Emerging evidences show that diminished activity of the vasoprotective axis of

Emerging evidences show 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). 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 GDC-0980 (RG7422) 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. promoter and the transcripts were stabilized by placing the untranslated region at the GDC-0980 (RG7422) 3′ end of the fusion genes (Figure 1A). To select the chloroplast transformed with the fusion genes aminoglycoside-3″-adenylyl-transferase gene (and flanking sequences.26 HindIII-digested chloroplast genomic DNA from 3 independent transplastomic lines for each transplastomic line showed 2 hybridizing fragments at 8.59 and 3.44 kb for CTB-ACE2 because of an internal Hind III site of ACE2 (Figure 1A) and a fragment at 9.71 kb for CTB-Ang-(1-7) which confirm the absence of untransformed chloroplast genomes (Figure 1B and 1C). Thus stable integration of the transgenes was confirmed and GDC-0980 (RG7422) the homoplasmic lines were used for further studies. The confirmed homoplasmic lines were multiplied using another round of antibiotic selection under aseptic conditions. Then they were cultivated in a controlled greenhouse for increasing biomass. CTB-ACE2 expression varied between GDC-0980 (RG7422) 1.69% and 2.14% of the total leaf proteins (Figure 1D) depending on the harvest time GDC-0980 (RG7422) because this transgene is regulated by light via the chloroplast promoter. Similarly the expression level of CTB-Ang-(1-7) varied between 6.0% and 8.7% of total leaf proteins (Figure 1E) at different durations of illumination reaching maximum expression at the end of SOX2 the day. Hence for performing in vivo experimental studies the therapeutic leaf materials were harvested at 6 PM and powdered in liquid nitrogen. 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 … Both the therapeutic proteins were fused to the transmucosal carrier CTB. The B subunit has a single intrasubunit disulfide bond that stabilizes the CTB monomer.25 The monomers then assemble to form ring-shaped pentameric structure via intersubunit interactions including hydrogen bonds salt bridges and hydrophobic interactions. Upon oral administration only the pentameric form of CTB binds to the gut epithelial GM1 receptor for internalization.27 Hence we investigated the proper formation of pentameric structure of the CTB-fused proteins and their binding affinity to GM1 receptor using GM1-ELISA. The binding affinity between CTB pentamers and the receptor was measured spectrophotometrically as a function of absorbance at 450 nm. The therapeutic proteins from the fresh leaf materials showed comparable absorbance to CTB (Figure 1F) confirming that chloroplasts form disulfide bridges fold and assemble these fusion proteins. We also lyophilized the leaves expressing ACE2 GDC-0980 (RG7422) and Ang-(1-7) and evaluated their affinity to the GM1 receptor (Figure 1F). Lyophilization not only maintained proper folding disulfide bond and pentamer assembly but also facilitated long-term storage at room temperature (Figure 1F). Furthermore the Western blot assay performed under.