**Background:** Diabetic retinopathy (DR) is a common complication of type 1 diabetes (T1D), affecting over 90% of individuals within two decades of disease onset. The retinal pigment epithelium (RPE) plays a critical role in retinal health, and its dysfunction contributes to DR pathogenesis. Dyslipidemia and impaired lipid metabolism are increasingly recognized as contributors to DR. The renin-angiotensin system (RAS) is dysregulated in diabetes, with reduced levels of angiotensin-converting enzyme 2 (ACE2) in multiple tissues, including the retina and intestine. The small intestine has the highest ACE2 expression in the body and is central to dietary lipid absorption via intestinal lymphatics (lacteals). Previous work by this group showed that T1D patients with DR exhibit gut barrier disruption, and that genetic depletion of ACE2 worsens retinal damage in diabetic mice. This study tested whether oral administration of a probiotic engineered to express soluble ACE2 (LP-ACE2) could improve intestinal lymphatic integrity, systemic lipid profiles, and retinal outcomes in a T1D mouse model.
**Methods:** Male Akita mice (a monogenic T1D model) and wild-type (WT) littermates were used. At 6 months of age, diabetic Akita mice were randomly assigned to receive either control probiotic (LP) or LP-ACE2 (200 μL, 1 × 10^10 CFU/mouse, 3 times/week) by oral gavage for 3 months. WT mice served as healthy controls. Intestinal tissues, plasma, and eyes were collected. Immunofluorescence staining was performed for lacteal marker LYVE1, endothelial barrier marker PLVAP1, epithelial tight junction proteins ZO-1 and p120-catenin, retinal ZO-1, VCAM-1 (inflammation), and ABCG1 (cholesterol efflux transporter). ABCA1 mRNA expression in retina was measured by qRT-PCR. Circulating lipids (LDL-cholesterol, HDL-cholesterol, triglycerides, free fatty acids) were measured by ELISA. Retinal function was assessed by electroretinography (scotopic a- and b-wave amplitudes) and visual acuity by optokinetic nystagmus (OKN). Acellular capillaries were quantified in retinal flatmounts. Statistical analysis used one-way ANOVA with Tukey's multiple comparison test; p < 0.05 was considered significant.
**Key Results:** LP-ACE2 treatment significantly restored intestinal lacteal integrity: LYVE1 expression increased from 2.07 ± 0.19 in Akita mice to 5.16 ± 0.43 (p < 0.001), reaching 89.96% of WT levels (5.74 ± 1.16). The endothelial barrier disruption marker PLVAP1, elevated in Akita mice (4.05 ± 1.03 vs. WT 1.05 ± 0.3, p < 0.05), was reduced to 1.54 ± 0.3 (p < 0.02) after LP-ACE2. Epithelial barrier proteins ZO-1 and p120-catenin, significantly decreased in Akita mice (ZO-1: 0.60 ± 0.15 vs. WT 3.22 ± 0.33, p < 0.001), were substantially restored by LP-ACE2. Circulating LDL-cholesterol was elevated in Akita mice (72.37 ± 7.62 vs. WT 45.28 ± 3.72, p < 0.01) and reduced by LP-ACE2 (p < 0.007). HDL-cholesterol was decreased in Akita mice (30.91 ± 4.19 vs. WT 65.41 ± 5.12, p < 0.0006) and increased by LP-ACE2 (43.94 ± 4.83, p < 0.01). Triglycerides were elevated in Akita mice (338.7 ± 22.03 vs. WT 190.2 ± 31.15, p < 0.0028) and reduced by LP-ACE2. Free fatty acids were higher in Akita mice (1971 ± 298.7 vs. WT 882.2 ± 130, p < 0.02) but showed a non-significant reduction after LP-ACE2. In the retina, ABCG1 expression was decreased in Akita mice (783.0 ± 87.94 vs. WT 1113.0 ± 64.24, p < 0.04) and restored by LP-ACE2 (1273.0 ± 124.5, p < 0.007). ABCA1 mRNA was significantly upregulated by LP-ACE2 compared to untreated Akita mice. Retinal ZO-1 expression showed a non-significant trend toward improvement. VCAM-1, a marker of retinal inflammation, was increased in Akita mice (12.33 ± 1.18 vs. WT 7.19 ± 1.35, p < 0.03) and reduced by LP-ACE2 (5.89 ± 0.53, p < 0.02). Retinal function: scotopic a-wave amplitude was reduced in Akita mice (127.8 ± 23.76 vs. WT 235.5 ± 29.89, p < 0.03) and restored by LP-ACE2 (252.7 ± 22.46, p < 0.01). Scotopic b-wave was reduced in Akita mice (275.6 ± 19.05 vs. WT 501.2 ± 73.48, p < 0.02) and restored by LP-ACE2 (534.9 ± 31.01, p < 0.009). Visual acuity (spatial frequency) was lower in Akita mice (0.33 ± 0.02 vs. WT 0.41 ± 0.007, p < 0.02) and improved by LP-ACE2 (p < 0.02). Acellular capillaries were increased in Akita mice (12.33 ± 1.18 vs. WT 7.19 ± 1.35, p < 0.03) and significantly reduced by LP-ACE2 (5.89 ± 0.53, p < 0.02).
**Clinical Implications:** This study demonstrates that oral administration of ACE2-expressing probiotics can simultaneously correct intestinal lymphatic dysfunction, systemic dyslipidemia, and retinal pathology in a T1D model. The findings suggest that the gut-retina axis, mediated by ACE2, plays a critical role in DR pathogenesis. By improving intestinal barrier function and lipid metabolism, LP-ACE2 treatment reduces circulating atherogenic lipids and enhances retinal cholesterol efflux via ABCG1/ABCA1, leading to improved retinal function and reduced vascular damage. These results support the potential of probiotic-based delivery of ACE2 as a novel therapeutic strategy for DR, particularly in patients with gut barrier dysfunction and dyslipidemia. Limitations include the use of only male mice, lack of direct measurement of lipid synthesis/turnover, and absence of ACE2 level confirmation in this study. Future studies should explore sex differences, lipid flux, and clinical translation.