Oral delivery of Angiotensin-converting enzyme 2 and Angiotensin-(1-7) bioencapsulated in plant cells attenuates pulmonary hypertension

Abstract

Emerging evidences indicate that diminished activity of the vasoprotective axis of the renin-angiotensin system, constituting angiotensin-converting enzyme 2 (ACE2) and its enzymatic product, angiotensin-(1-7) [Ang-(1-7)] contribute to the pathogenesis of pulmonary hypertension (PH). However, long-term repetitive delivery of ACE2 or Ang-(1-7) would require enhanced protein stability and ease of administration to improve patient compliance. Chloroplast expression of therapeutic proteins enables their bioencapsulation within plant cells to protect against gastric enzymatic degradation and facilitates long-term storage at room temperature. Besides, fusion to a transmucosal carrier helps effective systemic absorption from the intestine on oral delivery. We hypothesized that bioencapsulating ACE2 or Ang-(1-7) fused to the cholera nontoxin B subunit would enable development of an oral delivery system that is effective in treating PH. PH was induced in male Sprague Dawley rats by monocrotaline administration. Subset of animals was simultaneously treated with bioencapsulaed ACE2 or Ang-(1-7) (prevention protocol). In a separate set of experiments, drug treatment was initiated after 2 weeks of PH induction (reversal protocol). Oral feeding of rats with bioencapsulated ACE2 or Ang-(1-7) prevented the development of monocrotaline-induced PH and improved associated cardiopulmonary pathophysiology. Furthermore, in the reversal protocol, oral ACE2 or Ang-(1-7) treatment significantly arrested disease progression, along with improvement in right heart function, and decrease in pulmonary vessel wall thickness. In addition, a combination therapy with ACE2 and Ang-(1-7) augmented the beneficial effects against monocrotaline-induced lung injury. Our study provides proof-of-concept for a novel low-cost oral ACE2 or Ang-(1-7) delivery system using transplastomic technology for pulmonary disease therapeutics.

Trial registration: ClinicalTrials.gov NCT01884051.

Keywords: chloroplast; molecular farming; plant-made pharmaceuticals; pulmonary hypertension; renin–angiotensin system.

© 2014 American Heart Association, Inc.

Figures

Figure 1

Characterization, concentration, and evaluation of pentameric structure of cholera nontoxin B subunit (CTB)-angiotensin-converting enzyme 2 (ACE2) and CTB-angiotensin-(1-7) [Ang-(1-7)] expressed in plant chloroplasts. A, Schematic representation of CTB-ACE2 and CTB-Ang-(1-7) gene cassettes and flanking regions. Southern blot analysis of (B) CTB-ACE2 and (C) CTB-Ang-(1-7) transplastomic lines. HindIII-digested untransformed (UT) and transformed (lane 1, 2, and 3) genomic DNA was probed with P32-labeled flanking sequence. Quantification of (D) CTB-ACE2 and (E) CTB-Ang-(1-7) as a percentage of the total leaf proteins (TLP). F, GM1-binding assay of CTB-ACE2 and CTB-Ang-(1-7). G, Western blot analysis of CTB-Ang-(1-7) in nonreducing condition without boiling and dithiothreitol. Lanes 1, 2, and 3: 10, 15, and 20 ng of CTB; total homogenate of CTB-Ang-(1-7): 0.2, 0.4, 0.8, and 1.6 μg. The pentameric structures for the CTB alone and the fusion protein are indicated by arrow head and arrow, respectively. Data shown are mean ± SD of 3 independent experiments. F indicates fresh; L, lyophilized; and BSA, 1% wt/vol.

Figure 2

Oral administration of bioencapsulated angiotensin-converting enzyme 2 (ACE2) or angiotensin-(1-7) [Ang-(1-7)] prevents monocrotaline (MCT)-induced pulmonary hypertension. A, Measurement of right ventricular (RV) systolic pressure (RVSP) in normal controls and MCT-challenged rats that were either untreated or orally fed with wild-type (WT) leaf material or gavaged with bioencapsulated ACE2/ Ang-(1-7). B, RV hypertrophy, measured as the ratio of RV to left ventricle (LV) plus interventricular septum (S) weights [RV/(LV+S)]. Measurement of (C) RV end-diastolic pressure (RVEDP), (D) +dP/dt, and (E) −dP/ dt. Echocardiography data representing (F) ejection fraction, (G) ratio of the right to left end-diastolic area, signifying right heart dilation, and (H) the blood flow rate in the RV outflow tract (RVOT). Data shown are mean ± SEM. ***P<0.001 vs control rats and #P<0.05 vs untreated or WT leaf-fed MCT rats. n=6 to 8 animals/group.

Figure 3

Oral feeding of bioencapsulated angiotensin-converting enzyme 2 (ACE2) or angiotensin-(1-7) [Ang-(1-7)] exerts antifibrotic and antiremodeling effects in the prevention protocol. A, Interstitial collagen deposition in the right ventricle. B, Staining for α-smooth muscle actin to quantify medial wall thickness of the pulmonary arteries measuring <50 μm. Scale bar, 10 μm. C, ACE2 activity was measured in rat sera (10 μL) collected from different experimental groups Data represents mean ± SEM with *P<0.05 vs other groups, **P<0.01 compared with controls, whereas # representing P<0.05 vs untreated and wild-type (WT) plant material–fed monocrotaline (MCT) rats as assessed by 1-way ANOVA followed by Newman–Keuls test. AFU indicates arbitrary fluorescence units.

Figure 4

Oral treatment with angiotensin-converting enzyme 2 (ACE2) or angiotensin-(1-7) [Ang-(1-7)] arrests disease progression and attenuates cardiopulmonary remodeling. A, Individual values of the right ventricle systolic pressure (RVSP) from different experimental groups of the reversal protocol. B, Ratio of RV to left ventricle (LV) plus interventricular septum (S) weight [RV/(LV+S)] values from individual animals, denoting right heart hypertrophy. Echocardiography data representing (C) ratio of the right to LV end-diastolic area, (D) ejection fraction (EF), and (E) the blood flow rate in the RV outflow tract (RVOT) of the different experimental groups. F, Representative photographs and quantification of interstitial fibrosis. G, Measurement of vessel wall thickness after α-smooth muscle actin staining of the pulmonary arteries (<50 μm). Scale bar, 10 μm. Data shown are mean ± SEM. **P<0.01, ***P<0.001 vs control rats and #P<0.05 vs untreated or wild-type (WT) leaf-fed monocrotaline (MCT) rats. n=6 to 8 animals/group.

Figure 5

Combination therapy with angiotensin-converting enzyme 2 (ACE2) and angiotensin-(1-7) [Ang-(1-7)] rescues established pulmonary hypertension. A, Measurement of right ventricular systolic pressure (RVSP) in monocrotaline (MCT) rats treated with a combination of either 500 mg or 250 mg each of ACE2 and Ang-(1-7). B, Data representing right ventricular hypertrophy as a ratio of RV/(LV+S). Measurement of (C) right ventricular end-diastolic pressure (RVEDP), (D) +dP/dt, and (E) −dP/ dt from the combination study. Echocardiography data representing (F) ejection fraction (EF), (G) ratio of the right to left end-diastolic area, and (H) the blood flow rate in the right ventricular outflow tract (RVOT). Data shown are mean ± SEM. ***P<0.001 vs control rats and #P<0.05 vs untreated or wild-type (WT) leaf-fed MCT-rats. n=6 to 8 animals/group.

Figure 6

Combination of angiotensin-converting enzyme 2 (ACE2) and angiotensin-(1-7) [Ang-(1-7)] decreases ventricular fibrosis and attenuates pulmonary vascular remodeling. A, Representative photographs of collagen staining and quantitative analysis of right ventricular fibrosis after 2 week treatment with combination therapy. B, Measurement of vessel wall thickness of the pulmonary arteries (<50 μm). Scale bar, 10 μm. Data are expressed as mean ± SEM; **P<0.01; vs controls and #P<0.05 vs untreated and wild-type (WT) plant material–fed monocrotaline (MCT) rats. n=5 to 7 animals per experimental group.

Figure 7

Effects of angiotensin-converting enzyme 2 (ACE2) or angiotensin-(1-7) [Ang-(1-7)] treatment on the lung renin–angiotensin system, proinflammatory cytokines, and autophagy (prevention protocol). Relative change in lung mRNA levels of (A) ACE, (B) ACE2, (C) ACE/ACE2 ratio, (D) angiotensin type 1 receptor (AT1R), (E) angiotensin type 2 receptor (AT2R), and (F) AT1R/AT2R receptor. Relative mRNA levels of lung proinflammatory cytokines, G, tumor necrosis factor (TNF)-α, (H) transforming growth factor (TGF)-β, and (I) toll-like receptor-4 (TLR-4) from the monocrotaline (MCT) study. Autophagy marker, LC3-II is increased in the lungs of MCT-exposed animals. J, Immunoblot and densitometry analysis of the lung LC3I/II protein expression. Data are expressed as mean ± SEM. *P<0.05, **P<0.01, and ***P<0.001 vs control rats. #P<0.05 vs MCT group. WT indicates wild type.

Figure 8

Effects of monotherapy, as well as the combination therapy, on the lung renin–angiotensin system components, proinflammatory cytokines, and autophagy in the reversal protocol. Data represent relative changes in lung mRNA levels of (A) angiotensin-converting enzyme (ACE), (B) ACE2, (C) ACE/ ACE2 ratio, (D) angiotensin type 1 receptor (AT1R), (E) angiotensin type 2 receptor (AT2R), and (F) AT1R/AT2R ratio. Relative mRNA levels of lung proinflammatory cytokines, (G) tumor necrosis factor (TNF)-α, (H) transforming growth factor (TGF)-β, and (I) toll-like receptor-4 (TLR-4) from the same study. J, Immunoblot and densitometry quantification showing lung LC3I/II protein expression. Data are expressed as mean ± SEM. *P<0.05 and **P<0.01 vs control rats, whereas #P<0.05 vs monocrotaline (MCT) group. WT indicates wild type.