Early progression of pulmonary hypertension in the monocrotaline model in males is associated with increased lung permeability

Abstract

Background: The mechanisms involved in pulmonary hypertension (PH) development in patients and pre-clinical models are poorly understood. PH has a well-established sex dimorphism in patients with increased frequency of PH in females, and more severe disease with poor survival prognosis in males. Previously, we found that heme signaling plays an essential role in the development phase of the Sugen/Hypoxia (SU/Hx) model. This study is focused on the elucidation of sex differences in mechanisms of PH development related to heme action at the early stage of the monocrotaline (MCT) PH model.

Methods: Rats received MCT injection (60 mg/kg, i.p.) and followed for 14 days to investigate early disease changes. Hemodynamic parameters were recorded at the end of the study; plasma, lung homogenates, and nuclear fractions were used for the evaluation of protein levels.

Results: Our data indicate that on day 14, rats did not show any significant increase in the Fulton index due to the early disease phase. However, the right ventricular systolic pressure was significantly increased in male rats, while female rats showed only a trend. Interestingly, only males demonstrated an increased lung-to-bodyweight ratio that indicated lung edema. Indeed, lung histology confirmed severe perivascular edema in males. Previously, we have reported that the increased perivascular edema in SU/Hx model correlated with intravascular hemolysis and activated heme signaling. Here, we found that elevated free hemoglobin levels and perivascular edema were increased, specifically in males showing more rapid progress of PH. A high level of heme carrier protein 1 (HCP-1), which is involved in heme uptake from the bloodstream into the cells, was also found elevated in the lungs of males. The upregulation of heme oxygenase in males indicated increased intracellular heme catabolism. Increased heme signaling resulted in the activation of heme-mediated barrier-disruptive mechanisms. Thus, hemolysis in males can be responsible for increased permeability of the lungs and early disease development.

Conclusions: Our study indicates the importance of barrier-disruptive mechanisms as an earlier event in the induction of pulmonary hypertension. Importantly, males are more susceptible to hemolysis and develop PH earlier than females.

Keywords: Endothelial barrier function; Heme signaling; Lung permeability; Pulmonary hypertension; Sex difference.

Fig. 1

Hemodynamic changes in 14 days MCT model. Right ventricular (RV) systolic pressure found to be elevated significantly only in males treated with MCT. a Fulton index at this early stage of PH was unchanged between groups. b Contractility of the right heart (dP/dtmax) was significantly reduced in males, but not in females. c Relaxation parameter (dP/dtmin) did not reach statistical significance in males and was unaffected in females. d Mean ± SEM, N = 6, *p < 0.05 vs control, two-way ANOVA

Fig. 2

Lung edema in males treated with MCT. Lung weight normalized on the bodyweight showed a significant increase in males due to liquid accumulation in the lungs and indicated lung edema (a). Histology showed the formation of perivascular cuffs pronounced in male rats treated with monocrotaline (arrows) (b). Quantification of vessel wall thickness shows a significant increase in males (c) mean ± SEM, N = 6, **p < 0.01, ***p < 0.001 vs control, two-way ANOVA

Fig. 3

Hemolysis in early MCT model. Free hemoglobin signal in plasma showed a significant increase in male rats, but not female (a). Heme transporter, heme carrier protein 1 (HCP1), expression increased only in males (b). Inducible heme oxygenase (HO1) was activated in males (c). Mean ± SEM, N = 4–6, *p < 0.05,**p < 0.01 vs control, t test

Fig. 4

Translocation of transcriptional factors (TF). Nuclear fractions that were isolated from lung tissue were used to screen the activation of different transcription factors. It was found that the factor responsible for heme-mediated response, NFE2L2 (or NRF2), was activated in males only (a). Transcription suppressor BACH1 was unchanged (b). Energy regulating TF, NRF1, was also activated in males (c). Nuclear proteins were normalized over their respective total proteins (stain-free). Mean ± SEM, N = 4, *p < 0.05 vs control, t test

Fig. 5

Heme-mediated actin remodeling mechanisms in the MCT model. Our data indicate that the level of HSP27 involved in actin fibers formation was highly elevated in the lungs of male rats after MCT treatment (a). This correlated with increased phosphorylation of MLC (b). Barrier protective small GTPase, Rac1, was significantly decreased in MCT males (c). Mean ± SEM, N = 4, *p < 0.05,**p < 0.01 vs control, t test

Fig. 6

Heme-mediated effects on tight junctions. Proteins involved in the formation of tight junctions, claudins1/5, and ZO-1 were significantly reduced in MCT male rats (a, b, c). Mean ± SEM, N = 4, *p < 0.05,**p < 0.01 vs control, t test

Fig. 7

Overall mechanisms of heme-induced barrier dysfunction. During hemolysis, hemoglobin releases its co-factor, free heme. Through the heme carrier protein 1 (HCP1), free heme translocates into cells, leading to the activation of heme oxygenase and barrier disruptive mechanisms. Heme affects the barrier in two phases. First, heme induces actin fibers formation by upregulation of HSP27, inactivation of barrier protective MYPT and Rac1, leading to increased phosphorylation of MLC. The second phase is the dysregulation of tight junctions (TJ) between cells resulting in fluid leakage through the endothelial barrier. These effects of free heme contribute to the development of pulmonary hypertension via inflammation, vasoconstriction and local hypoxia due to the formation of perivascular cuffs