyrop-apoptosis.
- Received February 5, 2016.
- Revision received March 15, 2016.
- Accepted March 22, 2016.