Intratracheal Gene Delivery of SERCA2a Ameliorates Chronic Post-Capillary Pulmonary Hypertension: A Large Animal Model

Abstract

Background: Pulmonary hypertension (PH) is characterized by pulmonary arterial remodeling that results in increased pulmonary vascular resistance, right ventricular (RV) failure, and premature death. Down-regulation of sarcoplasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) in the pulmonary vasculature leads to perturbations in calcium ion (Ca(2+)) homeostasis and transition of pulmonary artery smooth muscle cells to a proliferative phenotype.

Objectives: We assessed the feasibility of sustained pulmonary vascular SERCA2a gene expression using aerosolized delivery of adeno-associated virus type 1 (AAV1) in a large animal model of chronic PH and evaluated the efficacy of gene transfer regarding progression of pulmonary vascular and RV remodeling.

Methods: A model of chronic post-capillary PH was created in Yorkshire swine by partial pulmonary vein banding. Development of chronic PH was confirmed hemodynamically, and animals were randomized to intratracheal administration of aerosolized AAV1 carrying the human SERCA2a gene (n = 10, AAV1.SERCA2a group) or saline (n = 10). Therapeutic efficacy was evaluated 2 months after gene delivery.

Results: Transduction efficacy after intratracheal delivery of AAV1 was confirmed by β-galactosidase detection in the distal pulmonary vasculature. Treatment with aerosolized AAV1.SERCA2a prevented disease progression as evaluated by mean pulmonary artery pressure, vascular resistance, and limited vascular remodeling quantified by histology. Therapeutic efficacy was supported further by the preservation of RV ejection fraction (p = 0.014) and improvement of the RV end-diastolic pressure-volume relationship in PH pigs treated with aerosolized AAV1.SERCA2a.

Conclusions: Airway-based delivery of AAV vectors to the pulmonary arteries was feasible, efficient, and safe in a clinically relevant chronic PH model. Vascular SERCA2a overexpression resulted in beneficial effects on pulmonary arterial remodeling, with attendant improvements in pulmonary hemodynamics and RV performance, and might offer therapeutic benefit by modifying fundamental pathophysiology in pulmonary vascular diseases.

Keywords: aerosol delivery; gene therapy; pig models; pulmonary vascular remodeling; right ventricular function.

FIGURE 1. Study Design and Timeline

The chronic pulmonary hypertension (PH) model was created surgically by selective banding of pulmonary veins in 10- to 15-kg piglets. Two months later, animals were randomized to aerosolized airway delivery of either recombinant adeno-associated virus serotype 1 carrying the human SERCA2a transgene (AAV1.SERCA2a) or saline. Changes in cardiopulmonary hemodynamics, right ventricular (RV) structure, and RV function were evaluated 2 months after randomization to treatment. Sham-operated animals (n = 4) served as controls. MRI = magnetic resonance imaging.

FIGURE 2. Pulmonary Hemodynamics

Individual changes from baseline (2 months [m]) to study’s end (4 months) are reported for each animal following right heart catheterization to evaluate the effect of adeno-associated virus serotype 1 carrying the human SERCA2a transgene (AAV1.SERCA2a) therapy on cardiopulmonary hemodynamics. (A) The mean pulmonary artery pressure (mPAP) and (C) pulmonary vascular resistance index (PVRi) reported as well as the corresponding mean changes for each study parameter (B and D) demonstrate improvement with AAV1.SERCA2a. Sham (n = 4), saline (n = 8), AAV1.SERCA2a (n = 8). *p < 0.05. PVR = pulmonary vascular resistance.

FIGURE 3. Pulmonary Vascular Remodeling

Distal vascular remodeling is ameliorated in adeno-associated virus serotype 1 carrying the human SERCA2a transgene (AAV1.SERCA2a)–treated animals compared with the saline group. Representative hematoxylin-eosin staining is shown for (A) sham (n = 4), (B) saline-treated (n = 8), and (C) AAV1.SERCA2a-treated (n = 8) animals. (D) Medial thickness was quantified for pulmonary arteries of <100 and >100 μm diameter. Data are mean SEM. *p < 0.05; **p < 0.01.

FIGURE 4. Lung and Pulmonary Artery SERCA2a Protein Expression

(A) Sarcoplasmic reticulum Ca²⁺-ATPase 2a (SERCA2a) protein expression was quantified in lung homogenates by Western blotting. Representative blots are shown with densitometry data provided below comparing sham (n = 3), adeno-associated virus serotype 1 carrying the human SERCA2a transgene (AAV1.SERCA2a)-treated (n = 6), and saline-treated (n = 6) animals. *p < 0.05 vs. saline. (B) Representative immunofluorescence images from confocal microscopy showing specific localization of SERCA2a protein (red) in pulmonary vessels and distal airway epithelium in AAV1.SERCA-treated (top) and saline-treated (bottom) animals. Green and blue staining localize to α-smooth muscle actin (SMA) and 4′,6-diamidino-2-phenylindole (DAPI), respectively. Bar = 50 μm.

FIGURE 5. RV Remodeling

Serial cardiac magnetic resonance imaging (CMR) was performed to examine the effect of aerosolized adeno-associated virus carrying SERCA2a (AAV1.SERCA2a) on RV structural remodeling. At the end of the 2-month treatment period, CMR-derived measures of (A) right ventricular ejection fraction (RVEF) and (B) indexed RV mass were preserved in the AAV1.SERCA2a-treated pigs (n = 8) but decreased in saline-treated pigs (n = 5). Mean change estimates with 95% confidence intervals are shown for (C) RVEF and (D) RV mass. *p < 0.05. Abbreviations as in Figure 1.

FIGURE 6. Indexes of RV Performance

At randomization and after the 2-month treatment period, RV pressure–volume relationships were assessed invasively and reported for animals randomized to saline (n = 6) or AAV1.SERCA2a (n = 8). End-systolic pressure–volume relationship (ESPVR) (A) and end-diastolic pressure–volume relationship (EDPVR) (B) slopes are shown, together with mean change estimate for EDPVR (C). (D) Representative pressure-volume loop volume reduction series from saline-treated (blue) and AAV1.SERCA2a-treated (gray) animals show steeper slopes for the saline group. (E) Relative RV weight from explanted hearts expressed as RV/left ventricle + septum (LV+S). (F) Myocardial interstitial fibrosis was assessed as collagen fractional area, which was quantified in RV free wall tissue by picrosirius red staining. *p < 0.05. Ea = arterial elastance; other abbreviations as Figure 1.

CENTRAL ILLUSTRATION. Inhaled AAV1.SERCA2a in PH

Chronic pulmonary hypertension (PH) is associated with increased right ventricular (RV) afterload and vascular remodeling, leading to right ventricular hypertrophy (RVH) that results from distal pulmonary artery hypertrophic remodeling. Airway gene delivery of recombinant adeno-associated virus serotype 1 carrying the human SERCA2a transgene (AAV1.SERCA2a) limits RV hypertrophy and failure by attenuating the progression of pulmonary artery hypertrophic remodeling in a swine model. EC = endothelial cell; SMC = smooth muscle cell.