Objective The role of receptors for endogenous metabolic danger signals-associated molecular patterns (DAMPs) has been characterized recently as bridging innate immune sensory systems for DAMPs to initiation of inflammation in bone marrow-derived cells such as macrophages. However, it remains unknown whether endothelial cells (ECs), the cell type with the largest numbers and the first vessel cell type exposed to circulating DAMPs in the blood, can sense hyperlipidemia. This report determined whether caspase-1 plays a role in ECs in sensing hyperlipidemia and promoting EC activation. Approach and Results Using biochemical, immunological, pathological and bone marrow transplantation methods together with the generation of new apoplipoprotein E (ApoE)−/−/caspase-1−/− double knock-out mice we made the following observations: 1) early hyperlipidemia induced caspase-1 activation in ApoE−/− mouse aorta; 2) caspase-1−/−/ApoE−/− mice attenuated early atherosclerosis; 3) caspase-1−/−/ApoE−/− mice had decreased aortic expression of pro-inflammatory cytokines and attenuated aortic monocyte recruitment; and 4) caspase-1−/−/ApoE−/− mice had decreased EC activation including reduced adhesion molecule expression and cytokine secretion. Mechanistically, oxidized lipids activated caspase-1 and promoted pyroptosis in ECs by a ROS mechanism. Caspase-1 inhibition resulted in accumulation of sirtuin 1 (Sirt1) in the ApoE−/− aorta, and Sirt1 inhibited caspase-1 upregulated genes via activator protein-1 (AP-1) pathway. Conclusions Our results demonstrate for the first time that early hyperlipidemia promotes EC activation before monocyte recruitment via a caspase-1-Sirt1-AP-1 pathway, which provides an important insight into the development of novel therapeutics for blocking caspase-1 activation as early intervention of metabolic cardiovascular diseases and inflammations.

Interleukin‐35 (IL‐35), a recently discovered heterodimeric cytokine with anti‐inflammatory/immunosuppressive properties, has a central role in limiting the immune response in various disease models including colitis, arthritis and asthma. However, it remains unknown whether IL‐35 is different from other anti‐inflammatory cytokines such as IL‐10 and transforming growth factor (TGF)‐β in terms of inhibition of inflammation initiation or suppression of full‐blown inflammation. In this study, we examined the tissue expression profiles and regulatory mechanisms of IL‐35 in comparison to other anti‐inflammatory cytokines. Our results suggest that in contrast to TGF‐β, IL‐35 is not constitutively expressed in human tissues but is inducible in response to inflammatory stimuli. We also provide structural evidence suggesting that AU‐rich element (ARE) binding proteins and microRNAs target IL‐35 subunit transcripts, which are responsible for quick degradation of IL‐35. Furthermore, we propose a new system to categorize anti‐inflammatory cytokines into two groups: (1) the housekeeping cytokines, such as TGF‐β, inhibit the initiation of inflammation whereas (2) the responsive cytokines including IL‐35 suppress inflammation in full‐blown stage. Our in‐depth analysis of molecular events that regulate the production of IL‐35 and new categorization system of anti‐inflammatory cytokines are important for the design of new strategies of immune intervention. This work was partially supported by the National Institutes of Health Grants HL094451 and HL108910 (XFY), HL67033, HL82774 and HL77288 (HW).

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. Conclusion 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.

IL-35 is induced during atherosclerosis development and inhibits mitochondrial reactive oxygen species-H3K14 acetylation-AP-1-mediated EC activation.

Background: Interleukin-35 is a novel inhibitory cytokine. Results: Interleukin-35 inhibits vascular endothelial cell activation by suppressing MAPK-AP1-mediated VACM-1 expression in LPS-induced acute inflammation. Conclusion: Interleukin-35 suppresses acute vascular endothelium response. Significance: Interleukin-35 may be an attractive reagent for anti-inflammatory therapy.