Oral Delivery of ACE2/Ang-(1–7) Bioencapsulated in Plant Cells Protects against Experimental Uveitis and Autoimmune Uveoretinitis

Abstract

Hyperactivity of the renin-angiotensin system (RAS) resulting in elevated Angiotensin II (Ang II) contributes to all stages of inflammatory responses including ocular inflammation. The discovery of angiotensin-converting enzyme 2 (ACE2) has established a protective axis of RAS involving ACE2/Ang-(1-7)/Mas that counteracts the proinflammatory and hypertrophic effects of the deleterious ACE/AngII/AT1R axis. Here we investigated the hypothesis that enhancing the systemic and local activity of the protective axis of the RAS by oral delivery of ACE2 and Ang-(1-7) bioencapsulated in plant cells would confer protection against ocular inflammation. Both ACE2 and Ang-(1-7), fused with the non-toxic cholera toxin subunit B (CTB) were expressed in plant chloroplasts. Increased levels of ACE2 and Ang-(1-7) were observed in circulation and retina after oral administration of CTB-ACE2 and Ang-(1-7) expressing plant cells. Oral feeding of mice with bioencapsulated ACE2/Ang-(1-7) significantly reduced endotoxin-induced uveitis (EIU) in mice. Treatment with bioencapsulated ACE2/Ang-(1-7) also dramatically decreased cellular infiltration, retinal vasculitis, damage and folding in experimental autoimmune uveoretinitis (EAU). Thus, enhancing the protective axis of RAS by oral delivery of ACE2/Ang-(1-7) bioencapsulated in plant cells provide an innovative, highly efficient and cost-effective therapeutic strategy for ocular inflammatory diseases.

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

“…CNTB fusion proteins expressed in chloroplasts form pentameric structures that are highly resistant to detergents, and this hampers solubilization due to tight interactions between CNTB monomers, mediated by 30 hydrogen bonds, seven salt bridges, and hydrophobic interactions (Miyata et al, 2012). In our previous studies (Boyhan and Daniell, 2011;Kwon et al, 2013;Kohli et al, 2014;Shil et al, 2014), multimeric forms exist even after treatment with DTT, detergents (SDS), and boiling. Also, acid (pH 2) could not completely dissociate CNTB pentamers due to the reformation of multimeric structures.…”

“…Therefore, transgenes inserted into chloroplast genomes are expressed at high levels, up to 70% of total leaf protein (De Cosa et al, 2001;Ruhlman et al, 2010). A wide range of proteins, from very small antimicrobial peptides (Lee et al, 2011) or hormones (Boyhan and Daniell, 2011;Kwon et al, 2013) to very large proteins encoded by bacterial, viral, fungal, animal, and human genes, have been expressed successfully in plant chloroplasts (DeGray et al, 2001;Daniell et al, 2009;Verma et al, 2010;Shenoy et al, 2014;Sherman et al, 2014;Shil et al, 2014). Most importantly, expressed proteins are highly stable when lyophilized plant cells are stored at ambient temperature (Kwon et al, 2013;Lakshmi et al, 2013;Kohli et al, 2014;Jin and Daniell, 2015).…”

Overexpression of Angiotensin-Converting Enzyme 2 Ameliorates Amyloid β-Induced Inflammatory Response in Human Primary Retinal Pigment Epithelium

“…21,36 On the other hand, ACE2 deletion exaggerated Ang II-induced expressions of inflammatory cytokines. 57 Here, we found that overexpression of ACE2 inhibited Ab-induced inflammatory responses in human RPE cells.…”

“…[27][28][29][30] Most components of RAS, including ACE2/Ang-(1-7)/Mas axis, have been confirmed in various tissues and organs, including the eye. 21,31,32 Our previous study showed that activation of ACE2 alleviated the lipopolysaccharide (LPS)-induced inflammatory response in both human RPE cells 33 and in endotoxin-induced uveitis (EIU) mouse/rat models. 34,35 Moreover, the most recent study from our laboratory demonstrated that adeno-associated virus (AAV)-mediated ACE2 gene delivery alleviated the ocular inflammation in experimental autoimmune uveitis (EAU) mouse model by activating the local ACE2/Ang-(1-7)/Mas axis.…”

“…RAS regulates homeostasis of the cardiovascular and renal systems, and also plays a crucial role in inflammatory responses and autoimmune dysfunction. 21,22 Two axes, a classic harmful and a newly discovered protective axis, have been identified. Activation of the angiotensin-converting enzyme (ACE)/angiotensin II (Ang II)/Ang II type 1 (AT1) receptor axis, the classic axis of RAS, mediates deleterious effects, including vasoconstriction, fibrosis, migration, fluid retention, and inflammation.…”