High mobility group box protein-1 preconditions isolated rat hearts and protects human pulmonary and renal cells against hypoxic injury

Introduction

High mobility group box protein-1 (HMGB) is released by activated monocytes/macrophages during sepsis. Levels correlated with mortality in patients and anti-HMGB antibodies prevented endotoxin-induced death in animal models [1]. In marked contrast to these findings are reports of cytoprotective effects because HMGB protected liver from ischemia/reperfusion (IR) [2] and HMGB-transgenic mice were resistant to myocardial infarction [3]. Despite these reports, little is known of the role or mechanism of HMGB. We aimed to further evaluate HMGB in rat hearts and in pulmonary and renal cell cultures.

Methods

Isolated hearts received HMGB/vehicle before 30 minutes ischemia and 120 minutes reperfusion. In separate studies, rats were rendered septic by caecal ligation and puncture (CLP) prior to IR. Left ventricular (LV) function was measured with a latex LV balloon. Infarct size was measured by TTC staining. For human cell culture studies, A549 alveolar cells or HK-2 renal tubular cells were treated with HMGB/vehicle before incubation in normoxia or hypoxia. Fluorimetric caspase-3 activity was used as measure of apoptosis. Cytochrome C was measured by ELISA. For HK-2 cells, the MTT assay was used to test viability.

Results

HMGB, prior to cardiac IR, preserved developed (d) LVP at 120 minutes reperfusion compared with controls (44 mmHg vs 27 mmHg, P < 0.05) and infarct size, expressed as %V weight ± SEM, was reduced (31 ± 5.4 vs 44 ± 4.2, P < 0.05), n = 6/group. Hearts from CLP rats had reduced baseline dLVP (59 mmHg ± 5.1 vs 97 ± 5.3, P < 0.05) but infarct sizes after IR were significantly reduced (27 ± 8.8, P < 0.05 vs 44 ± 6.2, P < 0.05). In A549 cells, HMGB inhibited apoptosis (49% reduced caspase-3 activity (8.6 vs 17.1 a.u., P < 0.01)). This was associated with reduced cytochrome C release (250 vs 389 pg/ml, P < 0.05). Using MTT assay, HMGB protected against hypoxia-induced cell death vs controls (viability 75% vs 61%, P < 0.05).

Conclusions

HMGB, or sepsis, similarly precondition the heart against IR injury. HMGB has anti-apoptotic effects that protect against hypoxic renal and alveolar cell injury. We conclude that HMGB has potent anti-ischemic effects in multiple organs and may function as an innate cytoprotective mediator in sepsis.