Pulmonary hypertension (PH) is a fatal disease characterized by increased pulmonary arterial pressure and abnormalities in blood vessel growth. While current therapies modestly improve functional capacity, they have marginal impact on long- term survival, perhaps because they neglect pathogenesis. Despite compelling evidence for thrombotic and inflammatory mechanisms in PH, no drugs have been developed to target these mechanisms. We recently reported that high mobility group box 1 (HMGB1) is elevated in human and murine PH contributing to the pathogenesis via toll like receptor 4 (TLR4)-dependent mechanisms. We also reported on the role of the complement system in PH. Activation of the complement cascade or TLR4 induces a pro-inflammatory, pro-adhesive, pro-coagulant phenotype and studies point to a cross talk between TLRs and the complement system. Preliminary data suggest that (1) HMGB1 stimulates TLR4 on platelets thus contributing to PH, (2) there is cross talk between platelet TLR4 and the complement system and (3) that platelets are a source of HMGB1 in PH. These data form the basis of our overarching hypothesis that HMGB1-dependent activation of platelet TLR4 causes platelet activation, secretion, platelet-dependent complement activation, and HMGB1 release thus contributing to the pathogenesis of PH.
In Aim 1 we will characterize the effect of HMGB1 on human and mouse platelet activation, secretion and aggregation as well as the HMGB1 isoform responsible for this effect. The TLR4- dependence of HMGB1-stimulated platelet activation will be explored. A role for mitochondrial ROS as a downstream signaling mechanism in HMGB1/TLR4-dependent platelet activation will be investigated. Transfusion of WT platelets into TLR4-/- mice will confirm a role for platelet TLR4 in hypoxia-induced PH. A novel HMGB1 inhibitor that specifically blocks HMGB1/TLR4 interaction will further identify the role of HMGB1/TLR4 interactions in PH.
In aim 2 we will examine if loss of platelet TLR4 attenuates complement activation in hypoxia-induced PH. We will test the ability of platelets activated by TLR4 agonists to activate complement and if these agonists can enhance platelet activation by complement components. Finally, we will explore the mechanism(s) by which TLR4 mediates C3a and C5a-dependent platelet activation.
In Aim 3 we will explore whether HMGB1 released from platelets contributes to the pathogenesis of PH using platelet specific HMGB1 knockout mice. Transfusion of WT platelets back into platelet specific HMGB1-/- mice will confirm a role for platelet HMGB1 in hypoxia-induced PH. Intracellular and platelet surface HMGB1 levels will be measured in platelets from patients to determine if platelets release HMGB1 in human PAH. It is our goal to define the pathophysiology of the platelet and immune system abnormalities in PH, and in so doing, apply the knowledge to improve the care of PH patients.
Pulmonary hypertension is a progressive and fatal disease with no known cure. Recent studies from our laboratory have identified a role for the innate immune mediator HMGB1 and its receptor, TLR4, in the pathogenesis of the disease. The proposed experiments are aimed at elucidating a role for HMGB1 and platelet TLR4 in mediating platelet and complement activation in pulmonary hypertension. Additional experiments will test whether platelets are a source of circulating HMGB1 in pulmonary hypertension.
