Expression of Human ACE2 in Lactobacillus and Beneficial Effects in Diabetic Retinopathy in Mice

Abstract

The angiotensin converting enzyme 2 (ACE2) catalyzes the degradation of Angiotensin II (Ang II) to generate Angiotensin-(1-7), which reduces inflammation and oxidative stress stimulated by Ang II. ACE2 has been shown to be protective in cardiovascular and metabolic diseases including diabetes and its complications. However, the challenge for its clinical application is large-scale production of high-quality ACE2 with sufficient target tissue bioavailability. We developed an expression and delivery system based on the use of probiotic species Lactobacillus paracasei (LP) to serve as a live vector for oral delivery of human ACE2. We show that codon-optimized ACE2 can be efficiently expressed in LP. Mice treated with the recombinant LP expressing the secreted ACE2 in fusion with the non-toxic subunit B of cholera toxin, which acts as a carrier to facilitate transmucosal transport, showed increased ACE2 activities in serum and tissues. ACE2-LP administration reduced the number of acellular capillaries, blocked retinal ganglion cell loss, and decreased retinal inflammatory cytokine expression in two mouse models of diabetic retinopathy. These results provide proof of concept for feasibility of using engineered probiotic species as live vector for delivery of human ACE2 with enhanced tissue bioavailability for treating diabetic retinopathy, as well as other diabetic complications.

Keywords: ACE2; Lactobacillus; diabetes; diabetic retinopathy; drug delivery; probiotics; renin angiotensin system.

Figure 1

ACE2 Activities in Lactobacillus paracasei Expressing Human ACE2 Protein with Three Different Options for Codon Optimization Error bars represent SD of three separate assays, each run duplicate. LP, Lactobacillus paracasei; c.o., codon optimization; RFU, relative fluorescent unit.

Figure 2

Construction of Lactobacillus Vector for Expression of Secreted Human ACE2 Protein Fused with CTB and Evaluation of In Vivo ACE2 Activities (A) Diagram of the Lactobacillus vector expressing codon optimized human ACE2. ACE2 is expressed as a secreted fusion protein with the CTB, which is separated by a furin cleavage site to release ACE2 once it is expressed. (B) ACE2 activities in different tissues in mice administered with LP-ACE2. RFU, relative fluorescent unit. N = 4/group.

Figure 3

Evaluation of Retinal Acellular Capillary in Diabetic eNOS^–/– and Akita Mice (A and C) Representative images of trypsin-digested retinal vascular preparations from non-diabetic (NDM) and diabetic (DM) eNOS^−/− mice treated with vehicle (PBS), WT-LP, and ACE2-LP (A); wild-type littermate control and Akita mice (C) treated with PBS, WT-LP, and ACE2-LP; and quantitative measurements of acellular capillaries of eNOS^−/− (B) and Akita (D) mice. Arrows indicate the acellular capillaries. *p < 0.01 (versus PBS-treated groups). ns^a, not significant (versus PBS-treated group). ns^b, not significant (versus control group). Error bars represent SD (n = 8/group). Treatments with ACE2-LP significantly reduced acellular capillaries in both eNOS^−/− and Akita mice. LP alone showed slight but not statistically significant reduction of capillary loss in both diabetic eNOS^−/− and Akita mice. Scale bar, 50 μm.

Figure 4

Evaluation of Retinal Ganglion Cell (RGC) Density Detected by Brn3a Immunostaining in Diabetic eNOS^–/– and Akita Mice Representative immunofluorescence images of Brn3a staining from non-diabetic (NDM) and diabetic (DM) eNOS^−/− mice treated with vehicle (PBS), WT-LP, and ACE2-LP (A); wild-type littermate control and Akita mice (C) treated with PBS, WT-LP, and ACE2-LP; and quantification of Brn3a-positive cells of eNOS^−/− (B) and Akita (D) mice. *p < 0.01 (versus PBS-treated groups). ns^a, not significant (versus PBS-treated group). ns^b, not significant (versus control group). Error bars represent SD (n = 6/group). Scale bar, 50 μm. Treatments with ACE2-LP prevented diabetes-induced RGC loss in both eNOS^−/− and Akita mice.

Figure 5

Real-Time RT-PCR Analysis of Retinal mRNA Levels of Inflammatory Cytokines in Diabetic eNOS^–/– and Akita Mice Retinal mRNA levels of inflammatory cytokines in diabetic eNOS^−/− (A) and Akita mice (B). Values on y axis represent relative expression levels compared to PBS-treated group for each gene. NDM, non-diabetic; DM, diabetic. *p < 0.01 (versus PBS groups). #p < 0.05 (versus WT-LP group). ns, not significant (versus PBS groups). Error bars represent SD (n = 4/group).