Angiotensin receptor expression revealed by reporter mice and beneficial effects of AT2R agonist in retinal cells.

The renin-angiotensin system (RAS) plays a vital role in cardiovascular physiology and body homeostasis. In addition to circulating RAS, a local RAS exists in the retina. Dysfunction of local RAS, resulting in increased levels of Angiotensin II (Ang II) and activation of AT1R-mediated signaling pathways, contributes to tissue pathophysiology and end-organ damage. Activation of AT2R on other hand is known to counteract the effects of AT1R activation and produce anti-inflammatory and anti-oxidative effects. We examined the expression of angiotensin receptors in the retina by using transgenic dual reporter mice and by real-time RT-PCR. We further evaluated the effects of C21, a selective agonist of AT2R, in reducing Ang II, lipopolysaccharide (LPS) and hydrogen peroxide induced oxidative stress and inflammatory responses in cultured human ARPE-19 cells. We showed that both AT1Ra and AT2R positive cells are detected in different cell types of the eye, including the RPE/choroid complex, ciliary body/iris, and neural retina. AT1Ra is more abundantly expressed than AT2R in mouse retina, consistent with previous reports. In the neural retina, AT1Ra are also detected in photoreceptors whereas AT2R are mostly expressed in the inner retinal neurons and RGCs. In cultured human RPE cells, activation of AT2R with C21 significantly blocked Ang II, LPS and hydrogen peroxide -induced NF-κB activation and inflammatory cytokine expression; Ang II and hydrogen peroxide-induced reactive oxygen species (ROS) production and MG132-induced apoptosis, comparable to the effects of Angiotensin-(1-7) (Ang-(1-7)), another protective component of the RAS, although C21 is more potent in reducing some of the effects induced by Ang II, whereas Ang-(1-7) is more effective in reducing some of the LPS and hydrogen peroxide-induced effects. These results suggest that activation of AT2R may represent a new therapeutic approach for retinal diseases.

A novel bispecific molecule delivered by recombinant AAV2 suppresses ocular inflammation and choroidal neovascularization.

Elevated vascular endothelial growth factor (VEGF) and complement activation are implicated in the pathogenesis of different ocular diseases. The objective of this study was to investigate the hypothesis that dual inhibition of both VEGF and complement activation would confer better protection against ocular inflammation and neovascularization. In this study, we engineered a secreted chimeric VEGF inhibitor domain (VID), a complement inhibitor domain (CID) and a dual inhibitor (ACVP1). Vectors expressing these three inhibitors were constructed and packaged into AAV2 (sextY-F) particles. The expression and secretion of the proteins were validated by Western blot. The effects of these inhibitors expressed from AAV2 vectors were examined in endotoxin-induced uveitis (EIU), experimental autoimmune uveoretinitis (EAU) and choroidal neovascularization (CNV) mouse models. The AAV2 vectors expressing the CID- and ACVP1-attenuated inflammation in EIU and EAU model, whereas the vector expressing VID showed improved retinal structure damaged by EAU, but not affect the infiltration of inflammatory cells in EAU or EIU eyes. Both VID and CID vectors improved laser-induced retinal and choroid/RPE injuries and CNV, whereas ACVP1 vector provided significantly better protection. Our results suggest that gene therapy targeting VEGF and complement components could provide an innovative and long-term strategy for ocular inflammatory and neovascular diseases.

Adeno-Associated Virus Overexpression of Angiotensin-Converting Enzyme-2 Reverses Diabetic Retinopathy in Type 1 Diabetes in Mice.

Angiotensin-converting enzyme (ACE)-2 is the primary enzyme of the vasoprotective axis of the renin angiotensin system that regulates the classic renin angiotensin system axis. We aimed to determine whether local retinal overexpression of adenoassociated virus (AAV)-ACE2 prevents or reverses diabetic retinopathy. Green fluorescent protein (GFP)-chimeric mice were generated to distinguish resident (retinal) from infiltrating bone marrow-derived inflammatory cells and were made diabetic using streptozotocin injections. Retinal digestion using trypsin was performed and acellular capillaries enumerated. Capillary occlusion by GFP(+) cells was used to measure leukostasis. Overexpression of ACE2 prevented (prevention cohort: untreated diabetic, 11.3 ± 1.4; ACE2 diabetic, 6.4 ± 0.9 per mm(2)) and partially reversed (reversal cohort: untreated diabetic, 15.7 ± 1.9; ACE2 diabetic, 6.5 ± 1.2 per mm(2)) the diabetes-associated increase of acellular capillaries and the increase of infiltrating inflammatory cells into the retina (F4/80(+)) (prevention cohort: untreated diabetic, 24.2 ± 6.7; ACE2 diabetic, 2.5 ± 1.6 per mm(2); reversal cohort: untreated diabetic, 56.8 ± 5.2; ACE2 diabetic, 5.6 ± 2.3 per mm(2)). In both study cohorts, intracapillary bone marrow-derived cells, indicative of leukostasis, were only observed in diabetic animals receiving control AAV injections. These results indicate that diabetic retinopathy, and possibly other diabetic microvascular complications, can be prevented and reversed by locally restoring the balance between the classic and vasoprotective renin angiotensin system.

Oral delivery of ACE2/Ang-(1-7) bioencapsulated in plant cells protects against experimental uveitis and autoimmune uveoretinitis.

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.

Oral delivery of bioencapsulated proteins across blood-brain and blood-retinal barriers.

Delivering neurotherapeutics to target brain-associated diseases is a major challenge. Therefore, we investigated oral delivery of green fluorescence protein (GFP) or myelin basic protein (MBP) fused with the transmucosal carrier cholera toxin B subunit (CTB), expressed in chloroplasts (bioencapsulated within plant cells) to the brain and retinae of triple transgenic Alzheimer's disease (3×TgAD) mice, across the blood-brain barriers (BBB) and blood-retinal barriers (BRB). Human neuroblastoma cells internalized GFP when incubated with CTB-GFP but not with GFP alone. Oral delivery of CTB-MBP in healthy and 3×TgAD mice shows increased MBP levels in different regions of the brain, crossing intact BBB. Thioflavin S-stained amyloid plaque intensity was reduced up to 60% by CTB-MBP incubation with human AD and 3×TgAD mice brain sections ex vivo. Amyloid loads were reduced in vivo by 70% in hippocampus and cortex brain regions of 3×TgAD mice fed with bioencapsulated CTB-MBP, along with reduction in the ratio of insoluble amyloid ß 42 (Aß42) to soluble fractions. CTB-MBP oral delivery reduced Aß42 accumulation in retinae and prevented loss of retinal ganglion cells in 3×TgAD mice. Lyophilization of leaves increased CTB-MBP concentration by 17-fold and stabilized it during long-term storage in capsules, facilitating low-cost oral delivery of therapeutic proteins across the BBB and BRB.

Expression and cellular localization of the Mas receptor in the adult and developing mouse retina.

PURPOSE: Recent studies have provided evidence that a local renin-angiotensin system (RAS) exists in the retina and plays an important role in retinal neurovascular function. We have recently shown that increased expression of ACE2 and angiotensin (1-7) [Ang (1-7)], two components of the protective axis of the RAS, in the retina via adeno-associated virus (AAV)-mediated gene delivery, conferred protection against diabetes-induced retinopathy. We hypothesized that the protective molecular and cellular mechanisms of Ang (1-7) are mediated by its receptor, Mas, and the expression level and cellular localization dictate the response to Ang (1-7) and activation of subsequent protective signaling pathways. We tested this hypothesis by examining the expression and cellular localization of the Mas receptor in adult and developing mouse retinas. METHODS: The cellular localization of the Mas receptor protein was determined with immunofluorescence of the eyes of adult and postnatal day 1 (P1), P5, P7, P15, and P21 mice using the Mas receptor-specific antibody, and mRNA was detected with in situ hybridization of paraffin-embedded sections. Western blotting and real-time reverse-transcription (RT)-PCR analysis were performed to determine the relative levels of the Mas protein and mRNA in adult and developing retinas, as well as in cultured retinal Müller glial and RPE cells. RESULTS: In the adult eye, the Mas receptor protein was abundantly present in retinal ganglion cells (RGCs) and photoreceptor cells; a lower level of expression was observed in endothelial cells, Müller glial cells, and other neurons in the inner nuclear layer of the retina. In the developing retina, Mas receptor mRNA and protein expression was detected in the inner retina at P1, and the expression levels increased with age to reach the adult level and pattern by P15. In the adult mouse retina, Mas receptor mRNA was expressed at a much higher level when compared to angiotensin II (Ang II) type I (AT1R) and type II (AT2R) receptor mRNA. CONCLUSIONS: The Mas receptor is expressed in developing and adult mouse retinas, and is more abundant in retinal neurons than in endothelial and Müller glial cells. These observations suggest that Mas receptor-mediated signaling may play important roles that extend beyond mediating the vascular effects of Ang (1-7) in developing and adult retinas. In addition, the relatively high expression of the Mas receptor when compared to AT1R suggests that they may play a more important role in maintaining normal retinal physiology than previously considered.

ACE2 and Ang-(1-7) confer protection against development of diabetic retinopathy.

Despite evidence that hyperactivity of the vasodeleterious axis (ACE/angiotensin II (Ang II)/AT1 receptor) of the renin-angiotensin system (RAS) is associated with the pathogenesis of diabetic retinopathy (DR) use of the inhibitors of this axis has met with limited success in the control of this pathophysiology. We investigated the hypothesis that enhancing the local activity of the recently established protective axis of the RAS, ACE2/Ang-(1-7), using adeno-associated virus (AAV)-mediated gene delivery of ACE2 or Ang-(1-7) would confer protection against diabetes-induced retinopathy. Genes expressing ACE2 and Ang-(1-7) were cloned in AAV vector. The effects of ocular AAV-ACE2/Ang-(1-7) gene transfer on DR in diabetic eNOS(-/-) mice and Sprague-Dawley (SD) rats were examined. Diabetes was associated with approximately tenfold and greater than threefold increases in the ratios of ACE/ACE2 and AT1R/Mas mRNA levels in the retina respectively. Intraocular administration of AAV-ACE2/Ang-(1-7) resulted in significant reduction in diabetes-induced retinal vascular leakage, acellular capillaries, infiltrating inflammatory cells and oxidative damage in both diabetic mice and rats. Our results demonstrate that DR is associated with impaired balance of retinal RAS. Increased expression of ACE2/Ang-(1-7) overcomes this imbalance and confers protection against DR. Thus, strategies enhancing the protective ACE2/Ang-(1-7) axis of RAS in the eye could serve as a novel therapeutic target for DR.

Comparison of the transduction efficiency of tyrosine-mutant adeno-associated virus serotype vectors in kidney.

Gene therapy has a distinct potential to treat kidney diseases. However, the efficient transduction of a significant number of renal cells by viral vectors has been difficult to accomplish. Previous studies indicate that adeno-associated virus (AAV) can transduce renal cells with variable and suboptimal efficiency. Because new and innovative mutants of AAV are now available, we compared their efficacy in transducing rat kidneys. We compared five types of AAV mutants (AAV2 mut-triple, AAV2 sextuple, AAV8 mut447, AAV8 mut733 and AAV9 mut446) carrying a green fluorescence protein (GFP) reporter gene. A pressure microinjection technique was used to inject either 1.5 × 10(11) vector genome (vg) AAV mutants or three dose of AAV2 sextuple into the renal cortex of rats. The microinjection approach has not been used in AAV-mediated renal gene transfer thus far. Slow and sustained microinjection enables continuous administration of the viral vector to the kidney cortex and limits any damage to the kidney, because the tip of a glass micropipette is very small. Three weeks after injection, the kidneys were collected and evaluated for GFP expression. Among the various mutated AAV serotypes studied, only AAV2 sextuple showed robust GFP expression in renal tissue. The AAV2 sextuple serotype appears to be an efficient gene transfer vector to preferentially target renal tubular epithelial cells. A combination of the AAV2 sextuple and the microinjection technique holds the key to the future of therapeutic treatments for kidney diseases.

Angiotensin (1-7) Delivered Orally via Probiotic in Combination With Exercise: Sex-Dependent Influence on Health Span.

Age-related declines in physical and cognitive function can have tremendous, negative impacts on health span and quality of life. Therefore, we investigated the potential of utilizing a probiotic treatment to target the renin-angiotensin system (RAS) in conjunction with moderate exercise to ameliorate age-related declines in cognitive and physical function in aged rats. Herein we utilized a genetically modified angiotensin (1-7), which activates a "complementary" arm of the RAS through binding Mas (AT7) receptors. This process induces several beneficial physiologic effects, including decreased inflammation and enhanced physical/cognitive function. Thus, in this short research report, we suggest the efficacy of this Ang(1-7) releasing Lactobacillus paracasei (LPA) as either an alternative strategy to exercise, or more likely as an adjuvant to moderate exercise, for the prevention of both physical and cognitive decline especially in female rats.

Multiomics profiling of the impact of an angiotensin (1-7)-expressing probiotic combined with exercise training in aged male rats.

Angiotensin (1-7) [Ang (1-7)] is an active heptapeptide of the noncanonical arm of the renin-angiotensin system that modulates molecular signaling pathways associated with vascular and cellular inflammation, vasoconstriction, and fibrosis. Preclinical evidence suggests that Ang (1-7) is a promising therapeutic target that may ameliorate physical and cognitive function in late life. However, treatment pharmacodynamics limits its clinical applicability. Therefore, this study explored the underlying mechanisms altered by a genetically modified probiotic (GMP) that expresses Ang (1-7) combined with and without exercise training in an aging male rat model as a potential adjunct strategy to exercise training to counteract the decline of physical and cognitive function. We evaluated cross-tissue (prefrontal cortex, hippocampus, colon, liver, and skeletal muscle) multi-omics responses. After 12 wk of intervention, the 16S mRNA microbiome analysis revealed a main effect of probiotic treatment within- and between groups. The probiotic treatment enhanced α diversity (Inverse Simpson (F[2,56] = 4.44; P = 0.02); Shannon-Wiener (F[2,56] = 4.27; P = 0.02)) and ß-diversity (F[2,56] = 2.66; P = 0.01) among rats receiving our GMP. The analysis of microbes' composition revealed three genera altered by our GMP (Enterorhabdus, Muribaculaceae unclassified, and Faecalitalea). The mRNA multi-tissue data analysis showed that our combined intervention upregulated neuroremodeling pathways on prefrontal cortex (i.e., 140 genes), inflammation gene expression in the liver (i.e., 63 genes), and circadian rhythm signaling on skeletal muscle. Finally, the integrative network analysis detected different communities of tightly (|r| > 0.8 and P < 0.05) correlated metabolites, genera, and genes in these tissues.NEW & NOTEWORTHY This manuscript uses a multiomics approach (i.e., microbiome, metabolomics, and transcriptomics) to explore the underlying mechanisms driven by a genetically modified probiotic (GMP) designed to express angiotensin (1-7) combined with moderate exercise training in an aged male rat model. After 12 wk of intervention, our findings suggest that our GMP enhanced gut microbial diversity while exercise training altered the transcriptional response in relevant neuroremodeling genes, inflammation, and circadian rhythm signaling pathways in an aging animal model.

Type II secretion system of Pseudomonas aeruginosa: in vivo evidence of a significant role in death due to lung infection.

BACKGROUND: The role of toxins secreted by the type II secretion system (T2SS) of Pseudomonas aeruginosa during lung infection has been uncertain despite decades of research. METHODS: Using a model of pneumonia in Toll-like receptor (TLR) 2,4(-/-) mice, we reexamined the role of the T2SS system. Flagellin-deficient mutants of P. aeruginosa, with mutations in the T2SS and/or T3SS, were used to infect mice. Mice were followed up for survival, with some killed at different intervals to study bacterial clearance, inflammatory responses, and lung pathology. RESULTS: Strains carrying either secretion system were lethal for mice. Double mutants were avirulent. The T3SS(+) strains killed mice within a day, and the T2SS(+) strains killed them later. Mice infected with a strain that had only the T2SS were unable to eradicate the organism from the lungs, whereas those infected with a T2SS-T3SS double deletion were able to clear this mutant. Death caused by the T2SS(+) strain was accompanied by a >50-fold increase in bacterial counts and higher numbers of viable intracellular bacteria. CONCLUSIONS: The T2SS of P. aeruginosa may play a role in death from pneumonia, but its action is delayed. These data suggest that antitoxin strategies against this organism will require measures against the toxins secreted by both T2SS and T3SS.

Probiotic Releasing Angiotensin (1-7) in a Drosophila Model of Alzheimer's Disease Produces Sex-Specific Effects on Cognitive Function.

BACKGROUND: While extensive research on the brain has failed to identify effective therapies, using probiotics to target the gut microbiome has shown therapeutic potential in Alzheimer's disease (AD). Genetically modified probiotics (GMP) are a promising strategy to deliver key therapeutic peptides with high efficacy and tissue specificity. Angiotensin (Ang)-(1-7) levels inversely correlate to AD severity, but its administration is challenging. Our group has successfully established a GMP-based method of Ang-(1-7) delivery. OBJECTIVE: Since Drosophila represents an excellent model to study the effect of probiotics on complex disorders in a high throughput manner, we tested whether oral supplementation with Lactobacillus paracasei releasing Ang-(1-7) (LP-A) delays memory loss in a Drosophila AD model. METHODS: Flies overexpressing the human amyloid-ß protein precursor and its ß-site cleaving enzyme in neurons were randomized to receive four 24-h doses of Lactobacillus paracasei alone (LP), LP-A or sucrose over 14 days. Memory was assessed via an aversive phototaxic suppression assay. RESULTS: Optimal dilution,1:2, was determined based on palatability. LP-A improved memory in trained AD males but worsened cognition in AD females. LP-supplementation experiments confirmed that Ang-(1-7) conferred additional cognitive benefits in males and was responsible for the deleterious cognitive effects in females. Sex-specific differences in the levels of angiotensin peptides and differential activation of the kynurenine pathway of tryptophan metabolism in response to supplementation may underlie this male-only therapeutic response. CONCLUSION: In summary, LP-A ameliorated the memory deficits of a Drosophila AD model, but effects were sex-specific. Dosage optimization may be required to address this differential response.

Expression and Function of Mas-Related G Protein-Coupled Receptor D and Its Ligand Alamandine in Retina.

A local renin-angiotensin system (RAS) exists in the retina and plays a critical role in retinal neurovascular function. The protective axis of RAS comprising of angiotensin-converting enzyme 2 (ACE2)/angiotensin-(1-7) [Ang-(1-7)]/Mas receptor attenuate the deleterious actions of increased levels of angiotensin II (Ang II), the main effector peptide of RAS. A new peptide, alamandine, and its receptor Mas-related G protein-coupled receptor D (MrgD) have been recently identified that share structural and functional similarity to Ang-(1-7) and its receptor, Mas, establishing another new protective axis of RAS. Here, we examined the expression and cellular localization of MrgD in the retina, the effect of MrgD deficiency on mouse retinal structure and function, as well as the biological function of alamandine in cultured retinal cells. We showed that MrgD is expressed in the retinal neurons, retinal vasculature, Müller glial and RPE cells, similar to Mas receptor expression. MrgD-deficient mice did not exhibit gross change in retinal morphology and thickness; however, these mice did show a progressive decrease in both scotopic and photopic a-wave and b-wave amplitudes, and increase in retinal capillary loss with age compared to age-matched wild-type mice. In vitro studies in human retinal cells showed that alamandine attenuated the Ang II and LPS-induced increases in inflammatory cytokine gene expression, NF-κB activation, Ang II and hydrogen peroxide-induced production of reactive oxygen species, comparable to that mediated by Ang-(1-7). These results support the notion that alamandine/MrgD may represent another new protective axis of RAS in the retina exerting anti-oxidative and anti-inflammatory effects.

Angiotensin-(1-7) Expressed From Lactobacillus Bacteria Protect Diabetic Retina in Mice.

Purpose: A multitude of animal studies substantiates the beneficial effects of Ang-(1-7), a peptide hormone in the protective axis of the renin angiotensin system, in diabetes and its associated complications including diabetic retinopathy (DR). However, the clinical application of Ang-(1-7) is limited due to unfavorable pharmacological properties. As emerging evidence implicates gut dysbiosis in pathogenesis of diabetes and supports beneficial effects of probiotics, we sought to develop probiotics-based expression and delivery system to enhance Ang-(1-7) and evaluate the efficacy of engineered probiotics expressing Ang-(1-7) in attenuation of DR in animal models. Methods: Ang-(1-7) was expressed in the Lactobacillus species as a secreted fusion protein with a trans-epithelial carrier to allow uptake into circulation. To evaluate the effects of Ang-(1-7) expressed from Lactobacillus paracasei (LP), adult diabetic eNOS-/- and Akita mice were orally gavaged with either 1 × 109 CFU of LP secreting Ang-(1-7) (LP-A), LP alone or vehicle, 3 times/week, for 8 and 12 weeks, respectively. Results: Ang-(1-7) is efficiently expressed from different Lactobacillus species and secreted into circulation in mice fed with LP-A. Oral administration of LP-A significantly reduced diabetes-induced loss of retinal vascular capillaries. LP-A treatment also prevented loss of retinal ganglion cells, and significantly decreased retinal inflammatory cytokine expression in both diabetic eNOS-/- and Akita mice. Conclusions: These results provide proof-of-concept for feasibility and efficacy of using engineered probiotic species as live vector for delivery of Ang-(1-7) with enhanced bioavailability. Translational Relevance: Probiotics-based delivery of Ang-(1-7) may hold important therapeutic potential for the treatment of DR and other diabetic complications.

Therapeutic Delivery of Ang(1-7) via Genetically Modified Probiotic: A Dosing Study.

In recent years a number of beneficial health effects have been ascribed to the renin-angiotensin system (RAS) that extend beyond lowering blood pressure, primarily mediated via the angiotensin-converting enzyme-2 (ACE2)/angiotensin (1-7) or Ang(1-7)/MAS receptor axis. Moreover, once thought as merely a systemic effector, RAS components exist within tissues. The highest tissue concentrations of ACE2 mRNA are located in the gut making it an important target for altering RAS function. Indeed, genetically engineered recombinant probiotics are promising treatment strategies offering delivery of therapeutic proteins with precision. An Ang(1-7) secreting Lactobacillus paracasei (LP) or LP-A has been described for regulation of diabetes and hypertension; however, we are the first to the best of our knowledge to propose this paradigm as it relates to aging. In this Research Practice manuscript, we provide proof of concept for using this technology in a well-characterized rodent model of aging: the Fisher344 x Brown Norway Rat (F344BN). Our primary findings suggest that LP-A increases circulating levels of Ang(1-7) both acutely and chronically (after 8 or 28 treatment days) when administered 3× or 7×/week over 4 weeks. Our future preclinical studies will explore the impact of this treatment on gut and other age-sensitive distal tissues such as brain and muscle.

Angiotensin (1-7) delivered orally via probiotic, but not subcutaneously, benefits the gut-brain axis in older rats.

To (1) investigate the efficacy of multiple doses of an orally delivered probiotic bacteria Lactobacillus paracasei (LP) modified to express angiotensin (1-7) (LP-A) in altering physiologic parameters relevant to the gut-brain axis in older rats and to (2) compare this strategy with subcutaneous delivery of synthetic Ang(1-7) peptide on circulating Ang(1-7) concentrations and these gut-brain axis parameters. Male 24-month-old F344BN rats received oral gavage of LP-A, or subcutaneous injection of Ang(1-7) for 0×, 1×, 3×, or 7×/week over 4 weeks. Circulating RAS analytes, inflammatory cytokines, and tryptophan and its downstream metabolites were measured by ELISA, electrochemiluminescence, and LC-MS respectively. Microbiome taxonomic analysis of fecal samples was performed via 16S-based PCR. Inflammatory and tryptophan-related mRNA expression was measured in colon and pre-frontal cortex. All dosing regimens of LP-A induced beneficial changes in fecal microbiome including overall microbiota community structure and α-diversity, while the 3×/week also significantly increased expression of the anti-inflammatory species Akkermansia muciniphila. The 3×/week also increased serum serotonin and the neuroprotective analyte 2-picolinic acid. In the colon, LP-A increased quinolinate phosphoribosyltransferase expression (1×/week) and increased kynurenine aminotransferase II (1× and 3×/week) mRNA expression. LP-A also significantly reduced neuro-inflammatory gene expression in the pre-frontal cortex (3×/week: COX2, IL-1ß, and TNFα; 7×/week: COX2 and IL-1ß). Subcutaneous delivery of Ang(1-7) increased circulating Ang(1-7) and reduced angiotensin II, but most gut-brain parameters were unchanged in response. Oral-but not subcutaneous-Ang(1-7) altered physiologic parameters related to gut-brain axis, with the most effects observed in 3×/week oral dosing regimen in older rats.

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

[This corrects the article DOI: 10.1016/j.omtm.2019.06.007.].

Gut Pathology and Its Rescue by ACE2 (Angiotensin-Converting Enzyme 2) in Hypoxia-Induced Pulmonary Hypertension.

Therapeutic advances for pulmonary hypertension (PH) have been incremental because of the focus on the pulmonary vasculature in PH pathology. Here, we evaluate the concept that PH is, rather, a systemic disorder involving interplay among multiorgan systems, including brain, gut, and lungs. Therefore, the objective of this study was to evaluate the hypothesis that PH is associated with a dysfunctional brain-gut-lung axis and that global overexpression of ACE2 (angiotensin-converting enzyme 2) rebalances this axis and protects against PH. ACE2 knockin and wild-type (WT; C57BL/6) mice were subjected to chronic hypoxia (10% FIO2) or room air for 4 weeks. Cardiopulmonary hemodynamics, histology, immunohistochemistry, and fecal 16S rRNA microbial gene analyses were evaluated. Hypoxia significantly increased right ventricular systolic pressure, sympathetic activity as well as the number and activation of microglia in the paraventricular nucleus of the hypothalamus in WT mice. This was associated with a significant increase in muscularis layer thickening and decreases in both villi length and goblet cells and altered gut microbiota. Global overexpression of ACE2 prevented changes in hypoxia-induced pulmonary and gut pathophysiology and established distinct microbial communities from WT hypoxia mice. Furthermore, WT mice subjected to fecal matter transfer from ACE2 knockin mice were resistant to hypoxia-induced PH compared with their controls receiving WT fecal matter transfer. These observations demonstrate that ACE2 ameliorates these hypoxia-induced pathologies and attenuates PH. The data implicate dysfunctional brain-gut-lung communication in PH and provide novel avenues for therapeutic interventions.

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

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.

Expression of surfactant protein D in human corneal epithelial cells is upregulated by Pseudomonas aeruginosa.

We reported previously that surfactant protein D (SP-D) was present in human tears and corneal epithelial cells, and that it contributed to tear fluid protection of those cells against Pseudomonas aeruginosa invasion. This suggested a role in ocular innate immunity. Here, we explored the effects of bacterial challenge on SP-D expression by human corneal epithelial cells. Results showed that these cells produced and secreted SP-D constitutively in culture, and that production (mRNA, protein) and secretion of SP-D were upregulated after exposure to heat-killed P. aeruginosa or to purified flagellin or lipopolysaccharide. To begin exploring the mechanism for flagellin-mediated SP-D induction, cells were exposed to purified flagellin or flagellin mutated in the TLR-5-binding domain (L94A, L88A) which reduces IL-8 secretion by A549 respiratory cells. Mutated flagellin did not upregulate IL-8 expression in corneal epithelial cells, but did induce SP-D responses. Mitogen-activated protein kinase inhibitors, especially the JNK inhibitor SP600125, reduced secretion of SP-D, but not production, in the presence of P. aeruginosa. These data show that while SP-D and IL-8 corneal responses are each induced by P. aeruginosa or its antigens, they can involve different regions of the same ligand. The data suggest that separate mechanisms may regulate SP-D secretion and production by human corneal epithelia.