Necrosis-Released HMGB1 (High Mobility Group Box 1) in the Progressive Pulmonary Arterial Hypertension Associated With Male Sex

Abstract

Damage-associated molecular patterns, such as HMGB1 (high mobility group box 1), play a well-recognized role in the development of pulmonary arterial hypertension (PAH), a progressive fatal disease of the pulmonary vasculature. However, the contribution of the particular type of vascular cells, type of cell death, or the form of released HMGB1 in PAH remains unclear. Moreover, although male patients with PAH show a higher level of circulating HMGB1, its involvement in the severe PAH phenotype reported in males is unknown. In this study, we aimed to investigate the sources and active forms of HMGB1 released from damaged vascular cells and their contribution to the progressive type of PAH in males. Our results showed that HMGB1 is released by either pulmonary artery human endothelial cells or human pulmonary artery smooth muscle cells that underwent necrotic cell death, although only human pulmonary artery smooth muscle cells produce HMGB1 during apoptosis. Moreover, only human pulmonary artery smooth muscle cell death induced a release of dimeric HMGB1, found to be mitochondrial reactive oxygen species dependent, and TLR4 (toll-like receptor 4) activation. The modified Sugen/Hypoxia rat model replicates the human sexual dimorphism in PAH severity (right ventricle systolic pressure in males versus females 54.7±2.3 versus 44.6±2 mm Hg). By using this model, we confirmed that necroptosis and necrosis are the primary sources of circulating HMGB1 in the male rats, although only necrosis increased circulation of HMGB1 dimers. Attenuation of necrosis but not apoptosis or necroptosis prevented TLR4 activation in males and blunted the sex differences in PAH severity. We conclude that necrosis, through the release of HMGB1 dimers, predisposes males to a progressive form of PAH.

Keywords: apoptosis; hypertension, pulmonary; hypoxia; necrosis; phenotype.

Figure 1.. Apoptosis induction in HPASMC and HPAEC.

The comparable level of apoptosis was induced in human pulmonary artery smooth muscle cells (HPASMC) by culturing them in a low-glucose media ((0.1g/L, A) and in human pulmonary artery endothelial cell (HPAEC) by culturing them in a serum-free media (B). The treatments did not induce significant necrotic cell death in any cell type. Data presented as Mean±SEM, **P<0.01, ***P<0.001 vs. untreated Control. N=6 in all groups.

Figure 2.. Necrosis and apoptosis of HPASMC and HPAEC in HMGB1 release and receptor activation.

Apoptotic HPASMC but not HPAEC release HMGB1. Necrosis produces a strong and comparable increase of the extracellular HMGB1 in both cell types (A, N=5). Conditioned media collected from apoptotic cells and used to treat the corresponding naïve cells induced transient activation of TLR4 in HPASMC but not HPAEC (B, N=5). Apoptotic media did not change the activity of RAGE in either cell type (C, N=5). Media collected from necrotic cells induced prolonged activation of TLR4 in HPASMC (D upper panel, N=5), while the activity of TLR4 in HPAEC was not changed (D lower panel, N=5). The stimulation of RAGE by necrotic media was early and transit in HPASMC (E upper panel, N=4) and delayed in HPAEC (E lower panel, N=4). Neutralization of HMGB1 prevents TLR4 or RAGE activation by conditioned media collected from necrotic HPASMC, confirming the importance of HMGB1 in receptor stimulation (F, N=13). Data presented as Mean±SEM, *P<0.05, **P<0.01, ***P<0.001 vs. untreated Control.

Figure 3.. The release of HMGB1 dimer from HPASMC is mitochondrial ROS dependent.

HPASMC but not HPAEC death induces a release of HMGB1 in a dimeric form (A, N=5). The elevated production of reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂, B, N=8) or mitochondrial superoxide (O₂⁻, C, N=8) found in HPASMC versus HPAEC, could be responsible for the oxidation of HMGB1 into dimers. However, attenuation of H₂O₂ levels by pretreatment HPASMC with NOX1/4 inhibitor, GKT137831, did not alter HMGB1 dimer/monomer levels in cells (D, N=6–8). In contrast, incubation of HPASMC in mitochondria-specific O₂⁻ scavenger, MitoTempol, reduced not only the levels of mitochondrial ROS but also HMGB1 dimer/monomer ratio (E, N=6–8). Data presented as Mean±SEM, *P<0.05, **P<0.01, ***P<0.001 vs. untreated Control; ^§P<0.05, ^§§§P<0.001 vs. necrosis in HPASMC.

Figure 4.. Model of progressive PAH in males.

To mimic the sex difference in PAH severity, rats were injected with a high dose of SU5416 (50 mg/kg s.c.) followed by 1 week of exposure to hypoxia (10% of O₂). There was a significant elevation of right ventricle (RV) systolic pressure (RVSP, A) and RV hypertrophy (B) in both sexes, although the changes in males were significantly more severe compared to females. At the same time, the initial sex difference in RV contractility (C) and relaxation function (D) was eliminated by PAH, suggesting that RV in males did not respond adequately to the demand. The contractility index (E) as an additional measure of RV performance was decreased in male but not female PAH rats. Data presented as Mean±SEM, ***P<0.001 vs. sex-matched Controls; ^§§P<0.01, ^§§§P<0.001 vs. Male Control. N=8 in all groups.

Figure 5.. An increased HMGB1 release in male PAH rats is fully attenuated only by anti-necrotic therapy.

PAH mediated a substantial increase in the levels of circulating HMGB1 in males, while in females the changes were significantly less pronounced and did not reach statistical significance compared to female Controls (A, N=6–8). The increased release of HMGB1 in males corresponded with significant activation of TLR4 in the lungs of male PAH rats, while females showed no changes in TLR4 in response to PAH (B, N=8). Both sexes had a significantly elevated expression of RAGE in the lungs of PAH rats (C, N=8). To evaluate the contribution of different types of cell death on the elevated HMGB1 release, male rats were treated with pan-caspase inhibitor Z-VAD(OMe)-FMK (Z-VAD), the selective blocker of necroptosis Necrostatin-1 (NCST), the inhibitor of necrosis Necrox-5 (NX-5), or vehicles (DMSO, used as vehicle in Z-VAD and NCST and ethanol (EtOH), a vehicle for NX-5). All treatments were given daily starting from the day of SU5416 injection and continued throughout the study (1 week). There were no significant changes in the amount of circulating HMGB1 monomers or dimers in Z-VAD treated rats (D, N=6). NCST significantly reduced the plasma levels of HMGB1 monomers but not dimers (E, N=6). NX-5 and its vehicle (EtOH) strongly attenuated the release of either HMGB1 monomers or dimers (F, N=6). Data presented as Mean±SEM, *P<0.05, **P<0.01, ***P<0.001 vs. sex-matched Controls; ^§P<0.05 vs. Male Su/Hx (A, B) or vs. untreated Su/Hx rats (F); ^§§P<0.01 vs. vehicle-treated rats (E, F).

Figure 6.. Hemodynamic changes and expression of TLR4 and RAGE in rats received anti-apoptotic, anti-necroptotic, and anti-necrotic therapy.

Treatment approaches that decrease necrotic cell death (Necrox-5 (NX-5) or its vehicle, ethanol (EtOH)) significantly attenuated RVSP in male PAH rats down to female level, thus eliminating the sex differences (A, N=6–8). RVSP in rats received anti-apoptotic (Z-VAD(OMe)-FMK (Z-VAD)), anti-necroptotic (Necrostatin-1 (NCST)) treatments or their vehicle, DMSO, remained unchanged. NX-5 and EtOH significantly attenuated RV hypertrophy (B, N=6–8) compared to untreated Su/Hx male rats and even below Su/Hx female level (NX-5). Although Z-VAD and NCST treatment slightly reduced RV/LV+S ratio compared to untreated Su/Hx male rats, the changes were not significantly different versus the group treated by the vehicle (DMSO). NX-5 and EtOH but not any other interventions significantly reduced RV contractility (dP/dt max) in male PAH rats (C, N=6–8). NX-5 attenuated RV dP/dt min (D, N=6–8), suggesting a reduced RV load in these rats. Induction of PAH in male rats corresponded with a significant upregulation of TLR4 and RAGE expression in the lungs. The expression of TLR4 (E, N=5) maintained significantly high in the rats treated by anti-apoptotic ((Z-VAD(OMe)-FMK (Z-VAD)), anti-necroptotic (Necrostatin-1 (NCST)) agents or their vehicle (DMSO), but stopped being significant and was found to be reduced down to female levels in the lung of rats treated by an anti-necrotic agent (Necrox-5 (NX-5)) or low dose of ethanol (EtOH). The expression of RAGE (F, N=6) remained significantly upregulated in all treatment groups and was increased in PAH females. Data presented as Mean±SEM, *P<0.05, **P<0.01, ***P<0.001 vs. untreated Su/Hx male rats (A-D) or vs. healthy male Controls (E, F); ^§P<0.05 vs. ^§§P<0.01, ^§§§P<0.001 vs. Su/Ha female rats (A-D).