View Abstract

A4627 - Therapeutic Targeting of High Mobility Group Box 1 in Pulmonary Hypertension
Author Block: N. M. Goldenberg1, Y. Hu2, A. Volchuk2, G. Wei2, Y. Zhao3, X. Hu2, K. J. Tracey4, Y. Al-Abed5, M. de Perrot6, B. E. Steinberg1, W. M. Kuebler7; 1Anesthesia and Pain Medicine, The Hospital For Sick Children, Toronto, ON, Canada, 2Keenan Research Centre, St. Michael's Hospital, Toronto, ON, Canada, 3Thoracic Surgery Labs, UHN, Toronto, ON, Canada, 4President and CEO, The Feinstein Institute for Medical Research, Manhasset, NY, United States, 5Centre for Molecular Innovation, The Feinstein Institute for Medical Research, Manhasset, NY, United States, 6Division of Thoracic Surgery, Toronto General Hospital, Toronto, ON, Canada, 7Institute of Physiology, Charite - Universitaetsmedizin, Berlin, Germany.
RATIONALE: There has been an increasing appreciation for the importance of inflammation in the pathogenesis of pulmonary hypertension (PAH). Previous work has investigated the role of the alarmin, high mobility group box 1 (HMGB1), in the pathogenesis of PAH. We sought to assess the importance of HMGB1 in human PAH patients and in rodent models. Furthermore, we tested a small molecule inhibitor of toll-like receptor-4 (TLR4)-mediated HMGB1 signalling for potential use as a novel therapeutic agent.
METHODS: Human studies were performed on explanted lungs at the time of transplant (PAH) or in lung resections for cancer (controls), and included Western blotting, RT-PCR and histology. Monocrotaline (MCT) and Sugen-hypoxia rat models were employed. Either anti-HMGB1 monoclonal antibody or peptide inhibitor were given at the assay start (prevention) or after established disease (treatment). Endpoint assays included right ventricular systolic pressure (RVSP), echocardiography, lung histology and Western blotting. In vitro work was performed on primary human macrophages, pulmonary artery endothelial cells (PAEC) or smooth muscle cells (PASMC).
RESULTS: Our human studies indicated that HMGB1 protein and mRNA expression are elevated in the lungs of patients with PAH sampled at the time of lung transplantation, as compared to control patients undergoing lung resection for cancer. Additionally, HMGB1 staining was increased in plexiform lesions in PAH patients, and colocalized significantly with perivascular macrophages. In the Sugen-hypoxia model, rats treated with anti-HMGB1 antibody had significant improvements in RVSP, lung vascular remodelling, and right ventricular function. Our small molecule, which blocks the specific interaction between HMGB1 and TLR4 via the adaptor MD2, was efficacious at both preventing the development of PAH in the MCT rat model, and in treating established disease in the same model. Additionally, the small molecule inhibitor was also effective at treating established disease in the Sugen-hypoxia model. In vitro studies showed that primary human macrophages release HMGB1 into the extracellular fluid in response to hypoxia. PAEC or PASMC do not exhibit this response, suggesting that macrophages may represent the source of circulating HMGB1 in our animal models. The addition of recombinant HMGB1 to PASMC and PAEC stimulated their proliferation and migration.
CONCLUSIONS: Taken together, these data suggest that HMGB1, released from macrophages, exerts a deleterious effect upon lung parenchyma, resulting in cellular changes consistent with the pulmonary vasculopathy seen in PAH. Importantly, PAH can be treated in animals by attenuating HMGB1 signalling using either anti-HMGB1 monoclonal antibody or peptide inhibitor.