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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.

SUBMITTER: Verma A

PROVIDER: S-EPMC6661465 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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

Verma Amrisha A Xu Kang K Du Tao T Zhu Ping P Liang Zhibing Z Liao Shengquan S Zhang Juantao J Raizada Mohan K MK Grant Maria B MB Li Qiuhong Q

Molecular therapy. Methods & clinical development 20190710

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 delive ...[more]

PMID: 31380462

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Project description:PurposeIncreased oxidative stress and inflammatory mediators are implicated in the development of diabetic retinopathy, and in rats, its development can be prevented by antioxidants. Carotenoids are some of the powerful antioxidants, and diabetes decreases lutein and zeaxanthin levels in the serum and retina. The aim of this study is to investigate the effect of carotenoid containing nutritional supplements (Nutr), which is in clinical trials for 'Diabetes Vision Function', on diabetic retinopathy.MethodsStreptozotocin-induced diabetic rats (Wistar, male) were fed Purina 5001 supplemented with nutritional supplements containing zeaxanthin, lutein, lipoic acid, omega-3 fatty acids and other nutrients, or without any supplementation. Retinal function was analyzed at ~4 months of diabetes by electroretinography. After 11 months of diabetes, capillary cell apoptosis (TUNEL-staining) and histopathology (degenerative capillaries) were quantified in trypsin-digested retinal vasculature. Retina was also analyzed for mitochondrial damage (by quantifying gene expressions of mtDNA-encoded proteins of the electron transport chain), VEGF and inflammatory mediators, interleukin-1β and NF-kB.ResultsDiabetes impaired retinal function decreasing the amplitudes of both a- and b-waves. In the same animals, retinal capillary cell apoptosis and degenerative capillaries were increased by 3-4 fold. Gene expressions of mtDNA encoded proteins were decreased, and VEGF, interleukin-1β and NF-kB levels were elevated. Supplementation with the nutrients prevented increased capillary cell apoptosis and vascular pathology, and ameliorated these diabetes-induced retinal abnormalities.ConclusionsNutritional supplementation prevents diabetic retinopathy, and also maintains normal retinal function, mitochondrial homeostasis and inflammatory mediators. Thus, this supplementation could represent an achievable and inexpensive adjunct therapy to also inhibit retinopathy, a slow progressing disease feared most by diabetic patients.

Project description:BackgroundWe examined components of systemic and intestinal renin-angiotensin system on gut barrier permeability, glucose homeostasis, systemic inflammation, and progression of diabetic retinopathy (DR) in human subjects and mice with type 1 diabetes (T1D).MethodsT1D individual with (n=18) and without (n=20) DR and controls (n=34) were examined for changes in gut-regulated components of the immune system, gut leakage markers (FABP2 [fatty acid binding protein 2] and peptidoglycan), and Ang II (angiotensin II); Akita mice were orally administered a Lactobacillus paracasei (LP) probiotic expressing humanized ACE2 (angiotensin-converting enzyme 2) protein (LP-ACE2) as either a prevention or an intervention. Akita mice with genetic overexpression of humanAce2 by small intestine epithelial cells (Vil-Cre.hAce2KI-Akita) were similarly examined. After 9 months of T1D, circulatory, enteral, and ocular end points were assessed.ResultsT1D subjects exhibit elevations in gut-derived circulating immune cells (ILC1 cells) and higher gut leakage markers, which were positively correlated with plasma Ang II and DR severity. The LP-ACE2 prevention cohort and genetic overexpression of intestinal ACE2 preserved barrier integrity, reduced inflammatory response, improved hyperglycemia, and delayed development of DR. Improvements in glucose homeostasis were due to intestinal MasR activation, resulting in a GSK-3β (glycogen synthase kinase-3 beta)/c-Myc (cellular myelocytomatosis oncogene)-mediated decrease in intestinal glucose transporter expression. In the LP-ACE2 intervention cohort, gut barrier integrity was improved and DR reversed, but no improvement in hyperglycemia was observed. These data support that the beneficial effects of LP-ACE2 on DR are due to the action of ACE2, not improved glucose homeostasis.ConclusionsDysregulated systemic and intestinal renin-angiotensin system was associated with worsening gut barrier permeability, gut-derived immune cell activation, systemic inflammation, and progression of DR in human subjects. In Akita mice, maintaining intestinal ACE2 expression prevented and reversed DR, emphasizing the multifaceted role of the intestinal renin-angiotensin system in diabetes and DR.

Project description:BackgroundHigh glycated-hemoglobin (HbA1c) levels correlated with an elevated risk of adverse cardiovascular outcomes despite renin-angiotensin system (RAS) inhibition in type-2 diabetic (T2DM) patients with reduced ejection fraction. Using the routine biopsies of non-T2DM heart transplanted (HTX) in T2DM recipients, we evaluated whether the diabetic milieu modulates glycosylated ACE2 (GlycACE2) levels in cardiomyocytes, known to be affected by non-enzymatic glycosylation, and the relationship with glycemic control.ObjectivesWe investigated the possible effects of GlycACE2 on the anti-remodeling pathways of the RAS inhibitors by evaluating the levels of Angiotensin (Ang) 1-9, Ang 1-7, and Mas receptor (MasR), Nuclear-factor of activated T-cells (NFAT), and fibrosis in human hearts.MethodsWe evaluated 197 first HTX recipients (107 non-T2DM, 90 T2DM). All patients were treated with angiotensin-converting enzyme inhibitor (ACE-I) or angiotensin receptor blocker (ARB) at hospital discharge. Patients underwent clinical evaluation (metabolic status, echocardiography, coronary CT-angiography, and endomyocardial biopsies). Biopsies were used to evaluate ACE2, GlycACE2, Ang 1-9, Ang 1-7, MasR, NAFT, and fibrosis.ResultsGlycACE2 was higher in T2DM compared tonon-T2DM cardiomyocytes. Moreover, reduced expressions of Ang 1-9, Ang 1-7, and MasR were observed, suggesting impaired effects of RAS-inhibition in diabetic hearts. Accordingly, biopsies from T2DM recipients showed higher fibrosis than those from non-T2DM recipients. Notably, the expression of GlycACE2 in heart biopsies was strongly dependent on glycemic control, as reflected by the correlation between mean plasma HbA1c, evaluated quarterly during the 12-month follow-up, and GlycACE2 expression.ConclusionPoor glycemic control, favoring GlycACE2, may attenuate the cardioprotective effects of RAS-inhibition. However, the achievement of tight glycemic control normalizes the anti-remodeling effects of RAS-inhibition.Trial registrationhttps://clinicaltrials.gov/ NCT03546062.

Dataset Information

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

Publications

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

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