Mitochondrial Transplantation from Bone Marrow Mesenchymal Stromal Cells Combined with Sildenafil Attenuated Vascular Remodeling and Improved Right Ventricular Dysfunction in Experimental Pulmonary Arterial Hypertension

TL;DR

Mitochondrial transplants from bone marrow cells, combined with sildenafil, reduced lung artery pressure and improved heart function in a rat model of pulmonary hypertension.

Contribution

Combining mitochondrial transplantation with sildenafil shows novel therapeutic potential for pulmonary arterial hypertension.

Findings

Mitochondrial therapy reduced right ventricular systolic pressure and vascular remodeling in PAH rats.

Combined mitochondrial and sildenafil treatment attenuated endothelial-mesenchymal transition and inflammation.

Mitochondrial therapy improved mitochondrial respiration and Complex IV activity.

Abstract

Pulmonary arterial hypertension (PAH) is characterized by progressive vascular remodeling and right ventricular (RV) dysfunction, processes that are increasingly associated with disturbances in cellular metabolism. We investigated whether transplantation of exogenous mitochondria derived from bone marrow mesenchymal stromal cells, alone or combined with sildenafil, could improve mitochondrial homeostasis and attenuate cardiopulmonary remodeling in monocrotaline-induced PAH. Male Wistar rats were assigned to control (CTRL, n = 8) or PAH (n = 32) groups. Fourteen days after induction of PAH, animals were randomized to receive saline, sildenafil (20 mg/kg/day for 14 days), intravenous mitochondrial transplantation (100 μg, days 14 and 21), or combined therapy. On day 28, echocardiography, invasive measurement of RV systolic pressure (RVSP), pulmonary vascular histology, gene expression analyses (vimentin, VE-cadherin, and mitochondrial metabolism–related genes), and high-resolution respirometry were performed. All treatments significantly reduced RVSP compared with untreated PAH. Mitochondrial therapy, alone or combined with sildenafil, decreased arteriolar α-smooth muscle actin content, whereas endothelial–mesenchymal transition was attenuated only with combined treatment. Mitochondrial transplantation and sildenafil increased Complex I–dependent respiration, whereas Complex IV activity improved exclusively with mitochondrial therapy. Combined treatment reduced plasma IL-6 and IL-1β levels compared with PAH. Thus, mitochondrial transplantation, particularly when combined with sildenafil, improved RV function, limited pulmonary vascular remodeling, reduced plasma inflammatory markers, and changed key mitochondrial pathways in experimental PAH.

Figures

Full text

1. Introduction

Pulmonary arterial hypertension (PAH) is a progressive and life-threatening disease characterized by structural remodeling of the pulmonary vasculature, leading to luminal narrowing, impaired pulmonary blood flow, and increased pulmonary vascular resistance [1]. Despite major advances in pharmacological therapy, the prognosis of patients with group 1 PAH, including idiopathic, heritable, drug-induced, and associated forms such as connective tissue disease and congenital heart disease, remains poor; median survival is approximately 6 years [2]. Currently approved therapies predominantly target vasoregulatory pathways and provide symptomatic and hemodynamic improvement, but they do not reliably prevent, halt, or reverse the structural vascular remodeling that drives disease progression [3,4,5,6,7].

Increasing evidence indicates that PAH is also a disorder of cellular metabolism, characterized by mitochondrial dysfunction and metabolic reprogramming within the pulmonary vasculature and the right ventricle [8]. Impaired oxidative phosphorylation, altered mitochondrial dynamics, increased oxidative stress, and changes in mitochondrial content and quality control have been described in both experimental models and human PAH [9]. These abnormalities contribute to a hyperproliferative, apoptosis-resistant vascular cell phenotype and are increasingly recognized as integral components of the remodeling process.

In this context, mitochondrial transplantation (mitotherapy) has emerged as an experimental strategy to improve cellular bioenergetics. After the discovery of spontaneous intercellular mitochondrial transfer [10], exogenous mitochondrial delivery has been shown to influence tissue homeostasis in models of neurodegenerative, ocular, and ischemic cardiac diseases [11,12,13]. However, whether mitochondrial transplantation can meaningfully affect pulmonary vascular remodeling and cardiopulmonary physiology in PAH, beyond isolated cellular energetic effects, remains insufficiently characterized.

Mitochondria-targeted interventions have been proposed as a means to influence both pulmonary vascular remodeling and RV dysfunction. Although experimental studies suggest that mitochondrial transfer may stabilize mitochondrial membrane potential, reduce reactive oxygen species production, and restore mitochondrial dynamics, several key questions remain unresolved. In particular, the effects of mitotherapy on pulmonary vascular structure, invasive hemodynamics, and mitochondrial metabolic pathways have not been systematically examined in established models of PAH [14,15]. Moreover, the importance of the source of mitochondria represents a critical and largely unexplored variable, given the marked tissue-specific differences in mitochondrial phenotypes and function.

Another unresolved issue is whether metabolic interventions, such as mitotherapy, might complement rather than replace established vasodilator therapies. Sildenafil, a phosphodiesterase-5 inhibitor, remains a cornerstone of PAH treatment through its effects on pulmonary vascular tone, but its impact on structural remodeling is limited. Whether targeting mitochondrial homeostasis can enhance or extend the biological effects of conventional hemodynamic therapy has not been tested.

Based on preliminary observations comparing mitochondria from different tissue sources, we hypothesized that mitochondria derived from bone marrow mesenchymal stromal cells (BM-MSCs), alone or in combination with sildenafil, could favorably improve mitochondrial homeostasis and attenuate pulmonary vascular remodeling in experimental PAH. We therefore designed this study to evaluate the effects of BM-MSC–derived mitochondrial transplantation, with or without sildenafil, on pulmonary hemodynamics, vascular remodeling, plasma levels of inflammatory markers, and mitochondrial metabolic pathways in a monocrotaline rat model of PAH.

2. Results

2.1. Biodistribution and Functional Characterization of Isolated Mitochondria

Fluorescence imaging demonstrated that intravenously administered mitochondria derived either from liver or BM-MSCs accumulated predominantly in the liver and kidneys at both 3 and 24 h after injection. In contrast, only minimal fluorescence signals were detected in the lungs and heart (Figure 1 and Figure S1), indicating limited pulmonary and cardiac biodistribution.

The median level of protein concentration in BM-MSC-derived mitochondria lysates was 52 mg/mL. BM-MSC-derived mitochondria had 86.3% ± 6.3% of particles positive for MitoTracker Deep Red FM. The functional properties of isolated mitochondria were assessed by high-resolution respirometry (Figure S2). Liver-derived mitochondria exhibited higher basal oxygen consumption and greater substrate-stimulated respiration than BM-MSC–derived mitochondria. After the addition of adenosine diphosphate (ADP), oxygen consumption increased markedly in liver-derived mitochondria (65.4 ± 4.8 pmol/s/mg), whereas only a modest increase was observed in BM-MSC–derived mitochondria (18.9 ± 2.8 pmol/s/mg), indicating preserved but limited phosphorylation capacity. Consistently, oligomycin-sensitive respiration revealed a substantially higher proton leak in liver-derived mitochondria (22.0 ± 3.0 pmol/s/mg) than in BM-MSC–derived mitochondria (4.9 ± 1.6 pmol/s/mg). Maximal uncoupled respiration induced by FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) was also markedly higher in liver-derived mitochondria (58.3 ± 9.7 pmol/s/mg) than in BM-MSC–derived mitochondria (22.6 ± 2.6 pmol/s/mg), indicating a greater respiratory reserve capacity. Despite their overall lower respiratory rates, BM-MSC–derived mitochondria retained measurable coupling efficiency.

2.1.1. Liver-Derived Mitochondria Induce Mortality in PAH Animals

All PAH animals receiving liver-derived mitochondria on day 14 died within a few days after injection (between days 20 and 26). In view of this unexpected and consistent lethality, subsequent experiments were performed exclusively using BM-MSC–derived mitochondria.

2.1.2. Effects on Right Ventricular Function and Hemodynamics

At day 14, PAH animals exhibited a reduced pulmonary artery acceleration time (PAT) to ejection time (PET) ratio and an increased right ventricular (RV) outflow tract diameter compared with control animals (Figure S3). At day 28, the PAT/PET ratio remained significantly reduced in all PAH groups, regardless of treatment (Figure 2A,B). RV outflow tract diameter remained increased in untreated PAH animals but was significantly reduced in animals treated with sildenafil alone or in combination with mitochondrial therapy (Figure 2A,C).

Right ventricular systolic pressure (RVSP) was markedly increased in PAH animals (50 ± 9 mmHg) compared with controls (20 ± 4 mmHg; p < 0.0001). All therapeutic interventions significantly reduced RVSP relative to untreated PAH animals (Figure 3). In contrast, the RV hypertrophy index was increased in PAH animals compared with controls and was not significantly modified by any treatment strategy (Figure 4).

2.1.3. Pulmonary Vascular Remodeling

Perivascular collagen deposition was significantly increased in PAH animals compared with controls. Treatment with sildenafil, BM-MSC–derived mitochondria, or their combination reduced collagen accumulation to a similar extent (Figure 5).

Pulmonary arterial muscularization, assessed by α-smooth muscle actin (α-SMA) expression, was markedly increased in PAH animals relative to controls. α-SMA expression was significantly reduced in animals treated with mitochondria alone or in combination with sildenafil, but remained unchanged in animals treated with sildenafil alone (Figure 6).

2.1.4. Markers of Endothelial-to-Mesenchymal Transition

Endothelial-to-mesenchymal transition (EndMT) was suggested by increased vimentin and reduced VE-cadherin mRNA expression in PAH animals compared with controls (Figure 7A,B). Vimentin expression was reduced in animals treated with sildenafil alone or in combination with mitochondria, but not in those treated with mitochondria alone. VE-cadherin expression was significantly increased only in the group receiving combined therapy, indicating a partial and treatment-specific attenuation of EndMT.

2.1.5. Mitochondrial Dynamics–Related Markers

Expression of succinate dehydrogenase complex, subunit A (Sdha) did not differ between PAH and control animals but was significantly reduced in animals treated with mitochondria alone or in combination with sildenafil (Figure 8A). Similarly, expression of mitochondrial transcription factor A (TFAM) was comparable between PAH and control animals but was decreased in all treated groups (Figure 8B).

Proteins involved in mitochondrial fusion displayed distinct patterns. Optic atrophy protein 1 (OPA1) expression was increased in PAH animals and was significantly reduced by all treatment strategies (Figure 8C). In contrast, mitofusin-1 (Mfn-1) expression did not differ between PAH and control animals but was consistently reduced across all treatment groups (Figure 8D).

2.2. Mitochondrial Respiratory Chain Activity

Mitochondrial oxygen flux through Complex I (CI + CIII + CIV) was similar in lung mitochondria from PAH and CTRL animals. However, Complex I–dependent respiration was significantly increased in mitochondria from animals treated with either sildenafil or mitochondrial therapy alone, whereas no significant change was observed in the combination therapy group (Figure S4A). No differences were detected in Complex II–dependent respiration among the groups (Figure S4B). Complex IV activity was significantly increased only in lung mitochondria from animals treated with mitochondria compared with controls (Figure S4C).

2.3. Plasma Levels of Inflammatory Markers

IL-6 and IL-1β plasma levels were higher in the PAH group than the CTRL group (p = 0.013 and p = 0.045, respectively) (Figure 9A,B). The combined therapy (PAH—sil & imt) decreased IL-6 and IL-1β plasma levels compared with the PAH group (p = 0.007 and p < 0.001, respectively). In addition, sildenafil combined with mitochondria transplantation showed lower IL-6 plasma level compared with PAH treated with sildenafil alone (p = 0.039) (Figure 9A). Figure S5A–C depicts a summary of the percentage changes of each therapy (Mitochondria alone, sildenafil alone, and combined therapy) compared with PAH.

3. Discussion

This study demonstrates that mitochondrial transplantation from BM-MSCs, either alone or in combination with sildenafil, attenuates several central features of experimental PAH. In monocrotaline-exposed rats, BM-MSC–derived mitochondrial administration reduced RVSP, perivascular collagen deposition, pulmonary arterial α-SMA expression, and the expression of genes related to mitochondrial homeostasis (Sdha, TFAM, OPA1, and Mfn-1), and selectively increased Complex IV activity in lung tissue. Sildenafil alone improved RV outflow tract diameter, reduced perivascular collagen accumulation, decreased vimentin and OPA1 expression, and increased Complex I activity. The combined treatment was associated with a broader pattern of biological effects, including reductions in RV outflow tract diameter, RVSP, perivascular collagen deposition, α-SMA, vimentin, Sdha, TFAM, OPA1, and Mfn-1, IL-6 and IL-1β plasma levels, together with restoration of VE-cadherin expression. These findings support the concept that metabolic modulation by mitochondrial transplantation complements established vasodilator therapy through partially overlapping and additive biological effects, rather than through true pharmacological synergy (Figure S5A–D).

The monocrotaline model was selected because of its reproducibility, cost-effectiveness, and ability to reproduce key features of human PAH, including endothelial dysfunction, pulmonary vascular inflammation, and progressive RV overload [16,17]. Although this model does not recapitulate complex plexiform lesions and is associated with systemic toxicity, it remains widely used to investigate mechanisms of pulmonary vascular remodeling and RV dysfunction [18]. PAT/PET and RV outflow tract diameter data from PAH animals at day 14 showed early signs of RV dysfunction. Translating to clinical practice, this would represent early echocardiographic manifestations of PAH, corresponding to a disease stage at which patients typically first seek medical evaluation. Sildenafil was chosen as a reference therapy because of its established clinical use as a phosphodiesterase-5 inhibitor that improves pulmonary hemodynamics through cGMP-mediated vasodilation [3]. The rationale for combining sildenafil with mitochondrial transplantation was to test whether targeting mitochondrial and metabolic abnormalities could enhance the effects of conventional hemodynamic therapy [12,13,14,15] (Figure S5A).

BM-MSC–derived mitochondria were selected based on their relative resistance to oxidative stress and their recognized immunomodulatory properties [19]. Although these mitochondria display lower maximal respiratory capacity than liver-derived mitochondria, they retain preserved coupling efficiency, a profile that may be advantageous in inflamed and oxidatively stressed microenvironments [20]. In a recent study from our group [21], mitochondria transplantation was evaluated in in vitro and in vivo models of acute lung injury induced by LPS. A trend toward reduction of ROS production (p = 0.072) was found after BM-MSC-derived mitochondria transplantation. In addition, BM-MSC-derived mitochondria transplantation reduced BALF neutrophils and total protein, denoting lung damage mitigation. Furthermore, previous studies have shown that stem cells are able to maintain their mitochondria structure, potentially through ATP hydrolyzation by ATP synthase, leading to high mitochondrial membrane potential [22,23,24]. A key and unexpected finding of the present study was that transplantation of liver-derived mitochondria was associated with death in PAH animals, despite their higher respiratory activity. This observation indicates that the biological effects of mitochondrial transplantation are not determined solely by bioenergetic performance. Mitochondria also function as signaling organelles involved in redox signaling, apoptosis regulation, calcium handling, and immune activation. Therefore, it is plausible that mitochondria with high metabolic activity may be poorly tolerated in the context of advanced pulmonary vascular disease, potentially due to metabolic mismatch, excessive redox signaling, or immune activation. Although the precise mechanism remains to be defined, this finding underscores the importance of the source and mitochondrial phenotype for both safety and efficacy [25,26].

Mitochondrial transplantation has emerged as a potential therapeutic strategy in diseases characterized by mitochondrial dysfunction [25,26,27,28]. This approach, initially explored in models of neurodegeneration, myocardial ischemia, and ocular disease, relies on the capacity of exogenous, respiration-competent mitochondria to be internalized by host cells and to influence cellular homeostasis through mechanisms that extend beyond the provision of ATP [13,24]. In PAH, where mitochondrial dysfunction contributes to abnormal vascular cell proliferation and apoptosis resistance, previous studies have suggested that mitochondrial transfer can attenuate vascular remodeling and improve right ventricular function. The present study extends these observations by demonstrating that BM-MSC–derived mitochondria exert biologically relevant effects in the monocrotaline model of PAH [14,15].

In vivo imaging showed that intravenously administered mitochondria predominantly accumulated in the liver and kidneys, with comparatively low signals in the lungs and heart. This distribution suggests that at least part of the benefit to the pulmonary vasculature observed here may be mediated indirectly, through systemic or immunomodulatory mechanisms, rather than exclusively through direct uptake by pulmonary vascular cells [29]. To corroborate this hypothesis, we analyzed plasma samples collected on day 28. The combined therapy decreased IL-6 and IL-1β plasma levels compared with PAH (Figure 9 and Figure S5D). A previous MCT-induced PAH study [30] showed similar results, using higher dosages of mitochondria transplantation (15,000 μg/rat and 1500 μg/rat). Furthermore, the study demonstrated that a low dose of mitochondria was superior to a high dose in protecting against MCT-induced PAH. Herein, we used 100 μg/animal on days 14 and 21 and observed a beneficial effect. This suggests that mitochondria dosage is an important issue when dealing with the PAH phenotype.

Pulmonary vascular remodeling was consistently attenuated, as indicated by reduced perivascular collagen deposition and decreased α-SMA expression. In parallel, the combined treatment reduced vimentin expression and restored VE-cadherin expression (Figure S5B), suggesting partial attenuation of EndMT, a process increasingly recognized as a contributor to pulmonary vascular remodeling in PAH [31,32,33]. Although these measurements were performed in whole-lung homogenates and therefore do not allow cell-specific conclusions, the results support an association between modulation of mitochondrial homeostasis and pathways related to endothelial plasticity. One of these pathways could be related to calcium homeostasis. Although we did not measure lung tissue calcium deposits, α-SMA lung tissue expression is a marker of endothelial–mesenchymal transition dependent on Ca^2+^ metabolism and was reduced after mitochondrial transplantation. For instance, reduced calcium overloading has been observed after mitochondrial transplantation therapy to correct the pathology in dystrophin-deficient mdx mice [34]. Aligned to this, normalization of the mitochondrial ultrastructure and sarcoplasmic reticulum/mitochondria interactions in mdx muscles was also observed.

The changes in mitochondrial markers should be interpreted cautiously. Sdha and TFAM are commonly used as indices of mitochondrial content and biogenesis [35], but their reduction after treatment does not necessarily imply impaired mitochondrial function. Instead, these changes may reflect normalization of a maladaptive mitochondrial program present in untreated PAH. Similarly, reductions in OPA1 and Mfn-1 expression likely indicate remodeling of mitochondrial dynamics rather than a simple loss of mitochondrial mass. These findings are more consistent with qualitative reprogramming of mitochondrial homeostasis than with a unidirectional increase in mitochondrial biogenesis.

Analysis of mitochondrial respiratory chain activity revealed relatively modest abnormalities in PAH lungs, with selective improvement of Complex I activity after sildenafil or mitochondrial therapy and increased Complex IV activity after solely mitochondrial transplantation (Figure S5C). The absence of additive effects with combination therapy suggests convergence on partially overlapping metabolic pathways. Given the limitations of assessing respiratory chain function in frozen tissue [36], these findings should be interpreted cautiously, but they are consistent with the concept that mitochondrial dysfunction in PAH is subtle and pathway-specific rather than global [14,15]. Previous studies have demonstrated that approximately 90–95% of maximal respiratory capacity is preserved in frozen samples, indicating that this approach can be applied to isolated mitochondria, permeabilized cells, and tissue homogenates with high sensitivity [36]. This limitation raises important questions regarding how mitochondrial respirometry can be reliably performed in multisite clinical studies and retrospective analyses. Further methodological advances are therefore needed to enable robust assessment of mitochondrial function in frozen tissues obtained after surgical resection, particularly from geographically distant clinical centers.

Despite significant improvements in pulmonary hemodynamics and indices of vascular remodeling, RV hypertrophy persisted across all treatment groups (Figure S5A). This dissociation between afterload reduction and structural RV remodeling is consistent with previous reports and may reflect the relatively short duration of treatment, the direct myocardial toxicity of monocrotaline, or the intrinsic resistance of established RV remodeling to rapid reversal [37]. These findings emphasize the need for longer-term studies specifically designed to address myocardial remodeling.

Sildenafil most likely exerted its predominant effects through vasodilation, as reflected by improvements in RVSP and RV outflow tract diameter, with more limited effects on pulmonary arterial muscularization. Although antiproliferative actions of PDE-5 inhibition have been reported [38,39], these appear to be dose- and context-dependent, and the dosing strategy used here was chosen to balance efficacy and tolerability.

This study has several limitations. The mitochondrial dose and administration schedule were extrapolated from previous studies in other disease models, and optimal dosing, timing, and frequency remain unknown [15,40,41]. No dose-response assessment was performed to determine the safety profile of liver-derived mitochondria. At the tested dose (100 μg/animal), liver-derived mitochondria were poorly tolerated in PAH animals. No direct measurements of oxidative stress, calcium deposits, or mitochondrial membrane potential were performed. Previous work has shown that mitochondria isolated from skeletal muscle can attenuate pulmonary hypertension in hypoxia-induced experimental models [15], whereas in the present study, mitochondria were derived from BM-MSCs and tested in the monocrotaline model. These findings support mitochondrial transplantation as a promising therapeutic strategy in cardiopulmonary disease and indicate that tissue-specific metabolic phenotypes may influence therapeutic efficacy and biological effects. The relatively short observation period limits conclusions regarding long-term outcomes, particularly with respect to RV remodeling. Mitochondrial biodistribution and persistence were only partially characterized. In addition, gene expression analyses performed in whole-lung homogenates preclude interpretation of cell-specific mechanisms. Future studies incorporating cell-specific approaches, lineage tracing, proteomics, and metabolomics will be required to define the precise mechanisms by which mitochondrial transplantation modulates pulmonary vascular disease.

4. Materials and Methods

4.1. Study Approval

The study protocol was approved by the Institutional Animal Care and Use Committee of the Health Sciences Center, Federal University of Rio de Janeiro (CEUA 159/21), on 22 June 2022, and complied with the Principles of Laboratory Animal Care and the US National Academy of Sciences Guide for the Care and Use of Laboratory Animals. The study followed the ARRIVE guidelines [42]. Male Wistar rats were housed under controlled conditions (23 °C, 12-h light–dark cycle) with free access to food and water. Only male animals were used to reduce biological variability in this mechanistic study.

4.2. Experimental Design, Randomization, and Blinding

The primary endpoint was RVSP. Secondary endpoints included echocardiographic indices, right ventricular hypertrophy, vascular remodeling, and metabolic measurements.

Animals were randomly allocated to experimental groups using a computer-generated random sequence prepared by an investigator not involved in data acquisition. The group assignment was concealed in sealed, opaque envelopes. Investigators performing echocardiography, hemodynamic measurements, histologic, immunohistochemical, molecular, and respirometry analyses were blinded to group allocation. Treatment administration was not blinded.

Sample size was based on prior experience with the monocrotaline model and on our group’s previous studies evaluating RVSP in interventional PAH experiments [16,17].

4.3. Mitochondrial Sources and Rationale

Two mitochondrial sources were used for distinct purposes. Liver-derived mitochondria were used exclusively for validation, functional characterization, and biodistribution experiments. The outcomes following transplantation of liver-derived mitochondria in PAH animals were unsatisfactory. Briefly, 2000 µg of liver-derived mitochondria were administered on day 14, and all PAH animals died within a few days. Subsequently, the dose was reduced to 100 µg, based on a previous preclinical study from our group [21]. PAH animals again died within a few days after mitochondrial transplantation. In a third experiment, the dose was maintained at 100 µg, but mitochondria derived from BM-MSCs were used. Under these conditions, PAH animals survived the protocol and exhibited beneficial effects. Accordingly, all in vivo therapeutic experiments were performed using BM-MSC–derived mitochondria at a dose of 100 µg per animal. Given the short-term retention of transplanted mitochondria [43], two intravenous jugular injections of 100 µg were administered 7 days apart to ensure full recovery.

4.3.1. Isolation of Mitochondria from Liver Tissue

Mitochondria were isolated from the liver of a healthy male Wistar rat (8 weeks, ~250 g). The liver was excised, minced, and homogenized in isolation buffer containing mannitol (225 mM), sucrose (75 mM), and EDTA (0.2 mM). The homogenate underwent differential centrifugation at 4 °C (1000× g, then 6200× g twice) to obtain a mitochondrial-enriched fraction [44].

4.3.2. Culture of Bone Marrow Mesenchymal Stromal Cells

BM-MSCs were isolated from femurs and tibias of healthy male Wistar rats (7 weeks, 220 ± 10 g). Cells were cultured in Iscove’s modified Dulbecco’s medium supplemented with 10% fetal bovine serum, 10% horse serum, and 1% penicillin/streptomycin at 37 °C in 5% CO_2_ [17].

4.3.3. Isolation of Mitochondria from BM-MSCs

BM-MSCs (passages 3–5) were harvested, and mitochondria were isolated using a commercial kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Briefly, the method uses sequential differential centrifugations to separate organelles and other subcellular particles based on their sedimentation rate. To obtain mitochondrial preparations with reduced interference from other cytosolic components, centrifugation for mitochondrial recovery was performed at 3000× g for 15 min at 4 °C. Mitochondrial preparations were made with 1 × 10^6^ BM-MSCs, with a typical yield of 600–800 μg of protein. For flow cytometry characterization, MSCs were pre-stained with 200 nM of Mitotracker Deep Red FM in 10 mL serum-free MEM in the dark for 45 min. Cells were washed with PBS, and the mitochondria were isolated. The final mitochondrial pellet was washed twice and resuspended in PBS. Gating of MSC mitochondria was performed based on FSC/SSC parameters. Analysis was conducted on a FACS CantoII flow cytometer using FACSDiva software, version 6.1.3, and data were analyzed with FlowJo software, version 10 (FlowJo, Ashland, OR, USA). Protein concentration was determined by BCA assay. Mitochondria were administered intravenously within 2 h of isolation to preserve viability [43].

4.3.4. Mitochondrial Oxygen Consumption

Oxygen consumption was measured using an OROBOROS O2k respirometer (Oroboros Instruments, Innsbruck, Austria) at 37 °C. Mitochondria (200 μg protein) were assessed using a standardized SUIT protocol with sequential addition of pyruvate, malate, ADP, cytochrome c, oligomycin, CCCP, and antimycin A. Data are expressed as oxygen flux (pmol O_2_·s^−1^·mL^−1^) [44,45].

4.3.5. Mitochondrial Biodistribution

Mitochondria were labeled with MitoTracker Deep Red and injected intravenously. Animals were euthanized at 3 and 24 h. Heart, lungs, liver, and kidneys were imaged ex vivo using an IVIS™ Lumina XRMS (X-Ray Multi-species optical imaging System, Perkin Elmer, Shelton, CT, USA) [46]. Fluorescence was quantified using Living Image software, version 4.5.2.

4.4. Animal Preparation and Experimental Protocol

For liver-derived mitochondria, forty male Wistar rats (196 ± 16 g, 7 weeks) were randomized to receive an intraperitoneal (i.p.) injection of monocrotaline (60 mg/kg, n = 32) or saline (n = 8). On day 14, animals were anesthetized with isoflurane 2.0% and PAH animals were randomized to receive (n = 8 per group): (1) saline (0.9% NaCl); (2) sildenafil (20 mg/kg/day), oral gavage for 14 days; (3) jugular intravenous injections of isolated liver-derived mitochondria (100 μg/animal on days 14 and 21); or (4) combined treatment with sildenafil and mitochondria.

For BM-MSC–derived mitochondria, a similar experimental protocol was done. Forty male Wistar rats (180 ± 22 g, 7 weeks) were randomized to receive an intraperitoneal (i.p.) injection of monocrotaline (60 mg/kg, PAH group, n = 32) or saline (CTRL group, n = 8) [16,17]. On day 14, animals were anesthetized with isoflurane 2.0% and PAH animals were randomized to receive (n = 8 per group): (1) saline (0.9% NaCl) (PAH); (2) sildenafil (20 mg/kg/day), oral gavage for 14 days (PAH—sil) [47]; (3) jugular intravenous injections of isolated mitochondria (100 μg/animal on days 14 and 21) (PAH—imt); or (4) combined treatment with sildenafil and BM-MSC-derived mitochondria (PAH—sil & imt). On day 28, echocardiography and invasive hemodynamics were repeated, followed by tissue collection [17].

4.4.1. Echocardiography

Transthoracic echocardiography was performed on days 1, 14, and 28 using a UGEO HM70A system (Samsung, San Jose, CA, USA). PAT and PET were measured, and the PAT/PET ratio was used as an indirect index of PAH severity [48,49].

4.4.2. Hemodynamic Measurements

On day 28, RVSP was measured under ketamine/midazolam anesthesia via direct RV puncture using a calibrated pressure transducer system. Pressure signals were recorded over multiple respiratory cycles and averaged for analysis. All measurements were validated by an investigator blinded to group allocation.

4.4.3. Right Ventricular Hypertrophy

The Fulton index (RV weight/[LV weight + septum weight]) was calculated and normalized to body weight [50].

4.4.4. Histology and Immunohistochemistry

Lung sections were stained with Masson’s trichrome for collagen quantification. α-SMA immunohistochemistry was performed to assess muscularization [51,52]. Quantification was performed by an investigator blinded to group allocation.

4.4.5. Reverse Transcription Polymerase Chain Reaction

RNA was extracted from lung tissue, reverse transcribed, and analyzed by qPCR. Expression levels of Sdha, vimentin, VE-cadherin, TFAM, Mfn-1, and OPA1 were normalized to the housekeeping gene GAPDH and calculated using the 2^−ΔΔCt^ method [53]. All analyses were performed by an investigator blinded to the group allocation. Primer sequences are presented in Table S1.

4.4.6. High-Resolution Respirometry in Lung Homogenates

Frozen lung samples were homogenized, and mitochondrial-enriched fractions were obtained. Oxygen consumption was measured to assess Complex I–, Complex II–, and Complex IV–dependent respiration, acknowledging limitations related to frozen tissue [36].

4.4.7. Plasma Levels of Inflammatory Markers by ELISA

Plasma was snap-frozen and stored at −80 °C. Protein levels were quantified via the Bradford assay. IL-6 and IL-1β concentrations were measured using commercial ELISA kits according to the manufacturer’s protocols (PeproTech, Cranbury, NJ, USA). All analyses were performed by investigators blinded to the group allocation.

4.5. Statistical Analysis

There was no exclusion of data points. Normality was assessed using the Kolmogorov–Smirnov test. Data are presented as means ± SD. Student’s t-test was used for day-14 comparisons. One-way ANOVA with Tukey’s post hoc test or Kruskal–Wallis with Dunn’s test was used for day-28 comparisons, as appropriate. Analyses were performed using GraphPad Prism version 9.5.1. p < 0.05 was considered significant.

5. Conclusions

BM-MSC–derived mitochondrial transplantation, particularly when combined with sildenafil, attenuates pulmonary vascular remodeling, reduces plasma inflammatory markers, improves mitochondrial homeostasis, and pulmonary hemodynamics in experimental PAH. These findings support further investigation of mitochondrial-based strategies as adjunctive approaches targeting metabolic dysfunction in pulmonary vascular disease.