Introduction

Pulmonary arterial hypertension (PAH) is a chronic and disabling disease characterized by vasoconstriction and pulmonary vascular remodeling, which leads to a progressive increase in pulmonary vascular resistance and increased mean pulmonary arterial pressure (mPAP) ≥25 mmHg at rest [1]. This high resistance could lead to a higher afterload imposed on the right ventricle (RV)—which develops hypertrophy—and, consequently, heart failure [2]. In experimental studies, the disease can be induced by the administration of monocrotaline (MCT), a pyrrolizidine alkaloid from plants of the genus Crotalaria; this compound mimics the PAH effects [3] and is capable of crossing the blood–brain barrier [4].

The development of PAH is strongly related to oxidative stress and mitochondrial dysfunction [5]. Oxidative stress occurs when there is a redox imbalance caused by an excess of oxidants and/or a decrease in the amount of antioxidants [6]. Some systems can generate reactive oxygen species (ROS), such as the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex in the plasma membrane and the electron transport chain in mitochondria [7]. The heart is an organ with a high energetic demand, and consequently, it contains many mitochondria, which can contribute even more to an oxidative cell state if these organelles are dysfunctional due to pathology [8]. In fact, there is a relationship between the development of PAH and dysfunctions in the energetic and mitochondrial metabolism of cells [9].

Furthermore, Paulin and Michelakis [10] proposed the theory that there is a global mitochondrial abnormality caused by PAH; this phenomenon would explain the involvement of several organs during the progression of the disease. In fact, there are studies showing that treatments that promote the improvement in mitochondrial health improve the pathological changes induced by the disease [11]. Therefore, the brain, an organ with a high rate of oxygen consumption, a large number of mitochondria, and high susceptibility to oxidative stress due to a myriad of factors—such as its modest antioxidant capacity [12], high iron and lipid contents, and oxygen consumption [13]—would have great potential to be affected by the disease.

Considering the signaling that occurs in the organism as PAH develops, one of the mechanisms that has been increasingly investigated in the literature is the role of extracellular vesicles (EVs). These cell-derived vesicles are nanosized and carry cytoplasmic and cellular membrane molecules, such as proteins, DNA, messenger RNA, and microRNA (miR) [14]. A study by Aliotta et al. [15] showed that EVs from mice with MCT-induced PAH promoted PAH in healthy mice, suggesting that EVs play a role in the development of the deleterious effects caused by the disease.

The aims of this study were to analyze the effects of MCT-induced PAH on oxidative stress and mitochondrial parameters in the RV and brain, two tissues enriched in mitochondria and high levels of ROS, and how circulating EVs are related to these effects by investigating their redox content.

Materials and methods

Animals and experimental protocol

All the experimental procedures were performed taking into consideration the welfare of the animals and were approved by the ethics committee of the Universidade Federal do Rio Grande do Sul (CEUA/UFRGS), with process number 33760. The animals were kept under standard conditions (a 12/12-h light–dark cycle, a temperature of 20–25 °C, and controlled humidity of 70%) with free access to water and food.

Male Wistar rats (170 ± 10 g) were divided into two experimental groups: control and MCT. First, the animals received a single intraperitoneal injection of saline (control group) or MCT (60 mg/kg body weight) to induce PAH [16]. Twenty-one days after this injection, the animals were weighed and killed by decapitation, and the RV, brain (whole), and lungs were collected.

Hemodynamic analysis

RV catheterization was performed in which a PE-50 polyethylene cannula was inserted through the jugular vein until it reached the RV. This cannula was then connected to a pressure transducer, and the RV systolic pressure (RVSP) and RV diastolic pressure (RVDP) were measured. The pressure values were recorded through Windaq-Data Acquisition System software and expressed as mmHg [17]. To estimate the mPAP using these data, the following formula [18] was used: mPAP = 0.61 × RVSP + 2.

Morphometric analysis

The control and MCT animals were weighed weekly to determine the effect of the disease on the body weight index. To determine RV hypertrophy, the Fulton index was calculated (the RV/[LV + septum] weight ratio) [19]. In addition, pulmonary congestion was calculated by the ratio of its wet to dry weight [20].

Sample preparation and protein concentration measurements

To determine the activities of respiratory chain complexes and citrate synthase (CS), RV and brain samples were homogenized (1:20 w/v) in SETH buffer (250 mmol/L sucrose, 2.0 mmol/L EDTA, 10 mmol/L Trizma base, and 50 IU/mL heparin, pH 7.4) and centrifuged at 750 g for 10 min at 4 °C. For the remaining analyses, the RV was homogenized in an Ultra-Turrax blender (1.15% w/v KCl and 20 mmol/L phenylmethylsulfonyl fluoride, pH 7.4). The brain was homogenized in a 1.5-mL tube by manually applying pressure with a ceramic pestle using the same buffer. The homogenates were centrifuged at 1000 g for 20 min at 4 °C [21].

After sample preparation, protein levels were measured using Lowry’s method [22], with bovine serum albumin (BSA) as the standard.

Isolation of circulating EVs

EVs were isolated with a technique based on vesicle precipitation using a commercial kit (miRCURYTM Exosome Isolation Kit, Exiqon, Vebæk, Denmark) according to the manufacturer’s instructions. Initially, 0.6 mL of the serum, obtained from truncal blood collected at the moment of decapitation, was centrifuged for 5 min at 10,000 g to remove cell debris, and the supernatant was transferred into a new tube. In the new tube, 0.2 mL of precipitation buffer was added, mixed by vortexing, and incubated for 60 min at 4 °C. Next, the samples were centrifuged again for 30 min at 1500 g at room temperature, and the supernatant was discarded. The resultant pellets were resuspended in 0.3 mL of phosphate-buffered saline using a vortex shaker for 5–15 min at room temperature and then stored at –20 °C. The circulating EVs were then characterized by size distribution through Nanosight Tracking Analysis (NTA) [23, 24] using the Nanosight characterization system LM10-HS (Nanosight Ltd., Amesbury, UK) and western blotting of flotillin-1 (FLOT1) [25, 26].

Western blot

Cell preparation, electrophoresis, and protein transfer for protein expression analysis were performed as described previously [27, 28]. Using a 10% polyacrylamide gel, proteins (80 µg) were separated by electrophoresis (sodium dodecyl sulfate–polyacrylamide gel electrophoresis, 1.5 mm, 130 V) and then transferred to polyvinylidene fluoride membranes (Merck-Millipore, Burlington, MA, USA). The membrane was incubated with nonfat dry milk (5%) in Tris-buffered saline with Tween 20 to block nonspecific protein binding. The membrane was processed for immunodetection using the following primary antibodies: manganese superoxide dismutase (MnSOD, 25 kDa), FLOT1 (47 kDa), proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α, 91 kDa), sirtuin 1 (Sirt1, 120 kDa), and hypoxia-inducible factor 1-alpha (HIF-1α, 132 kDa) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Primary antibodies were detected using appropriate secondary antibodies (anti-rabbit or anti-mouse IgG conjugated to horseradish peroxidase; Santa Cruz Biotechnology), and the protein bands were developed using a chemiluminescent substrate, detected with an ImageQuant LAS4000 system (GE Healthcare), and quantified by ImageJ software (National Institutes of Health, Bethesda, MD, USA). The results were normalized to β-tubulin (50 kDa) and Ponceau’s method [29].

ROS concentration

First described by Lebel et al. [30], the technique used in this study determines the total ROS concentration due to fluorescence emission of 2′,7′-dichlorofluorescein diacetate oxidation (Sigma-Aldrich, St. Louis, MO, USA). The reaction was measured in a fluorescence spectrophotometer in which the samples were excited at 488 nm and the fluorescence emission was measured at 525 nm. The results are expressed as nmol DCF formed/mg protein.

NADPH oxidase activity

NADPH oxidase activity was determined by measuring the consumption of NADPH at 340 nm according to Wei et al. [31]. Its activity is directly proportional to the production of the superoxide anion because this enzyme generates it through the transfer of electrons from NADPH to molecular oxygen. The results are expressed as nmol/min/mg protein.

Lipid peroxidation (LPO) levels

LPO was evaluated by a chemiluminescent reaction of the samples (0.5 mg/mL) with tert-butyl hydroperoxide (t-BOOH) in a beta counter (LKB Rack Beta Liquid Scintillation Spectrometer-1215; LKB Produkter AB, Brommma, Sweden). As described by Gonzalez Flecha et al. [32], the reaction medium consisted of phosphate buffer (30 mmol/L) and KCl (120 mmol/L) (pH = 7.4). First, the initial emission of the samples was measured; then, in the RV and EVs, the reaction was initiated by the addition of t-BOOH (3 mmol/L) and followed until it reached the peak (maximum emission). The brain has spontaneous emission, so t-BOOH was not added to these samples. The difference between the maximum and initial emission values was calculated to determine the lipid oxidative damage. The results are expressed as counts per second (cps)/mg protein.

Antioxidant enzyme activities

SOD activity was measured based on the inhibition of the superoxide radical reaction with pyrogallol in alkaline medium (pH = 10), as described by Marklund [33]. The reaction product was measured in a spectrophotometer at 420 nm, and the enzyme activity is expressed as U/mg protein.

Catalase (CAT) activity was quantified based on the fact that its activity is directly proportional to the hydrogen peroxide (H2O2) decomposition rate. H2O2 consumption was measured in a spectrophotometer at 240 nm, and the results are expressed as pmol/min/mg protein [34].

Glutathione peroxidase (GPx) activity was quantified based on the consumption of NADPH in the presence of reduced glutathione (GSH) and glutathione reductase. Sodium azide (NaN3) was used to inhibit CAT, and t-BOOH was used to initiate the reaction. The reaction was measured in a spectrophotometer at 340 nm, and the results are expressed as pmol/min/mg protein [35].

Determination of oxidized and reduced glutathione levels

To measure GSH and oxidized glutathione (GSSG) concentrations, the samples were deproteinized with 2 mol/L perchloric acid and centrifuged for 10 min at 1000 g. The supernatant was collected and neutralized with 2 mol/L potassium hydroxide. First described by Akerboom and Sies [36], the determination of GSH levels is based on the reaction with 2-nitrobenzoic acid (catalyzed by glutathione reductase). The determination of the GSSG concentration was made by the difference between total glutathione (TG) and GSH (GSSG = TG – GSH). Thus, to measure TG, n-ethylmaleimide was added to the neutralized supernatant to perform the reaction. The absorbance values were measured at 420 nm, and the result is expressed as the GSH/GSSG ratio.

Determination of the sulfhydryl content

The total amount of sulfhydryl (-SH) groups was determined by the reaction of 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) with sulfhydryl groups, according to Sedlak and Lindsay [37]. The optical density was read at 412 nm in a spectrophotometer against an appropriate blank. The concentration of total sulfhydryl groups is expressed as nmol 5-thio-2-nitrobenzoic acid (TNB)/mg protein.

Activity of respiratory chain complexes

Succinate dehydrogenase (complex II) and succinate/cytochrome c oxidoreductase (complexes II–III) activities were measured in reaction medium containing tissue supernatant, 40 mmol/L potassium phosphate buffer (pH 7.4), 16 mmol/L sodium succinate, 4 mmol/L NaN3, and 7 μmol/L rotenone. The reading was performed by following the reduction of 2,6-dinitrophenolindophenol (8.3 μmol/L) at 600 nm and of cytochrome c (50 μmol/L) at 550 nm [38]. Cytochrome c oxidase (complex IV) activity was evaluated by following cytochrome c oxidation (75 μmol/L) at 550 nm [39]. The reaction medium also contained tissue supernatant, 10 mmol/L potassium phosphate buffer (pH 7.0), and 0.6 mmol/L dodecyl maltoside. The results are expressed as nmol/min/mg protein.

Activity of CS

CS activity was determined according to Shepherd and Garland [40] by measuring DTNB reduction at 412 nm. The reaction medium contained 5 mmol/L potassium phosphate buffer (pH 7.4), 300 mmol/L sucrose, 1 mmol/L EGTA, 0.1% BSA, 5 mmol/L MOPS, 0.1% Triton X-100, 0.1 mmol/L DTNB, 0.1 mmol/L acetyl-CoA, and 0.2 mmol/L oxaloacetate. The results are expressed as μmol TNB/min/mg protein.

Pentose phosphate pathway (PPP) enzyme activities

Glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PDG) activities were evaluated according to Leong and Clark [41]. The reaction medium contained tissue supernatant, 100 mmol/L Tris-HCl buffer (pH 7.5), 1.0 mmol/L MgCl2, 0.05 mmol/L NADP+, and 0.1 mmol/L glucose-6-phosphate. NADPH formation was monitored at 340 nm. The results are expressed as U/mg protein.

Statistical analysis

The results are expressed as the mean ± standard deviation. First, the Shapiro–Wilk test was used to evaluate the data normality. The data were then analyzed by Student’s t-test for unpaired samples, and the correlations were assessed by Pearson’s correlation coefficient. The analyses that presented P < 0.05 were accepted as statistically significant. All statistical analyses were carried out using GraphPad Prism 8 software.

Results

Hemodynamic and morphometric analyses

The initial body weight was not different among the groups, unlike the final body weight, which was decreased in the MCT animals compared with the control animals (20%, P = 0.04). The RVSP and mPAP were increased in the MCT group compared with the control group (180%, P = 0.001 and 120%, P = 0.002, respectively). The MCT group presented RV hypertrophy because there was an increase in Fulton’s index in this group compared with the control group (109%, P < 0.0001). The MCT group also presented an increase in lung congestion compared with the control group (14%, P = 0.04; Table 1).

Table 1 Hemodynamic and morphometric parameters
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ROS concentration and oxidative damage in the RV and brain

The total ROS and LPO were both increased in the RV in the MCT group compared with the control group (43%, P = 0.006 and 137%, P = 0.007, respectively; Fig. 1A, B). In the brain, LPO was increased in the MCT group compared with the control group (34%, P = 0.001), but the ROS level was not different (Fig. 1D, E). Another oxidative stress marker examined was the activity of NADPH oxidase, a potent superoxide anion generator. The RV and brain showed higher activity of this enzyme in the MCT group (303%, P = 0.0001 and 79%, P = 0.001, respectively) than in the control group (Fig. 1C, F).

Fig. 1
figure 1

Total reactive oxygen species (ROS) concentration and oxidative damage in the right ventricle (RV) and brain of rats with monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH). A ROS levels in the RV; B lipid peroxidation (LPO) levels in the RV; C nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in the RV; D ROS levels in the brain; E LPO levels in the brain; F NADPH oxidase activity in the brain. Data are expressed as the mean ± standard deviation (n = 8). *P < 0.05 vs control; **P < 0.01 vs control; ***P < 0.001 vs control

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Antioxidant parameters in the RV and brain

To evaluate both enzymatic and nonenzymatic antioxidant defense parameters, SOD, CAT, and GPx activities and the GSH/GSSG ratio were determined. In the RV, the MCT group presented decreased SOD activity (19%, P = 0.006; Fig. 2A), no difference in CAT activity (Fig. 2B), and increased GPx activity (56%, P = 0.0001; Fig. 2C) compared with the control group. In the brain, the MCT group presented no difference in SOD activity (Fig. 2D), decreased CAT activity (25%, P = 0.001; Fig. 2E), and increased GPx activity (34%, P = 0.001; Fig. 2F) compared with the control group. The MCT group also presented a lower GSH/GSSG ratio (37%, P = 0.0007; Fig. 3A) in the RV than the control group, but there was no difference between groups in the brain (Fig. 3C). In addition, sulfhydryl groups also participate in the body’s antioxidant defense. The sulfhydryl group content was lower in the MCT group in the RV (53%, P = 0.0003; Fig. 3B) and higher in the MCT group in the brain (23%, P = 0.03; Fig. 3D) than in the control group.

Fig. 2
figure 2

Antioxidant enzyme activities in the right ventricle (RV) and brain of rats with monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH). A Superoxide dismutase (SOD) activity in the RV; B catalase (CAT) activity in the RV; C glutathione peroxidase (GPx) activity in the RV; D SOD activity in the brain; E CAT activity in the brain; F GPx activity in the brain. Data are expressed as the mean ± standard deviation (n = 8). *P < 0.05 vs control; **P < 0.01 vs control

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Fig. 3
figure 3

Nonenzymatic antioxidants in the right ventricle (RV) and brain of rats with monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH). A The glutathione (GSH)/glutathione disulfide (GSSG) ratio in the RV; B sulfhydryl (SH) group levels in the RV; C the GSH/GSSG ratio in the brain; D SH levels in the brain. Data are expressed as the mean ± standard deviation (n = 8). *P < 0.05 vs control; ***P < 0.001 vs control

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Mitochondrial parameters and PPP enzyme activities in the RV and brain

Mitochondrial parameters were assessed in the RV and brain. In the RV, the activities of mitochondrial respiratory complexes II and IV were decreased (41%, P = 0.007) and increased (9%, P = 0.01), respectively, in the MCT group compared with the control group, but there was no difference in complex II–III activity (Fig. 4A–C). Furthermore, CS activity was diminished in the MCT group in relation to the control group (25%, P = 0.04) in the RV (Fig. 4D). In the brain, the activities of all three respiratory complexes evaluated were increased in the MCT group compared with the control group (complex II: 42%, P = 0.01; complex II-III: 51%, P = 0.03; complex IV: 42%, P = 0.02; Fig. 4E–G). The CS activity was also higher in the MCT group than in the control group (9%, P = 0.02; Fig. 4H). For the PPP enzymes, G6PD and 6PGD activities were increased in the RV (175%, P < 0.0001 and 107%, P = 0.01, respectively; Fig. 5A, B), but there were no differences for either enzyme in the brain (Fig. 5C, D). When we evaluated MnSOD (the mitochondrial isoform of SOD) protein expression, it was lower in the RV but higher in the brain of the MCT group than in the control group (43%, P = 0.0008 and 33%, P = 0.009, respectively; Fig. 6A, B). Finally, HIF-1α expression was 37% (P = 0.03) lower in the brain of the MCT group than in the control group (Fig. 6C).

Fig. 4
figure 4

Activities of mitochondrial respiratory chain complexes and citrate synthase (CS) in the right ventricle (RV) and brain of rats with monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH). A Complex II activity in the RV; B complex II–III activity in the RV; C complex IV activity in the RV; D CS activity in the RV; E complex II activity in the brain; F complex II–III activity in the brain; G complex IV activity in the brain; H CS activity in the brain. Data are expressed as the mean ± standard deviation (n = 6). *P < 0.05 vs control; **P < 0.01 vs control

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Fig. 5
figure 5

Glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PDG) activities in the right ventricle (RV) and brain of rats with monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH). A G6PD activity in the RV; B 6PDG activity in the RV; C G6PD activity in the brain; D 6PDG activity in the brain. Data are expressed as the mean ± standard deviation (n = 6). *P < 0.05 vs control; ****P < 0.0001 vs control

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Fig. 6
figure 6

Manganese superoxide dismutase (MnSOD) immunocontent from the right ventricle (RV) and brain and hypoxia-inducible factor 1-alpha (HIF-1α) immunocontent from the brain of rats with monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH). A Graphic and illustrative figure of MnSOD protein expression in the RV; B graphic and illustrative figure of MnSOD protein expression in the brain; C graphic and illustrative figure of HIF-1α protein expression in the brain. Data are expressed as the mean ± standard deviation (n = 4). *P < 0.05 vs control; **P < 0.01 vs control; ***P < 0.001 vs control

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Characterization of EVs

To determine whether EVs were successfully isolated, we analyzed their size distribution using NTA. The mean size range was 262 ± 106 nm, which is consistent with a previous report from the literature [42]. The expression of the EV membrane protein FLOT1 was also evaluated to characterize the samples; it was equally expressed in both experimental groups, suggesting that the pathological process of PAH does not affect the number of EVs released into the circulation. The NTA graph and FLOT1 immunocontent are illustrated in Fig. 7A, B.

Fig. 7
figure 7

Characterization, oxidative stress markers, and immunocontent of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and sirtuin 1 (Sirt1) in blood-derived circulating extracellular vesicles (EVs) of rats with monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH). A Circulating EV size distribution (262 ± 106 nm) and concentration; B graphic and illustrative figure of flotillin-1 (FLOT1) protein expression in EVs; C reactive oxygen species (ROS) levels in EVs; D lipid peroxidation (LPO) levels in EVs; E nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in EVs; F superoxide dismutase (SOD) activity in EVs; G catalase (CAT) activity in EVs; H glutathione peroxidase (GPx) activity in EVs; I graphic and illustrative figure of PGC-1α protein expression in EVs; J graphic and illustrative figure of sirtuin 1 (Sirt1) protein expression in EVs. Data are expressed as the mean ± standard deviation (n = 4). *P < 0.05 vs control; **P < 0.01 vs control; ***P < 0.001 vs control

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Oxidative stress and mitochondrial parameters in circulating EVs

The same measurements of total ROS, oxidative damage, and activities of antioxidant enzymes performed in the RV and brain were evaluated in the circulating EVs of the rats. Although there was no change in total ROS between the groups (Fig. 7C), LPO and NADPH oxidase activity were increased in the MCT EVs compared with the control EVs (116%, P = 0.008 and 127%, P = 0.001, respectively; Fig. 7D, E). In addition, the MCT group presented decreased SOD activity (41%, P = 0.03; Fig. 7F), decreased CAT activity (60%, P = 0.001; Fig. 7G), and no difference in GPx activity (Fig. 7H) compared with the control group.

In the EVs, we also evaluated the protein expression of PGC-1α and Sirt1, which are mitochondrial biogenesis-related proteins. Both proteins were expressed at lower levels in the MCT group than in the control group (63%, P = 0.01 and 98%, P = 0.0004, respectively; Fig. 7I, J).

Discussion

This study revealed that MCT-induced PAH promotes substantial mitochondrial and oxidative stress changes in the RV and brain, and there is redox signaling mediated by circulating EVs that is probably linked to these effects. Although the main findings in the RV corroborate previously reported data, the effects of this disease in the brain have been barely explored until now.

First, the increased mPAP, RVSP, RV hypertrophy index, and lung congestion in MCT-treated rats showed that the induction of PAH was efficient in these animals [43]. In our work, some effects of oxidative stress parameters in the RV on MCT-induced PAH were in line with previous literature data. The RV of MCT rats presented increased NADPH oxidase activity, which contributes to the ROS buildup that causes LPO and is associated with the pathogenesis of PAH [44]. PAH can also promote redox changes in the brain, and although there was no increase in ROS levels in MCT rats, this group presented higher levels of LPO and NADPH oxidase activity. This can be considered a controversial result because, as observed in the right cardiac tissue, oxidative damage to lipids is usually caused by the abnormal generation of ROS, which can be derived from, among other sources, mitochondrial dysfunction and high NADPH oxidase activity [5]. However, the brain is rich in polyunsaturated fatty acids and has a modest antioxidant defense system, which renders it very susceptible to oxidative stress, making even small amounts of reactive species sufficient to cause oxidative damage to lipids and proteins [12, 45].

On the antioxidant side of the redox balance, the RV of MCT animals presented decreased SOD activity; because this enzyme is the first line of defense against ROS, it may be presumed that its decreased activity contributes to disease development [46]. In addition, the GSH/GSSG ratio and sulfhydryl content were lower in MCT rats, indicating a detriment to redox balance and an oxidative environment [47]. In contrast, it appears that there was a preservation of antioxidant defenses in the brains of diseased rats, as observed by the maintenance of SOD activity and the GSH/GSSG ratio. According to Ren et al. [48], one of the most relevant and abundant antioxidants involved in neuroprotection is GSH; thus, this molecule would possibly be better preserved in the brain than in the RV. Hence, the preservation of the GSH concentration prevents redox imbalance and increases the sulfhydryl content because GSH is a thiol-dependent molecule [49].

Recent data suggest that an important contributor to the development of PAH is mitochondrial respiratory chain dysfunction [50]. PAH creates hypoxic conditions in the cells, and the increase in complex IV activity in the MCT group may be an attempt to adapt to these conditions. Specifically, this complex may act as a primary oxygen sensor in cells [51], and it may represent upregulation at the gene level of the subunits of this complex to cope with impaired complex II activity [52, 53]. Furthermore, CS activity and MnSOD protein expression were decreased in the RV of MCT rats, indicating bioenergetic dysfunction in this tissue. CS is considered a mitochondrial mass marker [54] and was decreased even in the group that presented RV hypertrophy. Associated with the respiratory chain complex responses discussed above, this result suggests that there was a marked mitochondrial impairment in the RV cardiomyocytes of rats with MCT-induced PAH [54].

On the other hand, the elevated CS activity in the brain suggests an increase in the number of mitochondria in the brains of rats with MCT-induced PAH, a phenomenon that is consistent with the observed increase in the activities of respiratory chain complexes II, II–III, and IV and in MnSOD protein expression in this group. Although the mechanisms underlying the increase in mitochondrial function induced by PAH in the brain have not been established, we hypothesize that this response aims to protect neural cells against energetic failure and oxidative damage. A study by Furian et al. [55] helps to explain this hypothesis: The authors compared the oxygenation of the brains of people living in low and high altitudes who have pulmonary hypertension and suggested that there are compensatory mechanisms that protect the brain when less oxygen is available, such as in the case of PAH.

To understand the movement of proteins and molecules in different organs in the body, including those that are not commonly associated with the pathogenesis of PAH such as the brain, we asked whether circulating EVs could be involved in this process because they can reach different tissues via blood circulation, communicate with cells, and regulate intracellular responses [56]. Interestingly, circulating EVs of rats with MCT-induced PAH had more LPO, higher NADPH oxidase activity, and less activity of both antioxidant enzymes (SOD and CAT) in relation to healthy animals. Although we did not find a difference in the total ROS levels, the overall results indicate an oxidative state in the cargo of the EVs, similar to that found in the right cardiac tissue in this work and in the lung tissue of previous PAH studies [57, 58], suggesting that most of these vesicles may come from cells involved in pulmonary and RV remodeling. According to Henning et al. [59], cardiomyocyte EVs are rich in mitochondrial molecules involved in energy generation. PGC-1α is a transcription factor directly involved in biogenesis and mitochondrial energy metabolism [60]; it is regulated mainly by Sirt1 [54]. Thus, the impairment of mitochondrial function that occurs in PAH is associated with inhibition of the Sirt1/PGC-1α pathway in cardiac and pulmonary artery cells [61, 62]. We analyzed the protein expression of PGC-1α and Sirt1 in circulating EVs and found that both were expressed at lower levels in the MCT-treated animals than in the control animals, which provides more evidence that these vesicles are probably released by cells involved in the pathophysiology of PAH. A limitation of this work is that it was not possible to carry out analyses that could corroborate this possibility.

The EVs of diseased animals can contribute to the pathophysiological progression of the disease, mediating inflammatory processes, promoting endothelial dysfunction, and inducing oxidative stress [63, 64]. In fact, a study with EVs from MCT-treated mice showed that these vesicles induced the development of pulmonary hypertension in healthy mice [15]. Therefore, the oxidative profile observed in circulating EVs in the present study seems to be in accordance with this previously reported “prejudicial” paracrine effect. Although this communication within the cardiopulmonary system appears to be mainly harmful, EVs are also able to regulate beneficial pathways, promoting angiogenic and antiapoptotic effects [65].

Therefore, we wondered whether EVs could act as messengers and whether there is a pathological process taking place in the body because it has been shown that EVs mediate crosstalk between different cells and organs [66, 67]. In this study, the increased expression of MnSOD and decreased expression of HIF-1α in the brains of animals with PAH indicate protective signaling in this tissue. This may sound somewhat contradictory because HIF-1α is often identified as a protective factor against cellular hypoxia [68]. However, several studies have related the activation of this transcription factor with the pathogenic progression of PAH through the upregulation of proteins involved in the glycolytic shift responsible for the Warburg effect [69, 70], as well as in the processes of cell proliferation that cause pulmonary vascular remodeling and RV hypertrophy [71] and inflammation [72]. In addition, treatments that act by inhibiting factors in the HIF-1α signaling pathway have been shown to be effective against the development of PAH [72,73,74]. In addition, previous studies have reported that MnSOD downregulation is implicated in the activation of HIF-1α [75], and its activation due to hypoxia in the brain leads to the suppression of the PPP, a phenomenon that would impair redox homeostasis and favor an oxidative cellular environment [76]. Thus, in the present study, MnSOD upregulation, HIF-1α downregulation, and the maintenance of the activities of G6PD and 6PGD, key PPP enzymes, combined with the unchanged GSH/GSSG ratio, indicate the preservation of the antioxidant defense system and redox homeostasis in the brain, and this is probably related to the MnSOD-HIF-1α signaling observed.

This response can be associated with EV communication; indeed, there is a correlation between MnSOD protein expression in the RV and brain and PGC-1α and Sirt1 protein expression in circulating EVs of rats with MCT-induced PAH. The decreased MnSOD expression in the RV was positively correlated with the low expression of PGC-1α and Sirt1 in the EVs (r = 0.8422, P = 0.0087 and r = 0.9619, P = 0.0001, respectively); however, the higher expression of MnSOD in the brain was negatively correlated with low levels of PGC-1α and Sirt1 (r = –0.8235, P = 0.0120 and r = –0.8455, P = 0.0082, respectively). Considering the Sirt1/PGC-1α/MnSOD signaling pathway, we hypothesized that the positive correlation between EVs and the RV indicates that most of these vesicles come from cells directly affected by the disease, such as cardiomyocytes and pulmonary endothelial cells. However, the low expression of Sirt1/PGC-1α in EVs appears to be inversely proportional to the upregulation of MnSOD protein expression in the brain. This phenomenon probably occurs because after being released into the circulation, these vesicles act as signals to brain cells, triggering an adaptive response to upregulate mitochondrial activity, mainly stimulating the increase in MnSOD expression.

Considering the aforementioned information, we hypothesize that the protective effects that occur in the brain during the development of PAH are triggered by upregulation of MnSOD and downregulation of HIF-1α through miR signaling from circulating EVs derived from cardiac and pulmonary cells. However, further studies are needed to test this relationship, focusing on the action of miRs that have already been identified in PAH-EVs [15, 77] and regulate several pathways involved in the redox and mitochondrial systems, as well as hypoxia factors and apoptosis [78,79,80].

Conclusion

Based on our results, we can conclude that there are many pathological changes caused by PAH in the cardiovascular system, especially regarding the oxidative stress and mitochondrial dysfunction that occur in the RV. However, these events may also signal the release of circulating EVs that communicate with cells of other organs in the body, such as the brain, helping to trigger transcriptional signals to increase the antioxidant defenses and regulatory proteins of mitochondrial function in this tissue to preserve its function and attempt to prevent the damage caused by the disease.