Interference With TNFα Using Long-Term Etanercept In S+S^Antilles Sickle Transgenic Mice Ameliorates Abnormal Endothelial Activation, Vasoocclusion, and Pulmonary Hypertension Including Its Pulmonary Arterial Wall Remodeling

A systemic inflammatory state is a prominent feature of sickle cell anemia. Using sickle transgenic mice, we previously identified a blood monocyte-TNF-endothelium activation axis using multiple complementary approaches. For example, endothelial activation (identified by abnormal expression of VCAM-1 and tissue factor (TF) by pulmonary vessel endothelium) was evident in unstressed BERK and S+S^Antilles sickle mice. In NY1DD sickle mice, this was triggered by transient enhanced sickling and vasoocclusion resulting from transient exposure to hypoxia/reoxygenation (H/R). Endothelial activation depended upon Egr-1 and NFkB(p50) in peripheral blood mononuclear cells, and transfusion experiments clarified that the peripheral blood monocytes (PBM) were the responsible effecter cells. Finally, interference with TNFa using etanercept (Etn) prevented this PBM-induced endothelial activation. Based on these background data, we have now tested the “clinical” efficacy of long-term, anti-inflammatory TNFa interference via treatment of S+S^Antilles sickle mice with Etn (started at weaning, 3 mg/kg sc, once/wk x 6 wk). In parallel, littermate controls received equimolar scrambled peptide version of Etn's TNF-receptor moiety. In response we observed significant improvement in multiple parameters relevant to inflammation-induced vessel wall disease in this model, as follows (data shown in inset). Etn decreased blood markers of inflammation: plasma levels of sVCAM-1 and SAP (by ELISA), and MCP-1 and TNFa (by FACS cytobead assay). Etn also decreased the % of PBM positive for TNFa (by FACS). Etn diminished the abnormal pulmonary vessel endothelial expression of VCAM-1 and TF (by histochemical and immunofluorescent staining). Etn eliminated the enhanced vasoocclusion seen as dermal microvascular stasis in the live mouse (using the dorsal skin fold chamber) measured at 1 and 4 hours post-H/R (7%O₂x1hr, followed by 21%O₂x 1-4hr). Instructively, Etn did not significantly improve the similar stasis induced by heme infusion, a TNF-independent direct endothelial stressor. Finally, Etn diminished evidence for pulmonary hypertension, assessed via three of its hallmark features: elevated right ventricular mean systolic pressure (by RV catheterization), abnormal peri-vascular inflammatory cell aggregates (by immuno-histochemical staining for CD11b), and abnormal pulmonary arterial wall muscularization (by immuno-histochemical staining for SM actin). Remarkably, Etn actually normalized RVSP, and Etn's partial improvement in pulmonary arterial muscularization resulted not only from prevention of its progression over duration of this study but also from some reversal of the abnormal remodeling that had already developed by the time Etn was started. In parallel, Etn reduced the number of abnormal peri-vascular aggregates of CDllb+ cells in the pulmonary arterial system.

These results enable several conclusions. In the S+S^Antilles sickle model, endothelial activation and aspects of vascular wall pathobiology derive from TNFa produced by activated PBM, a monocyte-TNF-endothelial activation axis. Interference with TNFa using etanercept exerts multiple salubrious vascular effects that predict possible clinical benefit. And results suggest that inflammation involving TNFa plays a proximate, generative role in development of pulmonary hypertension in this model. Assuming these benefits are independently corroborated, a controlled test of etanercept's efficacy for aspects of human sickle vascular pathobiology perhaps should be considered.

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