Influence of gender in monocrotaline and chronic hypoxia induced pulmonary hypertension in obese rats and mice

Pulmonary hypertension (PH) is a disease characterized by increased pulmonary vascular resistance, alteration of the normal vascular cellular processes and inflammation in the lungs, which leads to right heart failure and death [1]. Obesity, a condition with the excessive adipose tissue accumulation in the body, has been previously shown to be associated with cardiovascular and pulmonary diseases. Obesity and obesity related complications are regarded to have impact on developing PH and early mortality [2, 3]. However, no clear data exists in connection to PH and obesity [4, 5]. Interestingly, conflicting effects of obesity were seen in patients diagnosed with pulmonary arterial hypertension (PAH) after first or third year of PAH diagnosis. The mortality in the first year after PAH diagnosis was higher, while it was significantly lower in obese patients after third year of diagnosis [6]. The role of obesity in PH is still unclear suggesting it neither as a friend nor as a foe. Systematic research, therefore, is needed to establish an apparent association between these two conditions.

Cardiac dysfunction and pulmonary vascular remodeling in PH have also been shown to be affected by gender and female sex hormones [7, 8]. Previously known to be predominantly present in younger females few decades ago, PAH recently has an even occurrence ratio in males and females [9]. Interestingly, female survival rate after the first diagnosis of PH is higher than in males [10]. Similarly, pre-clinical studies in rats with monocrotaline induced PH have shown less severe condition in female rats which has been attributed to their higher antioxidant defense capacity compared to male [8]. A recent study shows better long-term survival rate in females who have chronic thromboembolic PH, albeit the short term survival rates were identical in both genders [11]. This noticeable heterogeneity of females over males also requires further research to explore the gender specific alteration concerning obesity and PH.

Furthermore, a broad spectrum of inflammatory mediators and oxidative stress plays a critical role in both pre-clinical and clinical forms of PH [12, 13]. Similarly, obesity is responsible to trigger inflammation and oxidative stress leading to increased endothelial dysfunction and contributing to cardiovascular disease [14]. This suggests that a possible intervention on inflammatory pathway might be an effective future target for the treatment of PH.

Up-to-date, research evidences are not conclusive enough to understand the mechanisms and relationship between obesity and PH. This study addresses both the relation between obesity and PH and the differences between male and female obesity associated with PH. We have used well-established mouse and rat models of PH with both genders.

Globally, around 107.7 million children and 603.7 million adults were obese in 2015 with a prevalence of 5 and 12% in children and adults, respectively [17]. Though the data are insufficient about the prevalence of PH in obese people, it has been shown that 5% of people with body mass index (BMI) > 30 kg/m² had PH with pulmonary artery systolic pressure (PASP) > 40 mmHg, as measured echocardiographically [18]. In addition Wong et al. showed the increased severity of right ventricular (RV) dysfunction with increasing BMI [19]. Although obesity is a growing worldwide problem and is associated with many diseases, there is an ongoing debate if some underlying factors of obesity have beneficial effect in heart disease.

Obesity is featured by excessive adipose tissue accumulation in the body. These adipose tissues release adipokines [20, 21]. Recently, it has been shown that there is a relation between adipokine dysregulation and hemodynamic disorders in PAH [22]. Obesity is associated with low mortality in some PH patients indicating it might have a protective effect [23]. Diong et al. have demonstrated that sympathetic nerve activity which facilitates the pulmonary vasodilatation was increased in obesity or chronic hypoxia. This hyperactivity may help to decrease the severity of PH [24].

The role of obesity is obviously still unclear in relation to PH. A retrospective case control study regarding the association between obesity and PH did not show any correlations with class I obesity. However, there was a slight indication of possible connection with class II or class III obesity [25]. Animal experiments in relation to gender have shown that estrogens have protective role in animal models of PH [26].

Mild increment of pulmonary arterial pressure, RV hypertrophy and pulmonary artery thickening have been shown in Zucker diabetic fatty rats [27]. Irwin and coworkers demonstrated the development of PH in chow-fed Zucker rats. Adipose tissue and liver acted as a source for circulating free fatty acids and triglycerides respectively [4]. When the clearance of lipoproteins by the liver is hindered, there is increase in fat delivery to the pulmonary arterial wall affecting vascular structure and function. The inefficient oxidation of fatty acids results in the production of reactive oxygen species [28, 29]. Likewise Irwin et al. also showed that PH in Zucker rats showed no inflammatory cytokines but rather the obese rat model with high fat diet showed the inflammatory cytokines increment [4]. However, in the present study, we used MCT to induce PH and demonstrated that obesity did not influence the disease severity in MCT-induced PH in male Zucker rats. In details, our data revealed that the parameters of right ventricular hypertrophy/remodeling (RVID, RVWT and RV/(LV + S)) and function (TAPSE) were comparable between male MCT obese Zucker rats and their lean controls. We have shown previously that PAAT was reduced in MCT rat model and this parameter negatively correlated with RVSP [16]. In the present study, obesity did not change the value of PAAT/PAET in MCT treated male rats. Similarly, there was no influence of obesity in male MCT rats with regard to the pulmonary vascular remodeling, which represents the hallmark of PH pathology, as evident from the medial wall thickness and muscularization measurements. Finally, our hemodynamic data indicated that obesity did not affect one of the most important characteristic features of PH, such as RVSP, in MCT treated animals. In addition, there were no prominent changes in SAP due to obesity. There was only slight reduction of SAP in MCT obese rats in comparison to their saline control, and this might appear because MCT injected animals develop a severe pulmonary vascular disease. However, after MCT injection there was more prominent inflammation (CD68-positive cells) present in the lungs of obese male rats compared to the lean counterparts. Inflammatory cytokines and chemokines in PAH are important attributes of PH. In obese individuals, the cytokines produced by the adipose tissues might add-up to the already present inflammation in the lung tissues aggravating pulmonary vascular pathology [4, 30].

Female obese Zucker rats, on the other hand, showed high severity of the disease but less effects of MCT-induced PH. In details, our data demonstrated that obesity prominently worsened the right ventricular hypertrophy/remodeling (RVID, RVWT and RV/(LV + S)) and function (TAPSE) in female MCT Zucker rats in comparison to their lean controls. Furthermore, obesity reduced the value of PAAT/PAET and significantly enhanced the pulmonary vascular remodeling in MCT treated female rats. Importantly, one of the main characteristics of PH, such as elevated RVSP, was noticeably increased in obese female MCT rats compared to their lean counterparts. Finally, there were no alterations in SAP among the experimental groups. The difference of MCT-induced PH in male and female rats have been shown to be due to female hormones and oxidative stress [8, 31]. Our results indicate that obesity might represent an important factor modulating the PH development in the context of females. Therefore, obese Zucker female rats are more susceptible to pulmonary vascular remodeling and right ventricular dysfunction after MCT injection. As in males, upon the MCT injection there was increased accumulation of CD68-positive cells in the lungs of obese female rats compared to the lean counterparts. In contrast to the males, the female obese rats injected with saline were characterized by augmented number of CD68-positive cells in comparison to their lean controls.

Interestingly, there was a significant reduction of BMI in male Zucker rats upon the induction of PH with MCT. This phenomenon was not observed in the context of female Zucker rats. In general, the weight loss may appear due to harsh pulmonary vascular disease in MCT model, and such discrepancy may be attributed to already mentioned fact that MCT caused more severe disease in male rats as compared to the females.

The REVEAL and COMPERA registry have shown that PAH predominantly affects females [9, 32]. Therefore, the hormone might have influence on PAH development. However, irrespective to the disease severity, the prevalence of male mortality with PAH was twice as compared to females [32, 33].

Chronic hypoxia induced model in mice is used as a PH model in several studies [34, 35]. Although some data with regard to the right ventricular remodeling and function (RVID and TAPSE), PAAT/PAET and medial wall thickness showed a mild “improvements” of these PH parameters in obese versus lean male mice under hypoxia, other important measures demonstrated either a slight worsening of the right ventricular hypertrophy (RVWT) or no change (RV/(LV + S)). In addition, there were no significant differences in the muscularization between obese and lean male mice exposed to hypoxia. Furthermore, invasive hemodynamic measurement of RVSP, as one of the main feature of PH, revealed the increase of this parameter due to hypoxia exposure in both lean and obese male mice. Surprisingly, there were significant differences under the baseline conditions and reduction of RVSP even in obese male mice under normoxia compared to their lean counterparts. In the case of female mice, some parameters indicated that right ventricular structure (RVID and RVWT) and function (TAPSE) were a bit worse in obese female mice in comparison to the lean controls under hypoxic conditions. Conversely, the medial wall thickness was reduced, while there were no differences in the muscularization between obese and lean female mice exposed to hypoxia. Importantly, the main PH parameters derived from the invasive measurements of the hemodynamics and right ventricular hypertrophy, such as RVSP and Fulton’s index, did not show any effect of obesity in female mice in chronic hypoxia-induced PH. In both male and female mice, there was no change of SAP among the experimental groups. Overall, in the case of hypoxia-induced PH, obesity was not convincingly demonstrated to play an important role in both genders. In contrast, it was shown that mild form of PH is higher in obese patients living at higher altitude with low oxygen [36]. Therefore, the future studies are crucially needed for final establishment of whether or not obesity modifies chronic hypoxia-induced PH.

Both obese male and obese female Zucker rats showed higher degree of inflammation (CD68-positive cells) in the lung tissues following MCT injection. Lungs from both idiopathic PAH patients as well as MCT-injected rats, have higher infiltration of peri-vascular inflammatory cells [37]. Our studies contribute to the fact that obesity accelerates the degree of inflammation and suggests that obesity might have crucial inflammatory effects to add up the disease severity.

Obesity and PH are characterized by increased oxidative stress and inflammation. Though many studies have shown the possible correlation between obesity and PH, the exact pathology is not known yet. This study provides a clear role of inflammation in the MCT model of PH in obese Zucker rats with significant differences between genders. Further studies are required to substantiate these findings.