Pulmonary artery denervation by noninvasive stereotactic radiotherapy: a pilot study in swine models of pulmonary hypertension

Cai, Xingxing; Wei, Zhixing; Shen, Yichen; Qian, Li; Cai, Jing; Yang, Yuli; Chi, Runmin; Wang, Wei; Yu, Shunxuan; Li, Keke; Fei, Yudong; Li, Cheng; Han, Yaqin; Liu, Ming; Zhang, Jianguo; Wang, Dengbin; Jiang, Mawei; Li, Yi-Gang

doi:10.1038/s41467-025-55933-8

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Published: 09 January 2025

Pulmonary artery denervation by noninvasive stereotactic radiotherapy: a pilot study in swine models of pulmonary hypertension

Xingxing Cai ORCID: orcid.org/0009-0002-2150-9618¹^na1,
Zhixing Wei¹^na1,
Yichen Shen²^na1,
Li Qian³,
Jing Cai⁴,
Yuli Yang¹,
Runmin Chi⁴,
Wei Wang²,
Shunxuan Yu²,
Keke Li²,
Yudong Fei¹,
Cheng Li¹,
Yaqin Han¹,
Ming Liu⁴,
Jianguo Zhang³,
Dengbin Wang ORCID: orcid.org/0000-0001-9529-4870⁴,
Mawei Jiang² &
…
Yi-Gang Li ORCID: orcid.org/0000-0003-3007-7212¹

Nature Communications volume 16, Article number: 558 (2025) Cite this article

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Abstract

Catheter-based pulmonary artery denervation (PADN) has achieved promising outcomes to treat pulmonary hypertension (PH). We herein present stereotactic body radiotherapy (SBRT) as a novel noninvasive approach for PADN. A single fraction of 15 Gy, 20 Gy or 25 Gy was delivered for PADN in a thromboxane A2 (TxA2) - induced acute PH swine model. We demonstrated that PADN by 20-Gy SBRT reduced mean pulmonary artery (PA) pressure during the TxA2 challenge. All SBRT dosages led to a deeper denervation area compared with radiofrequency ablation (RFA) and reduced sympathetic neural norepinephrine synthesis in the ablation zone. Probable radiation related side effects were mostly found in animals treated with 25-Gy. In subsequent monocrotaline-induced chronic PH animals, PADN by 20-Gy SBRT resulted in more significant improvement in pulmonary hemodynamics and PA remodeling in comparison to RFA. In summary, our findings suggest that appropriate SBRT scheme could balance the efficacy and safety for PADN, potentiating to be a novel strategy to treat PH.

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Introduction

Pulmonary hypertension (PH) is a progressive disorder characterized by increased pulmonary vascular resistance (PVR) and pulmonary artery (PA) pressure, leading to subsequent right ventricular failure and ultimately death. Despite significant advances in pharmacological treatment, PH patients continue to have high symptom burden and poor long-term prognosis^1,2. Extensive research has shown that overactivation of the sympathetic nervous system and subsequent PA vasoconstriction and remodeling plays a critical role in the pathogenesis of PH^3,4. PA denervation (PADN), as an innovative therapy mostly employing catheter-based radiofrequency ablation (RFA) to destroy baroreceptors or sympathetic nervous fibers around the bifurcation area of the main PA, has gradually proved beneficial to improve pulmonary hemodynamics in animal and initial human studies^{5,6,7,8,9,10,11}. Most recently, in a sham-controlled, multicenter randomized trial, PADN improved exercise capacity and clinical outcomes during 6-month follow-up in patients with WHO Group I PH¹².

In humans, the PAs are predominantly innervated by sympathetic nerves concentrated in the adipose and connective tissue around the main PA trunk and bifurcation, with >40% of nerves at a depth of >4 mm^13,14. Therefore, the efficacy of transluminal RFA-based PADN may be limited due to the unachievable ablation depth^15,16. In addition, pulmonary embolism and PA wall injuries associated with RFA including dissection, focal hemorrhage, reduced medial thickness, intimal disruption and thrombosis have been revealed in pre-clinical experiments^7,16. Moreover, procedure-related chest pain has been frequently observed when applied in the real world^5,17.

Stereotactic body radiotherapy (SBRT) is a common utilized noninvasive technology to treat solid tumors by delivering high-dose radiation precisely to the target while minimizing injury to surrounding tissues with a rapid dose falloff. Additionally, SBRT has been successfully used to treat a limited number of patients with cardiac arrhythmias, including ventricular tachycardia and atrial fibrillation^18,19. Recently, it has been demonstrated in swine models that SBRT can be deployed to renal denervation safely and effectively with appropriate radiation dosage. Compared with conventional radiofrequency, SBRT offers the advantages of a noninvasive approach and complete, circumferential denervation^20,21.

To date, the potential value of noninvasive SBRT for PADN is unknown. Here, we show SBRT adopting the appropriate scheme can noninvasively impair the sympathetic innervation of the PAs, with subsequent improved pulmonary hemodynamics in both acute vasoconstriction-based and chronic monocrotaline (MCT)-induced swine models of PH.

Results

SBRT procedure

All animals survived the expected-in-life phase of the study. SBRT was successfully performed for PADN in the treatment groups (Fig. 1), and no acute complications were observed during the perioperative period. Treatment parameters are provided in Supplementary Table 1. Radiation dose and volume restriction to the organs at risk (OARs) led to a dose distribution that did not have perfect conformity with the target volume in some animals. A typical dose-volume histogram is presented on Supplementary Fig. 1, showing that the designated dose precisely targeted the bifurcation area of the main PA with a rapid dose falloff to minimize radiation to surrounding tissues.

Hemodynamic performance

At baseline, both PADN and control animals developed PH as demonstrated by maximum mean PA pressure (PAP) greater than 40 mmHg, and hemodynamic response to thromboxane A2 (TxA2) agonist infusion was similar between groups. In the 4-month follow-up, PADN by RFA and 20-Gy SBRT reduced mean and systolic PAP during the TxA2 challenge (P < 0.05), while no significant differences were found in the 15-Gy and 25-Gy groups when compared with controls. In addition, diastolic PAP post-PADN in response to TxA2 infusion showed a similar trend like mean and systolic PAP, but did not reach statistical significance in comparison to the untreated animals (Fig. 2). No significant effect was observed on heart rate or systemic blood pressure (Supplementary Fig. 2).

Plasma norepinephrine (NE) and renin-angiotensin-aldosterone system (RAAS) quantification

There were no significant differences in plasma NE, renin, angiotensin II, and aldosterone absolute concentration or fold change between control and treatment groups at baseline and 4-month follow-up (all P > 0.05, Supplementary Table 2).

Effects of SBRT in the ablation zone

Apart from mild tissue adherence occasionally found in the 25-Gy group, no definite lesions were observed along the route of treated PAs during gross anatomical examination (Supplementary Fig. 3).

In resemblance to nerve injury caused by intravascular radiofrequency or ultrasound ablation, SBRT could effectively destroy the PA nerves and present as degenerative or necrotic endoneural changes including vacuolization, nuclear pyknosis, formation of digestion chambers and coagulative necrosis, as well as mild to moderate perineural fibrosis and collagen deposition (Fig. 3A). Overall nerve injury scores were significantly higher in RFA, 20-Gy and 25-Gy groups (2.5 ± 0.2, 2.5 ± 0.4, 2.7 ± 0.2 vs. 1.2 ± 0.5, P < 0.01), but not in the 15-Gy group (1.9 ± 0.5 vs. 1.2 ± 0.5, P > 0.05) as compared with controls. Furthermore, dosage at 25 Gy led to more pronounced nerve damage than 15 Gy (P < 0.05, Fig. 3B). Similarly, PADN with 20 Gy and 25 Gy resulted in significant nerve injuries in both regions within and beyond 5 mm from lumen (P < 0.05), while no statistical differences were found in the 15-Gy group in comparison to controls (P > 0.05). However, PADN by RFA induced no significant damage to nerves beyond 5 mm from the lumen (P > 0.05) notwithstanding apparent injury to those within 5 mm (P < 0.01, Fig. 3C, D).

**Fig. 3: Pathological nerve injury evaluation.**

All the radioablative lesions displayed by depth from the intima to the deepest site of tissue injury were > 10 mm, and SBRT led to a deeper denervation area compared with RFA (P < 0.05). The endothelium of the targeted PAs remained intact in all subjects, and minimal neointima, formed on the internal elastic lamina, was observed occasionally in the animals ablated by 20 Gy and 25 Gy. PA medial injury, as demonstrated by minimal to moderate smooth muscle cell loss with proteoglycan replacement, increased in a dosage-dependent manner without reaching statistical significance (P > 0.05). Nutrient arteriolar damage characterized by minimal to mild perivascular inflammation and smooth muscle cell loss was less in the 15-Gy group in comparison to the RFA and 25-Gy groups (P < 0.05). Additionally, the severity of soft tissue injury, which was characterized by the presence of denatured collagen, inflammation, fat necrosis, and fibrosis, gradually increased with escalating SBRT dosage, and RFA caused more serious soft tissue injury than SBRT with 15 Gy and 20 Gy. The specific data and representative images are presented in Supplementary Table 3 and Fig. 4.

**Fig. 4: Representative Images of the Ablation Area.**

Denervation efficiency assessed by immunohistochemical assay

Tyrosine hydroxylase (TH) staining scores for nerves within the ablation zone were significantly less in PADN groups than in the untreated controls (1.2 ± 0.3 for RFA, 1.4 ± 0.3 for 15 Gy, 1.2 ± 0.3 for 20 Gy, 1.1 ± 0.3 for 25 Gy vs. 2.3 ± 0.4, P < 0.05). In contrast, there were no significant differences between treatment groups (P > 0.05, Fig.5A). A trend towards lower TH scores proximal to the 20-Gy and 25-Gy - ablation zone was evidenced (P > 0.05, Fig.5B). PADN with 20 Gy and 25 Gy resulted in significant weaker TH expression in PA sections distal to the target site (P < 0.05). In contrast, the staining intensity of RFA and 15-Gy group were comparable to the controls (P > 0.05, Fig. 5C). Representative images of functionally impaired nerves after immunostaining are shown in Fig. 5D.

**Fig. 5: Immunohistochemical analysis of denervation.**

Effects of SBRT on PA remodeling and RAAS activity in the lung tissue

As shown in Fig. 6, in small (<100 μm) and medium (100–300 μm) pulmonary vessels, ratios of the vascular wall thickness and area appeared to be lower in the RFA, 15-Gy, and 20-Gy groups, and a trend towards thicker vessel wall and larger wall area was observed in the 25-Gy group. However, there were no significant differences between groups (P > 0.05). In addition, although a similar trend was shown in fluorescence densities and protein expressions of angiotensin II type 1 receptor (AT1R) and mineralocorticoid receptor (MR) in the lung tissue adjacent to the ablation area, the PADN groups showed comparable RAAS activities to the untreated animals (P > 0.05, Fig. 7).

**Fig. 6: Effects of Stereotactic Radioablative PADN on PA Remodeling.**

**Fig. 7: Effects of stereotactic radioablative PADN on RAAS activity of lung tissue.**

Treatment related side effects

Despite the absence of obvious gross morphological injury to the surrounding organs, such as the heart, lungs, trachea, esophagus, and aorta, radiation related side effects were occasionally observed on computed tomography (CT) images. Compared with the control group, PA diameters of the targeted distal bifurcation area were reduced in animals receiving 25 Gy (P < 0.05), whereas no significant difference was found in the other ablation groups (P > 0.05, Supplementary Table 4). Furthermore, radiation pneumonia was observed in 4 animals treated with 25 Gy (Fig. 8). No evident damage to coronary arteries and the other OARs was seen up to 4 months post SBRT.

**Fig. 8: Terminal computed tomography (CT) evaluation.**

In consistent with CT findings, histopathological manifestation of probable radiation related lung injuries, including various degrees of chronic inflammation, hyperplasia of interalveolar lymphoid and fibrous tissue, were mostly observed in animals treated with 25 Gy (Supplementary Fig. 4). Focal degeneration and calcification of the myocardium was found adjacent to the ablation area in 1 animal receiving 25 Gy. No apparent microscopic evidence of esophageal, trachea, or aorta injury was found (Supplementary Fig. 5).

Efficacy of SBRT-based PADN in a MCT-induced chronic PH model

The hemodynamic data of three time points (week 0, week 6, and week 12) among the four groups were shown in Table 1. The baseline hemodynamic variables were similar. In the 6th week, an elevation of mean PAP was observed in the PH groups compared with the control group (P < 0.05), as well as increased systolic PAP, diastolic PAP, and PVR, which revealed that the PH model was successfully established. PADN was associated with significant reductions of mean PAP, systolic PAP, and diastolic PAP, leading to a significant reduction of PVR compared with the PH-Control group (P < 0.05). In addition, SBRT led to a more pronounced improvement of mean PAP, systolic PAP, and PVR in comparison to RFA (P < 0.05).

Table 1 Hemodynamic changes in the chronic PH model

Full size table

Although MCT induced severe PA remodeling, as reflected by the higher percentage of medial wall thickness and the lower percentage of none muscularization, PADN resulted in a significant decrease in full muscularization and overall medial wall thickness when compared with the PH-Control group (P < 0.05). Moreover, PADN by 20-Gy SBRT led to more significant reduction in medial wall thickness (P < 0.05), and showed a trend towards a lower rate of full muscularization than RFA (Fig. 9).

Discussion

PADN by intravascular radiofrequency or ultrasound has gradually arisen as a novel intervention to treat pulmonary arterial hypertension, and other PH subtypes^{5,8,9,17,22,23,24,25}. To the best of our knowledge, this is the first systemic study to evaluate the functional, anatomic, and histopathologic performance of external photo beams therapy for PADN. The major findings are: (1) PADN by SBRT applying appropriate radiation dosage and treatment plan could balance the efficacy and safety to destroy the PA nerves and suppress the sympathetic activity, inhibiting the increase of mean PAP in an acute swine model of PH; (2) In the MCT-induced chronic PH model, PADN by 20-Gy SBRT resulted in more significant improvement in pulmonary hemodynamics and PA remodeling in comparison to RFA.

The pulmonary vasculature is highly innervated, releasing and metabolizing > 40% of circulating catecholamines²⁶. Over the past few decades, a large body of evidence has revealed that sympathetic overactivation and its direct effects on PA vasoconstriction play a critical role in the pathogenesis of PH^3,4. Baroreceptor structures have been described in or near the main PA bifurcation, and the efferent branch of the vasoconstrictive baroreceptor reflex is predominantly mediated by the sympathetic tone²⁷. The current study confirmed that PADN by noninvasive SBRT with 20 Gy targeting the bifurcation of the main PA trunk could improve pulmonary hemodynamics as demonstrated by a reduction in the maximum mean PAP induced by TxA2 and a decrease in mean PAP and PVR of chronic PH animals. Confirmation of the efficacy of this technique was also observed by apparent histological nerve damage and a marked reduction in TH stain. As previously reported in the literature about radiation injury to peripheral nerves following intraoperative radiotherapy, the incidence of neuropathy increased with escalating doses, beginning at 15 Gy²⁸. Although 15 Gy induced relatively mild morphological changes in our study, it impaired NE synthesis of nerves in the ablation zone. Nevertheless, circulating plasma NE concentrations remained unchanged after PADN by delivering prespecified radiation doses. This might be explained by the fact that plasma catecholamines are affected by neuronal release, reuptake, metabolism, and clearance, and therefore are insensitive markers of sympathetic nerve outflow²⁹.

To date, limited data revealed that the human PAs are predominantly innervated by sympathetic nerves concentrated on the bifurcation, with > 40% of nerves at a depth of > 4 mm, tracking the arterial course distally^13,14. Due to the average penetration depth of 3–4 mm³⁰, transluminal radiofrequency energy caused limited histological damage to nerves around thicker-walled PAs and produced incomplete PADN^15,16. The present study demonstrates radioablative lesion depth up to more than 10 mm, thereby covering the vast majority of nerves. Furthermore, remarkable nerve injuries in the regions within and beyond 5 mm isodose line of the planning target volume delivered by radiation of ≥ 20 Gy indicates adequate denervation.

Chronic activation of the RAAS in PH patients is well described. Sympathetic overactivation may promote abnormal vasoconstriction, cell proliferation and migration, and fibrosis through modulation of RAAS activity, leading to pulmonary vascular remodeling³¹. PADN has been shown to downregulate the local and systemic RAAS activity, and then attenuate PA remodeling in chronic PH studies^13,32. However, PH in the TxA2 model was induced by acute vasoconstriction and not by progressive pulmonary vascular remodeling, of which parameters, such as plasma RAAS levels, expressions of local AT1R and MR and degrees of PA muscularization, remained constant after PADN by 15 Gy or 20 Gy. In the chronic PH model, PADN reversed PA remodeling, and SBRT-based PADN induced less vessel muscularization than RFA, which is consistent with more improvement in pulmonary hemodynamics. This may be explained by the fact that SBRT led to a deeper denervation area, thereby targeting the vast majority of nerves. Another possible explanation for this is that low-dose radiation therapy may reduce proliferating cells and attenuate adverse remodeling³³.

Despite more efficiency of denervation by higher doses, it is to note that excessive irradiation of the lung may lead to pulmonary vascular remodeling with subsequent PH, depending on numerous variables including irradiated volume, dose, dose rate and the specific radiation technique³⁴. Although not well documented for SBRT, vascular complications following cranial radiosurgery have been documented after doses as low as 25 Gy³⁵, and vascular remodeling was more pronounced with an increase in irradiated volume despite the lower radiation doses³⁴. Consistent with the literature, this study found that by counteracting the benefit of PADN, the direct effects of radiation at 25 Gy showed a tendency to upregulate expressions of AT1R and MR and PA remodeling in the lungs adjacent to the ablation zone, providing the possible explanation for the neutral effect on pulmonary hemodynamics during TxA2 challenge after PADN by 25 Gy.

So far, toxicity data for high-dose radiation to the limited area of the PA is scarce. From the animal experiments in the field of intraoperative radiotherapy, the great vessels may tolerate up to 30 Gy without apparent complications, such as thrombosis, fibrotic occlusion and aneurysm formation²⁸. However, dose-related PA intimal hyperplasia, medial wall injury, and especially decreased diameters of target PAs in the 25-Gy group were observed in this research, indicating that the tolerance limit for the PA appeared to be lower than anticipated. A possible explanation for this might be that the PA is relatively vulnerable to hypofractionated high doses due to its thinner wall than aorta. Even so, the larger caliber of the human PA, compared with that of the swine, likely has a somewhat higher tolerance for major complications.

Radiation pneumonia, as a common collateral damage observed in current research, is of particular concern when performing SBRT for PADN. Previous work about thoracic tissue tolerance to single-fraction high-dose intraoperative radiotherapy revealed that doses up to 20 Gy were well tolerated, whereas 30 Gy or more resulted in significant injury³⁶. The increased incidence of radiation pneumonia in the animals treated with 25 Gy in this study corroborates these earlier findings. The dose and total volume of critical normal structures that receive radiation can be used to predict the potential likelihood of toxicity. In this study, the maximum dose and volume of the target PA and surrounding organs in the 15-Gy and 20-Gy groups were all below the tolerance limits for SBRT³⁷, and the safety profile was confirmed by CT scan, autopsy and histological examination. In addition, in a pooled analysis of 88 clinical studies, the overall rate of radiation-induced lung toxicity is relatively low after thoracic SBRT³⁸. Hence, it could conceivably be hypothesized that the safety could have been realized with 15 Gy to 20 Gy without sacrificing the efficacy of denervation, since the percentage volume of bilateral lungs receiving 20 Gy, which is recognized as an important parameter in predicting severe radiation pneumonia and early death for SBRT to treat thoracic tumors³⁹, can be limited to safe levels with this novel technique for PADN in human. In addition, collateral injury is likely to result from inaccurate targeting. Therefore, procedural details including target delineation, motion compensation through respiratory gating, or tracking and alignment should be paid more attention to ensure accuracy. Notwithstanding the acceptable benefit-risk ratio, mediastinal, central, and ultra-central lung SBRT is one of the most high-risk areas for radiation treatment. More data need to be obtained before moving forward to the clinic cautiously.

PH is a lethal disease with poor prognosis and high mortality. Various invasive strategies aimed at modulating the autonomic nervous system have been continuously explored for the treatment of PH. It was reported that accidental low-dose irradiation of the main stems of the PAs reversed the elevated pulmonary arterial resistance of an idiopathic PH patient who was first misdiagnosed with lung cancer or lymphoma. Therefore, it can be hypothesized that the reduction of sympathetic efferent signals by low-dose radiation could have contributed to the reduction of PVR⁴⁰. In the future, with the improvement of radiotherapy techniques, noninvasive SBRT potentiates accurate and efficient PADN, thus providing a promising treatment option for PH patients. However, considering the possibility of secondary malignancy, the application of this strategy for PH patients at young age should be carefully evaluated according to the benefits and potential harm.

Our study has several limitations. First, this pilot study was to explore the feasibility of SBRT as a novel strategy for PADN, the improved hemodynamics demonstrated in the swine models may not be directly extrapolated to humans. Second, the hemodynamic measurements were performed under anaesthesia which could modify sympathetic tone. Third, although the swine shares a similar cardiovascular system with humans, anatomy and position of OARs around the ablation zone are different. Meanwhile, it is possible that pathological PAs and nerves may exhibit a different response to radioablation in PH patients. Last, long-term effects of irradiation to the target and surrounding tissues were not evaluated.

In conclusion, our proof-of-principle study demonstrates that SBRT adopting appropriate scheme can noninvasively impair the sympathetic innervation of the PAs, with subsequent improved pulmonary hemodynamics in both acute vasoconstriction-based and chronic MCT-induced swine models of PH. Targeting (ie, the necessary volume leading to the desired effect, motion compensation), optimal dosing, and long-term efficacy and safety require further investigation before clinical translation.

Methods

Study design of acute PH animals

Based on availability of animals and previous investigators’ experience, the experimental study comprised 30 Bama miniature swine of both sexes, weighing from 20 to 25 kg. All procedures and protocols were approved by Institutional Ethics Committee of Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine and performed in accordance with the Guide for the Care and Use of Laboratory Animals.

All swine underwent the first hemodynamic measurement at the beginning of study and then were randomized into 5 groups: a control group (n = 6), a RFA-based PADN group (n = 6) and 3 SBRT-based PADN groups receiving pre-defined dosages (15 Gy, 20 Gy, 25 Gy, n = 6 each). Group assignment was performed by Excel’s function “=randbetween()” to select a random swine in the room. Rebalancing was performed if ≥2 swine from the same treatment group were selected in one room. The animal allocation was blind and researchers performing operations were aware of group assignment. During serum and tissue collection, researchers were blinded to each animal, a supervisor was aware of the sample positions of Western Blot and ELISA experiments. The PADN groups underwent RFA or SBRT procedure, in which the bifurcation areas of the main PAs were accurately targeted. Four months later, chest and cardiac CT images, the second hemodynamics, histological changes in the PAs and adjacent tissues, and parameters of the sympathetic nervous system and RAAS were analyzed.

Animal husbandry

Animal husbandry was conducted in accordance with each facilities’ SOPs. Swine were housed in rooms under a temperature (20–24 °C) and humidity (40–60%) with a 12-h light/dark cycle each day and were fed with commercial formula feed satisfying the standard nutritional requirements and water ad libitum. Animal well-being was checked daily, including observation for behavioral differences. Swine suffering from severe infections with non-effective respond to drug therapy, multi-organ system failure or pain that was difficult to relieve by drug therapy were considered to reach the humane endpoints.

Hemodynamic measurements

Anesthesia was induced by intramuscular injection of Telazol (4 mg/kg) and maintained with isoflurane 2% in 100% O2 via endotracheal tube. A bolus of 100 U/Kg heparin was administered after the insertion of the 6 F femoral arterial and venous sheaths (Cordis, Florida, USA). Additional 1000- to 2000-U heparin boluses were administered if the procedural time was > 1 hour. Right heart catheterization was performed using a 6 F pigtail catheter (Cordis, Florida, USA) with radiographic guidance. The hemodynamic parameters including systolic PAP, diastolic PAP, mean PAP, heart rate, systemic blood pressure were assessed. Acute PH was induced with continuous infusion of TxA2 agonist (D0400, Sigma-Aldrich, USA). The dose of TxA2 was increased at 5-minute intervals in accordance with previous literature (at 15, 20, 25, 30 μg/kg per hour)^7,14, providing a dose-dependent increase in PAP. Once maximum PAP was attained, TxA2 was discontinued. Vital signs, electrocardiography, systemic blood pressure, and oximetry were continuously monitored during the procedure.

RFA procedure for PADN

The RFA procedure was performed under electroanatomical guidance (CARTO 3, Biosense Webster, USA) with three-dimensional reconstruction of the PA trunk, its bifurcation, and proximal parts of the right and left PAs. The ablation area included leision sets encircling the main PA trunk at a level about 5 mm proximal to the bifurcation and the branch PAs at a level 2 to 3 mm distal to the bifurcation (Supplementary Fig. 6). Radiofrequency energy was delivered point-by-point in a power control mode at 30 W, with an irrigation rate of 15 ml/min and duration of 30 seconds per application.

SBRT procedure for PADN

The swine were routinely anesthetized by intramuscular Telazol (4 mg/kg) and then maintained intravenously via marginal ear vein with 1/2 induction dosage of Telazol every hour. After immobilization in lateral position, the CT reference points were marked on the skin by use of wall-mounted lasers. Chest contrast-enhanced CT and respiration-gated 4-dimensional native CT scans were acquired on a Brilliance 16-slice CT scanner (Philips Medical Systems, Cleveland, USA) with 1 mm slice thickness. For enhanced scans to facilitate definition of target PAs, 50 ml contrast medium was injected (6 s delay; flow rate 2.5 ml/s, Omnipaque 350 mg I/ml, GE Healthcare, USA) through cannulated ear vein. By using the Varian Real-time Position Management Respiratory Gating System (Varian Medical Systems, California, USA), 4-dimensional CT scans were obtained and binned into ten motion phases (0–90%).

The process of contouring and treatment planning was conducted in the Eclipse External Beam Planning System (Varian Medical Systems, California, USA). The ablation lesion defined as an area between 10 mm proximal and 5 mm distal to the PA bifurcation was contoured on five respirational phases (30–70%) of 4-dimensional CT. Accumulated target structures were subsequently created on the phase-averaged CT image that was used for treatment planning. Meanwhile, OARs including heart, lungs, trachea, esophagus, aorta, and spinal cord were delineated on the averaged CT. Finally, an isotropic margin of 5 mm was added to create the planning target volume to account for radiation delivery uncertainties. The treatment plan was computed using 2 arcs to concentrate the prescribed dose to the planning target volume in a single fraction. For OARs, predefined radiation dose and volume limits were referenced to minimize the probability of radiation-related toxicity⁴¹.

At the time of treatment, the sedated animals were initially aligned to the room lasers using skin tattoos. Before radiation delivery, a cone beam CT was acquired and compared to the reference CT from the treatment plan. Automatic fusion was performed and then followed by manual refining to ensure precise beam delivery. Once the swine were accurately positioned after image registration, respiration gated beam of 10 MV photons was delivered to the target area in less than 1 hour with a linear accelerator (Varian Edge, Varian Medical Systems, California, USA).

Follow-up CT studies

Four months later, contrast-enhanced chest and cardiac CT scans were performed on a 256-slice CT scanner (Philips Medical Systems, Cleveland, USA) to assess potential radiation injuries to PAs and adjacent tissues. Contrast medium (60–80 ml, Omnipaque 350 mg I/ml, GE Healthcare, USA) was injected through cannulated ear vein at a rate 2.5 ml/s. Bolus tracking was used in the region of interest in the aorta with a preset threshold of 100 HU to trigger cardiac image acquisition.

All acquired data were processed on Extended Brilliance Workspace (Philips Medical Systems, Cleveland, USA). The post-processing techniques, such as multiplanar reformation, maximum intensity projection, volume rendering and curved planar reconstruction, were used for image analysis. The minimum diameters were measured for the main, the left and the right PA around the bifurcation.

Plasma NE and RAAS quantification

NE, renin, angiotensin II and aldosterone were measured from plasma obtained at baseline and 4-month follow-up using enzyme-linked immunosorbent assay kit (Cloud-Clone, Wuhan, China).

Autopsy and histological analysis

After the final hemodynamic assessment, all animals were euthanized with intracardiac injection of potassium chloride. Thoracic organs were excised en bloc and grossly evaluated for treatment-related side effects. Thereafter, the PA and adjacent tissue block was perfusion fixed and then immediately immersed in 4% paraformaldehyde. PA and its branches were transversely cut at 3–5 mm intervals with 10–15 segments, and OARs surrounding the ablation area were also partially cut into segments. All sections were embedded in paraffin for microscopic evaluation. The slides were cut with a rotary microtome at 4 μm thickness and stained with hematoxylin and eosin, Movat pentachrome and Masson trichrome if necessary.

Treatment effects on PA nerves in the target area were determined using a standard scoring system (0=none, 1=minimal, 2=mild, 3=moderate, and 4=severe)^14,20,42 based on the extent of perineural inflammation or fibrosis and endoneural injury including vacuolization, pyknotic nuclei, digestion chambers, and necrosis. With minimal injury (Grade 1), the nerve would be considered largely intact, and likely functional, but exhibit subtle signs of damage which may include trivial perineuronal inflammation or hemorrhage and limited endoneuronal damage (perineuronal proteoglycan deposition, fibroplasia with little to no vacuolization, pyknosis or increase in cellularity). With mild injury (Grade 2), changes are more conspicuous and/or involving more of the nerve bundle and may include increased cellularity, perineuronal inflammation, fibrosis and/or endoneuronal changes (vacuolization, pyknotic nuclei, or digestion chambers). Nerves with moderate injury (Grade 3) would be interpreted to exhibit more notable changes which may include perineuronal inflammation, fibrosis as well as endoneuronal damage (frequent pyknotic nuclei, digestion chambers, vacuolization, or swelling of endoneuronal tissue). Severe injury (Grade 4) changes are typically overwhelming and may consist of marked perineuronal inflammation and/or fibrosis, and endoneurium damage including effacement of nerve architecture, necrosis, and axonal retraction.

To investigate the denervation efficiency at depth, the following parameters were analyzed: (1) nerve injury grade in separate regions of <5 mm and > 5 mm from arterial lumen; (2) maximum distance between arterial lumen and ablation zone.

In addition, pathological changes of PAs, including endothelial loss, medial injury, and damage to surrounding nutrient arterioles and soft tissues, were also semiquantified using above standard system^14,20,41. Arterial endothelium damage was evaluated circumferentially as 0 = no endothelial loss; 1 = endothelial loss <25% of the vessel’s circumference; 2 = endothelial loss 25–50% of the vessel’s circumference; 3 = endothelial loss 51–75% of the vessel’s circumference; 4 = endothelial loss > 75% of the vessel’s circumference. The medial injury was assessed separately by the depth and circumference of the involved segments: 0 = no medial change; 1 = medial change <25% of the medial depth/circumference; 2 = medial change 25–50% of the medial depth/circumference; 3 = medial change 51–75% of the medial depth/circumference; 4 = medial change > 75% of the medial depth/circumference. Nutrient arteriolar injury around the PAs was evaluated using a grading system of 0 to 4, which was applied with respect to perivascular inflammation, smooth muscle cell loss, and fibrinoid necrosis. Additionally, the severity of soft tissue injury characterized by the presence of denatured collagen, inflammation, fat necrosis, and fibrosis were also semiquantified as 0=none, 1=minimal, 2=mild, 3=moderate, and 4=severe.

Elastic-Van Gieson staining and immunofluorescence double-staining with von Willebrand Factor (1:100, ab6994, Abcam, MA, USA) to identify endothelial cells and α-smooth muscle actin (1:100, ab7817, Abcam, MA, USA) to identify smooth muscle cells were performed on lung tissue sections to evaluate pulmonary vessel muscularization. Ten randomly chosen PAs (100–300 μm diameter) and another ten small PAs (<100 μm) per section were analyzed. The ratios of the vascular wall thickness and area were determined using CaseViewer version 2.0 (3DHISTECH Ltd, Budapest, Hungary).

Immunohistochemical assay of peri-vascular nerves

The PA sections were immunohistochemically stained against S-100 protein (1:150, ab41548, Abcam, MA, USA), a marker for detection of nerve fibers and TH (1:200, ab112, Abcam, MA, USA), a functional indicator for NE synthesis. The intensity and distribution of nerve staining against TH of the ablation zone, proximal and distal to the target site, were analyzed separately using the semi-quantitative scoring system (0 = no reaction, 1 = patchy/very weak reaction, 2 = weak to moderate reaction and 3 = strong reaction)^20,42.

RAAS expression in lung tissue adjacent to the ablation area

The expression of RAAS in lung tissue adjacent to the ablation area was evaluated by immunofluorescence (IF) and Western Blot (WB) using AT1R (1:100 (IF) / 1:1000 (WB), bs-23774R, Bioss, Beijing, China), and MR (1:100 (IF) / 1:1000 (WB), bs-21356R, Bioss, Beijing, China) as primary antibodies. The fluorescence and protein band intensities were analyzed using Image J software (National Institute of Health, Bethesda, Maryland, USA).

Verification of SBRT efficacy for PADN in a MCT-induced chronic PH model

Another 24 swine were randomly assigned to 2 groups after the first hemodynamic measurement: control (n = 4, intraperitoneal injection of 75% alcohol) and test (n = 20, intraperitoneal injection of MCT dissolved in 75% alcohol twice at an interval of 1 week, 12 mg/kg). Six weeks after the first injection, repeat hemodynamic measurements were performed. Animals in the test group with mean PAP > 25 mmHg were randomly divided into PH-Control group (n = 4), PH-SBRT group (n = 4, PADN procedure was performed by 20 Gy - SBRT), PH-RFA group (n = 4, PADN procedure was performed by RFA). After an additional 6 weeks, the final hemodynamic measurement was performed and all animals were sacrificed. The lung tissues were fixed, paraffin – embedded, sectioned and stained with hematoxylin and eosin. Elastic-Van Gieson staining was performed to evaluate pulmonary vessel muscularization, which was classified as none (0–25%), partial (25–50%), or full (50–100%). Ten randomly chosen PAs (<150 μm) per section were analyzed. The ratio of the vascular wall thickness to its outer diameter was determined by the same method in the acute model.

The protocol of hemodynamic measurement in the chronic PH model has been described in previous literature^6,15. Briefly, after stable anesthesia, a Swan-Ganz catheter (Edwards Lifesciences, Irvine, California) was positioned at the distal PA for the measurement of mean PAP, systolic PAP, diastolic PAP, pulmonary artery wedge pressure (PAWP) and cardiac output (CO). Then, PVR was calculated using the formula: PVR = (mean PAP- PAWP)/CO.

Statistical analysis

The treatment parameters are expressed as median (range), while the other data are presented as mean ± SD. Normality of distribution was tested with Shapiro-Wilk test. Comparisons of continuous variables with normal distribution were accomplished by one-way ANOVA with Bonferroni post hoc analysis, whereas variables with skewed distribution and ordinal data were compared with Kruskal-Wallis test followed by all pairwise multiple comparisons. Statistical significance was defined as a two-sided p value < 0.05. All analysis were performed with SPSS version 24 (IBM corp., NY, USA) and Prism 9 (GraphPad Software, California, USA).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The data supporting the findings of this study are included in the article and the Supplementary Information or are available from the corresponding authors upon request. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the State Key Program of the National Natural Science Foundation of China (82130009 to Y.G.L.), National Natural Science Foundation of China (82070515 to Y.G.L., 82100528 to X.C., 82470528 to X.C.), Shanghai City Committee of Science and Technology Research Projects (201409005600 to Y.G.L., 23Y11902900 to X.C.), Shanghai Rising-Star Program (21QA1405900 to X.C.), Interdisciplinary Program of Shanghai Jiaotong University (YG2021QN55 to X.C.), and the Nonprofit Central Research Institute Fund of Chinese Academy of Medical Science (2021-JKCS-030 to X.C.).

Author information

These authors contributed equally: Xingxing Cai, Zhixing Wei, Yichen Shen.

Authors and Affiliations

Department of Cardiology, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, China
Xingxing Cai, Zhixing Wei, Yuli Yang, Yudong Fei, Cheng Li, Yaqin Han & Yi-Gang Li
Department of Oncology, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, China
Yichen Shen, Wei Wang, Shunxuan Yu, Keke Li & Mawei Jiang
Department of Pathology, Affiliated Hospital of Nantong University, Nantong, China
Li Qian & Jianguo Zhang
Department of Radiology, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, China
Jing Cai, Runmin Chi, Ming Liu & Dengbin Wang

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Xingxing Cai
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Contributions

Conception, design, and study direction: Y.G.L., M.J., D.W., X.C.; Animal studies: X.C., Z.W., Y.Y., Y.F., C.L., Y.H.; Treatment procedure: X.C., Y.S., W.W., S.Y., K.L.; C.T. analysis: J.C., R.C., M.L.; Pathological analysis: L.Q., J.Z., X.C.; Manuscript write-up: X.C., Z.W., Y.S.

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Correspondence to Dengbin Wang, Mawei Jiang or Yi-Gang Li.

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Cai, X., Wei, Z., Shen, Y. et al. Pulmonary artery denervation by noninvasive stereotactic radiotherapy: a pilot study in swine models of pulmonary hypertension. Nat Commun 16, 558 (2025). https://doi.org/10.1038/s41467-025-55933-8

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Received: 09 February 2023
Accepted: 06 January 2025
Published: 09 January 2025
Version of record: 09 January 2025
DOI: https://doi.org/10.1038/s41467-025-55933-8