PH is classically described as a disease of endothelial dysfunction involving pulmonary vasoconstriction and pulmonary vascular remodeling, leading to increased pulmonary vascular resistance. However, clinical data demonstrate that despite advances in therapeutics aimed at promoting pulmonary vasodilation, survival remains poor (30
). These data highlight the necessity of novel approaches to treating PH. There is mounting evidence of a role for the immune system in the pathogenesis of PH. A role for inflammation in human PH is based on the identification of inflammatory cells in vascular lesions of PH patients (31
), elevated circulating levels of inflammatory cytokines in PH patients (32
) and case studies in PH patients demonstrating improvement with antiinflammatory drugs (33
). In the mouse model of CH-induced PH, there is an influx of inflammatory cells within the first 48 h that diminishes by 2 wks. This increase in inflammatory cells coincides with increased secretion of proinflammatory cytokines, including TNFα, interleukin (IL)-1β, IL-6 and MCP-1 (35
). In addition, IL-6 overexpression promotes, and genetic deletion of IL-6 attenuates, PH in this model (36
A key facet of the innate immune response is self-/nonself recognition. Through the use of PRRs, the innate immune system is capable of detecting conserved pathogen motifs, termed pathogen-associated molecular patterns (PAMPs), thereby stimulating an immune response (38
). In recent years, however, it was acknowledged that the self-/nonself paradigm fails to explain innate immune activation in diseases involving sterile inflammation (39
). The discovery of DAMPs, which have a similar function to PAMPs, has provided a context for understanding innate immune activation in the absence of foreign pathogens (9
). HMGB1 fulfills the functions of a DAMP, being involved in inflammation caused by both infectious and noninfectious stimuli (40
). Once released, HMGB1 signals through various PRRs to activate immune and parenchymal cells involved in the immune process (11
Our studies clearly establish a role for extracellular HMGB1 in the pathogenesis of CH-induced PH in mice. In particular, data showing translocation of HMGB1 into the extracellular milieu and that HMGB1-neutralizing antibody attenuates CH-induced PH show the necessity of extracellular HMGB1 to fully develop the disease. HMGB1 may have additional intracellular roles in PH; however, the strategies used in this study likely do not address these roles. The clinical relevance of these data is indicated by the observations that (a) there is a diffuse extranuclear staining pattern of HMGB1 in pulmonary vascular lesions of PH patients and (b) there is increased circulating HMGB1 in PH patients, which correlates with mean pulmonary arterial pressure.
In addition to our findings in mice and humans, it was also reported, in the form of an a published abstract, that there is increased circulating HMGB1 in monocrotaline rats and that HMGB1 is released from smooth muscle cells and alveolar macrophages in this model (41
). The consistency of this phenomenon (increased circulating HMGB1) across mice, rats and humans suggests that HMGB1 is an important mediator of PH across species. Future studies using similar strategies (that is, HMGB1-neutralizing antibodies) in other experimental PH models, such as monocrotaline and the SU5416/hypoxia model in rats, will be important to support this hypothesis.
A role for HMGB1-TLR4 interaction in driving immunopathology was initially described in 2005. In that study, it was found that an HMGB1-neutralizing antibody protects WT mice (C3H/HeouJ) but not Tlr4−/−
mice (C3H/HEJ) from liver ischemia reperfusion injury (42
). Since then, several studies have demonstrated a role for HMGB1–TLR4 interactions in the immunopathology of diseases, including inflammation-induced seizures (43
), inflammation-induced skin cancer (44
), end organ injury after tissue trauma (45
) and ischemic kidney injury (46
). Our study establishes a role for HMGB1 and TLR4 interaction in the pathogenesis of PH. Genetic deletion of TLR4 attenuated CH-induced PH, as assessed by measuring RVSP, RVH and pulmonary vascular remodeling. Furthermore, exogenous HMGB1 exacerbated CH-induced PH in WT but not Tlr4−/−
mice, establishing a link between HMGB1 and TLR4 in PH. Consistent with a role for HMGB1 and TLR4 in endothelial activation and inflammation, HMGB1 neutralizing antibody or loss of TLR4 also prevented hypoxia-induced increases in endothelin-1 and soluble ICAM-1. Interestingly, however, treating mice with exogenous HMGB1 during hypoxia exacerbated the increase in ET-1 but not sICAM-1 in WT mice. This result suggests that HMGB1 may directly contribute to the elevation of ET-1 in PH. Indeed, human pulmonary artery endothelial cells treated with HMGB1 secreted more ET-1 than untreated cells, which was attenuated by TLR4-neutralizing antibody. TLR4 agonists have also been shown to induce production of ET-1 by dendritic cells (47
). The effect of the HMGB1-neutralizing antibody or genetic deletion of TLR4 on sICAM levels would appear to be indirect, since exogenous HMGB1 did not further increase sICAM. Alternatively, it is possible that sICAM expression is maximally induced by endogenous HMGB1 and cannot be further induced by addition of the exogenous protein.
In addition to its effects on ET-1 and ICAM-1 expression, recent data from our laboratory demonstrate that HMGB1 is a potent inhibitor of endothelial cell migration, suggesting one mechanism may be the inhibition of endothelial repair mechanisms (17
). In addition, in that study, we show that hypoxia induces the release of HMGB1 from endothelial cells, suggesting that endothelial cells could be one source of increased circulating HMGB1 in PH. The HMGB1/TLR4 signaling axis has been shown to stimulate neutrophil NADPH oxidase (NOX2) in both neutrophils and lung microvascular endothelial cells, and NOX2 is thought to be important in the pathogenesis of PH (48
). TLR4 is also expressed on multiple other cell types important in the pathogenesis of PH. HMGB1 induces macrophages to secrete proinflammatory cytokines in a TLR4-dependent manner (49
). Studies also demonstrate that TLR4 is found on platelets and that activation of platelet TLR4 promotes platelet aggregation (50
), which may promote in situ
thrombosis, a feature of human IPAH. Future experiments using bone marrow chimeric mice and tissue-specific knockouts will help reveal cell-specific mechanisms by which TLR4/HMGB1 signaling promotes PH.
We also explored the possibility that RAGE contributes to the development of CH-induced PH. RAGE is highly enriched in the lung (51
) and has been reported to be a important receptor for HMGB1 (53
). Interestingly Rage−/−
mice developed the same degree of PH (as determined by RVSP) as WT mice when exposed to CH, suggesting that RAGE does not contribute to increased pulmonary vascular resistance in CH- induced PH. This was corroborated by data showing that CH induced a similar degree of pulmonary vascular remodeling in WT and Rage−/−
mice. Despite these observations, CH-induced RVH was significantly attenuated in Rage−/−
mice when compared with WT mice. These data suggest that RAGE is involved in the hypertrophic response of the right heart to increased pulmonary arterial pressure. This result is consistent with a previous study demonstrating a role for RAGE in mediating cardiac hypertrophy in mice fed a Western diet (56
). Whereas the attenuated RVH in Rage−/−
mice is an important observation, experiments interrogating a role for RAGE in RVH are beyond the scope of the current study.