Structure of the essential inner membrane lipopolysaccharide-PbgA complex.

Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function1,2. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics3,4. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains5, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter6-9, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme10-12. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery.

The angiotensin-(1-7)/MasR axis improves pneumonia caused by Pseudomonas aeruginosa: Extending the therapeutic window for antibiotic therapy.

Pseudomonas aeruginosa is a frequent cause of antimicrobial-resistant hospital-acquired pneumonia, especially in critically ill patients. Inflammation triggered by P. aeruginosa infection is necessary for bacterial clearance but must be spatially and temporally regulated to prevent further tissue damage and bacterial dissemination. Emerging data have shed light on the pro-resolving actions of angiotensin-(1-7) [Ang-(1-7)] signaling through the G protein-coupled receptor Mas (MasR) during infections. Herein, we investigated the role of the Ang-(1-7)/Mas axis in pneumonia caused by P. aeruginosa by using genetic and pharmacological approach and found that Mas receptor-deficient animals developed a more severe form of pneumonia showing higher neutrophilic infiltration into the airways, bacterial load, cytokines, and chemokines production and more severe pulmonary damage. Conversely, treatment of pseudomonas-infected mice with Ang-(1-7) was able to decrease neutrophilic infiltration in airways and lungs, local and systemic levels of pro-inflammatory cytokines and chemokines, and increase the efferocytosis rates, mitigating lung damage/dysfunction caused by infection. Notably, the therapeutic association of Ang-(1-7) with antibiotics improved the survival rates of mice subjected to lethal inoculum of P. aeruginosa, extending the therapeutic window for imipenem. Mechanistically, Ang-(1-7) increased phagocytosis of bacteria by neutrophils and macrophages to accelerate pathogen clearance. Altogether, harnessing the Ang-(1-7) pathway during infection is a potential strategy for the development of host-directed therapies to promote mechanisms of resistance and resilience to pneumonia.

HuM195 and its single-chain variable fragment increase Aß phagocytosis in microglia via elimination of CD33 inhibitory signaling.

CD33 is a transmembrane receptor expressed on cells of myeloid lineage and regulates innate immunity. CD33 is a risk factor for Alzheimer's disease (AD) and targeting CD33 has been a promising strategy drug development. However, the mechanism of CD33's action is poorly understood. Here we investigate the mechanism of anti-CD33 antibody HuM195 (Lintuzumab) and its single-chain variable fragment (scFv) and examine their therapeutic potential. Treatment with HuM195 full-length antibody or its scFv increased phagocytosis of ß-amyloid 42 (Aß42) in human microglia and monocytes. This activation of phagocytosis was driven by internalization and degradation of CD33, thereby downregulating its inhibitory signal. HumM195 transiently induced CD33 phosphorylation and its signaling via receptor dimerization. However, this signaling decayed with degradation of CD33. scFv binding to CD33 leads to a degradation of CD33 without detection of the CD33 dimerization and signaling. Moreover, we found that treatments with either HuM195 or scFv promotes the secretion of IL33, a cytokine implicated in microglia reprogramming. Importantly, recombinant IL33 potentiates the uptake of Aß42 in monocytes. Collectively, our findings provide unanticipated mechanistic insight into the role of CD33 signaling in both monocytes and microglia and define a molecular basis for the development of CD33-based therapy of AD.

Novel Angiogenesis Role of GLP-1(32-36) to Rescue Diabetic Ischemic Lower Limbs via GLP-1R-Dependent Glycolysis in Mice.

BACKGROUND: Restoring the capacity of endothelial progenitor cells (EPCs) to promote angiogenesis is the major therapeutic strategy of diabetic peripheral artery disease. The aim of this study was to investigate the effects of GLP-1 (glucagon-like peptide 1; 32-36)-an end product of GLP-1-on angiogenesis of EPCs and T1DM (type 1 diabetes) mice, as well as its interaction with the classical GLP-1R (GLP-1 receptor) pathway and its effect on mitochondrial metabolism. METHODS: In in vivo experiments, we conducted streptozocin-induced type 1 diabetic mice as a murine model of unilateral hind limb ischemia to examine the therapeutic potential of GLP-1(32-36) on angiogenesis. We also generated Glp1r-/- mice to detect whether GLP-1R is required for angiogenic function of GLP-1(32-36). In in vitro experiments, EPCs isolated from the mouse bone marrow and human umbilical cord blood samples were used to detect GLP-1(32-36)-mediated angiogenic capability under high glucose treatment. RESULTS: We demonstrated that GLP-1(32-36) did not affect insulin secretion but could significantly rescue angiogenic function and blood perfusion in ischemic limb of streptozocin-induced T1DM mice, a function similar to its parental GLP-1. We also found that GLP-1(32-36) promotes angiogenesis in EPCs exposed to high glucose. Specifically, GLP-1(32-36) has a causal role in improving fragile mitochondrial function and metabolism via the GLP-1R-mediated pathway. We further demonstrated that GLP-1(32-36) rescued diabetic ischemic lower limbs by activating the GLP-1R-dependent eNOS (endothelial NO synthase)/cGMP/PKG (protein kinase G) pathway. CONCLUSIONS: Our study provides a novel mechanism with which GLP-1(32-36) acts in modulating metabolic reprogramming toward glycolytic flux in partnership with GLP-1R for improved angiogenesis in high glucose-exposed EPCs and T1DM murine models. We propose that GLP-1(32-36) could be used as a monotherapy or add-on therapy with existing treatments for peripheral artery disease. REGISTRATION: URL: www.ebi.ac.uk/metabolights/; Unique identifier: MTBLS9543.

STAP-2-Derived Peptide Suppresses TCR-Mediated Signals to Initiate Immune Responses.

Signal-transducing adaptor protein-2 (STAP-2) is an adaptor protein that contains pleckstrin and Src homology 2-like domains, as well as a proline-rich region in its C-terminal region. Our previous study demonstrated that STAP-2 positively regulates TCR signaling by associating with TCR-proximal CD3ζ ITAMs and the lymphocyte-specific protein tyrosine kinase. In this study, we identify the STAP-2 interacting regions of CD3ζ ITAMs and show that the STAP-2-derived synthetic peptide (iSP2) directly interacts with the ITAM sequence and blocks the interactions between STAP-2 and CD3ζ ITAMs. Cell-penetrating iSP2 was delivered into human and murine T cells. iSP2 suppressed cell proliferation and TCR-induced IL-2 production. Importantly, iSP2 treatment suppressed TCR-mediated activation of naive CD4+ T cells and decreased immune responses in CD4+ T cell-mediated experimental autoimmune encephalomyelitis. It is likely that iSP2 is a novel immunomodulatory tool that modulates STAP-2-mediated activation of TCR signaling and represses the progression of autoimmune diseases.

A naturally occurring 22-amino acid fragment of human hemoglobin A inhibits autophagy and HIV-1.

Autophagy is an evolutionarily ancient catabolic pathway and has recently emerged as an integral part of the innate immune system. While the core machinery of autophagy is well defined, the physiological regulation of autophagy is less understood. Here, we identify a C-terminal fragment of human hemoglobin A (HBA1, amino acids 111-132) in human bone marrow as a fast-acting non-inflammatory inhibitor of autophagy initiation. It is proteolytically released from full-length HBA1 by cathepsin E, trypsin or pepsin. Biochemical characterization revealed that HBA1(111-132) has an in vitro stability of 52 min in human plasma and adopts a flexible monomeric conformation in solution. Structure-activity relationship studies revealed that the C-terminal 13 amino acids of HBA1(120-132) are sufficient to inhibit autophagy, two charged amino acids (D127, K128) mediate solubility, and two serines (S125, S132) are required for function. Successful viruses like human immunodeficiency virus 1 (HIV-1) evolved strategies to subvert autophagy for virion production. Our results show that HBA1(120-132) reduced virus yields of lab-adapted and primary HIV-1. Summarizing, our data identifies naturally occurring HBA1(111-132) as a physiological, non-inflammatory antagonist of autophagy. Optimized derivatives of HBA1(111-132) may offer perspectives to restrict autophagy-dependent viruses.

The impact of heparin and direct thrombin inhibitors on cell-penetrating polymer siRNA transfection.

Gene therapy using siRNA has become a promising strategy to achieve targeted gene knockdown for treatment of cardiovascular pathologies. However, efficient siRNA transfection often relies on cationic delivery vectors such as synthetic cell-penetrating polymers which are susceptible to interference by negatively charged molecules. Anticoagulants such as heparin, which is negatively charged and widely used in cardiovascular applications, may pose a significant barrier to effective siRNA delivery. We therefore conducted in vitro studies utilizing human smooth muscle and endothelial cells transfected with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ß2-microglobulin (B2M) siRNA in the presence of heparin, argatroban, and bivalirudin in order to determine which anticoagulant therapy is most compatible for siRNA delivery. We observed that while heparin, at clinical doses, decreases the efficiency of siRNA targeted mRNA knockdown, mRNA knockdown is not inhibited in the presence of either argatroban or bivalirudin. Our data suggests that heparin should be avoided during siRNA therapy with cationic transfection agents, and argatroban and bivalirudin should be used in its stead.

Renin-angiotensin system-mediated nitric oxide signaling in podocytes.

Nitric oxide (NO) is widely recognized for its role in regulating renal function and blood pressure. However, the precise mechanisms by which NO affects renal epithelial cells remain understudied. Our previous research has shown that NO signaling in glomerular podocytes can be initiated by Angiotensin II (ANG II) but not by ATP. This study aims to elucidate the crucial interplay between the renin-angiotensin system (RAS) and NO production in podocytes. To conduct our research, we used cultured human podocytes and freshly isolated rat glomeruli. A variety of RAS peptides were used, alongside confocal microscopy, to detect NO production and NO/Ca2+ cross talk. Dynamic changes in the podocyte cytoskeleton, mediated by RAS-NO intracellular signaling, were observed using fluorescent labeling for F-actin and scanning probe microscopy. The experiments demonstrated that ANG II and ANG III generated high levels of NO by activating the angiotensin II type 2 receptor (AT2R). We did not detect functional MAS receptor presence in podocytes, and the moderate NO response to ANG 1-7 was also mediated through AT2R. Furthermore, NO production impacted intracellular Ca2+ signaling and correlated with an increase in podocyte volume and growth. Scanning probe experiments revealed that AT2R activation and the corresponding NO generation are responsible for the protrusion of podocyte lamellipodia. Taken together, our data indicate that AT2R activation enhances NO production in podocytes and subsequently mediates changes in Ca2+ signaling and podocyte volume dynamics. These mechanisms may play a significant role in both physiological and pathophysiological interactions between the RAS and podocytes.NEW & NOTEWORTHY The renin-angiotensin system plays a crucial role in the production of intracellular nitric oxide within podocytes. This mechanism operates through the activation of the angiotensin II type 2 receptor, leading to dynamic modifications in intracellular calcium levels and the actin filament network. This intricate process is vital for linking the activity of angiotensin receptors to podocyte function.

Direct thrombin inhibitors fail to reverse the negative effects of heparin on lung growth and function after murine left pneumonectomy.

Neonates with congenital diaphragmatic hernia (CDH) frequently require cardiopulmonary bypass and systemic anticoagulation. We previously demonstrated that even subtherapeutic heparin impairs lung growth and function in a murine model of compensatory lung growth (CLG). The direct thrombin inhibitors (DTIs) bivalirudin and argatroban preserved growth in this model. Although DTIs are increasingly used for systemic anticoagulation clinically, patients with CDH may still receive heparin. In this experiment, lung endothelial cell proliferation was assessed following treatment with heparin-alone or mixed with increasing concentrations of bivalirudin or argatroban. The effects of subtherapeutic heparin with or without DTIs in the CLG model were also investigated. C57BL/6J mice underwent left pneumonectomy and subcutaneous implantation of osmotic pumps. Pumps were preloaded with normal saline, bivalirudin, or argatroban; treated animals received daily intraperitoneal low-dose heparin. In vitro, heparin-alone decreased endothelial cell proliferation and increased apoptosis. The effect of heparin on proliferation, but not apoptosis, was reversed by the addition of bivalirudin and argatroban. In vivo, low-dose heparin decreased lung volume compared with saline-treated controls. All three groups that received heparin demonstrated decreased lung function on pulmonary function testing and impaired exercise performance on treadmill tolerance testing. These findings correlated with decreases in alveolarization, vascularization, angiogenic signaling, and gene expression in the heparin-exposed groups. Together, these data suggest that bivalirudin and argatroban fail to reverse the inhibitory effects of subtherapeutic heparin on lung growth and function. Clinical studies on the impact of low-dose heparin with DTIs on CDH outcomes are warranted.NEW & NOTEWORTHY Infants with pulmonary hypoplasia frequently require cardiopulmonary bypass and systemic anticoagulation. We investigate the effects of simultaneous exposure to heparin and direct thrombin inhibitors (DTIs) on lung growth and pulmonary function in a murine model of compensatory lung growth (CGL). Our data suggest that DTIs fail to reverse the inhibitory effects of subtherapeutic heparin on lung growth and function. Clinical studies on the impact of heparin with DTIs on clinical outcomes are thus warranted.

Prion protein prevents the inhibition of large-conductance calcium-activated potassium channel by Tau peptide K18 oligomers.

Alzheimer's disease (AD) is a tauopathy characterized by the deposition of amyloid aggregates of hyperphosphorylated Tau protein and amyloid-ß peptide (Aß) in the brain. Nevertheless, a soluble, oligomeric forms of Tau and Aß are considered to be the most neurotoxic species responsible for neurodegenerative processes in AD. The mechanism of action of these oligomers remains largely unclear. Previously, we demonstrated the inhibition of the large-conductance calcium-activated potassium channel (BKCa) by Aß. Therefore, in the present study we investigated the effect of Tau protein on the BKCa activity. Furthermore, since prion protein (PrP) interacts with Tau and the N-terminal fragment of PrP, called N1, can be neuroprotective in tauopathies, we checked whether N1 can also act at the level of BKCa channel. In the studies we used monomers, oligomers and amyloid fibrils of aggregation-prone Tau fragment, called K18, carrying tauopathy-associated mutation - deletion of Lys280 (K18Δ280). Additionally, to induce formation of neurotoxic oligomers, K18Δ280 was phosphorylated by protein kinase A (PKA). The activity of the plasma membrane BKCa of hippocampal neurons was recorded using single-channel patch-clamp technique in both inside-out and outside-out modes, exposing the cytosolic or extracellular surface of the membrane, respectively. In the outside-out mode - performing the extracellular application of the neurotoxic oligomers of phosphorylated K18Δ280, we observed a significant and concentration-dependent decrease in the probability of opening (Po) of BKCa. The Po of BKCa was fully recovered after washing the oligomers out. In the case of the inside-out patch-clamp configuration, we found that the Po of BKCa was not affected by the oligomers. In contrast to the oligomers, the monomers and amyloid fibrils of K18Δ280 had no effect on the channel activity, analyzed in inside-out as well as outside-out modes. Noteworthy, upon incubation with N1, the oligomers did not inhibit BKCa channel. The BKCa channel inhibition, dependent on the outside-out membrane orientation, implies specific interaction of the oligomers with the extracellular part of the channel. Moreover, our results suggest that N1 can convert the neurotoxic oligomers of Tau into a form which is not able to inhibit the channel, and indicate novel possible neuroprotective mechanism of PrP action in AD and other tauopathies.

Therapeutic role of interferon-γ in experimental autoimmune encephalomyelitis is mediated through a tolerogenic subset of splenic CD11b+ myeloid cells.

Cumulative evidence has established that Interferon (IFN)-γ has both pathogenic and protective roles in Multiple Sclerosis and the animal model, Experimental Autoimmune Encephalomyelitis (EAE). However, the underlying mechanisms to the beneficial effects of IFN-γ are not well understood. In this study, we found that IFN-γ exerts therapeutic effects on chronic, relapsing-remitting, and chronic progressive EAE models. The frequency of regulatory T (Treg) cells in spinal cords from chronic EAE mice treated with IFN-γ was significantly increased with no effect on Th1 and Th17 cells. Consistently, depletion of FOXP3-expressing cells blocked the protective effects of IFN-γ, indicating that the therapeutic effect of IFN-γ depends on the presence of Treg cells. However, IFN-γ did not trigger direct in vitro differentiation of Treg cells. In vivo administration of blocking antibodies against either interleukin (IL)-10, transforming growth factor (TGF)-ß or program death (PD)-1, revealed that the protective effects of IFN-γ in EAE were also dependent on TGF-ß and PD-1, but not on IL-10, suggesting that IFN-γ might have an indirect role on Treg cells acting through antigen-presenting cells. Indeed, IFN-γ treatment increased the frequency of a subset of splenic CD11b+ myeloid cells expressing TGF-ß-Latency Associated Peptide (LAP) and program death ligand 1 (PD-L1) in a signal transducer and activator of transcription (STAT)-1-dependent manner. Furthermore, splenic CD11b+ cells from EAE mice preconditioned in vitro with IFN-γ and myelin oligodendrocyte glycoprotein (MOG) peptide exhibited a tolerogenic phenotype with the capability to induce conversion of naïve CD4+ T cells mediated by secretion of TGF-ß. Remarkably, adoptive transfer of splenic CD11b+ cells from IFN-γ-treated EAE mice into untreated recipient mice ameliorated clinical symptoms of EAE and limited central nervous system infiltration of mononuclear cells and effector helper T cells. These results reveal a novel cellular and molecular mechanism whereby IFN-γ promotes beneficial effects in EAE by endowing splenic CD11b+ myeloid cells with tolerogenic and therapeutic activities.

METTL14 inhibits Aß1-42-induced neuronal injury through regulating the stability of CBLN4 mRNA in Alzheimer's disease.

Previous studies have suggested that N6-methyladenosine (mA) modification of RNA affects fundamental aspects of RNA metabolism, and mA dysregulation is implicated in various human diseases, including Alzheimer's disease (AD). This study is designed to explore the role and mechanism of methyltransferase-like 14 (METTL14) in the pathogenesis of AD. SK-N-SH cells were treated with Aß1-42 to establish an in vitro model of AD. Cerebellin 4 (CBLN4) and METTL14 expression levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Cell viability and apoptosis were analyzed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometry assay. B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), C-caspase-3, total-caspase-3, C/EBP homologous protein (CHOP), and glucose-related protein 78 (GRP78) protein levels were determined using Western blot. Interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNF-α) levels were analyzed using ELISA. Reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) products were examined using special assay kits. Interaction between CBLN4 and METTL14 was verified using methylated RNA immunoprecipitation (MeRIP) and dual-luciferase reporter assays. CBLN4 and METTL14 expression was decreased in Aß1-42-treated SK-N-SH cells. Upregulation of CBLN4 relieved Aß1-42-induced SK-N-SH cell apoptosis, inflammation, oxidative stress, and endoplasmic reticulum (ER) stress in vitro. At the molecular level, METTL14 could improve the stability and expression of CBLN4 mRNA via m6A methylation. Our findings indicated that m6A methylase METTL14-mediated upregulation of CBLN4 mRNA stability could repress Aß1-42-triggered SK-N-SH cell injury, providing a promising therapeutic target for AD treatment.

Central angiotensin-(1-7) attenuates hypoglycemia in sepsis-like conditions via reducing systemic and hepatic inflammation.

Sepsis is understood as the result of initiating systemic inflammation derived from an inadequate host response against pathogens. In its acute phase, sepsis is marked by an exacerbated reaction to infection, tissue damage, organ failure, and metabolic dysfunction. Among these, hypoglycemia, characterized by disorders of the gluconeogenesis pathway, is related to one of the leading causes of mortality in septic patients. Recent research has investigated the involvement of sympathetic efferent neuroimmune pathways during systemic inflammation. These pathways can be stimulated by several centrally administered drugs, including Angiotensin-(1-7) (Ang-(1-7)). Therefore, the present study aims to evaluate the effects of central treatment with Ang-(1-7) on hypoglycemia during endotoxemia. For this, male Wistar Hannover rats underwent stereotaxic surgery for intracerebroventricular (i.c.v.) administration of Ang-(1-7) and cannulation of the jugular vein for lipopolysaccharide (LPS) injection. Our results demonstrate that LPS was capable of inducing hypoglycemia and that prior central treatment with Ang-(1-7) attenuated this effect. Our data also show that Ang-(1-7) reduced plasma concentrations of TNF-α, IL-1ß, IL-6, and nitric oxide, in addition to the decrease and increase of hepatic IL-6 and IL-10 respectively, in animals subjected to systemic inflammation by LPS, resulting in the reduction of systemic and hepatic inflammation, thus attenuating the deleterious effects of LPS on phosphoenolpyruvate carboxykinase protein content. In summary, the data suggest that central treatment with Ang-(1-7) attenuates hypoglycemia induced by endotoxemia, probably through anti-inflammatory action, leading to reestablishing hepatic gluconeogenesis.

CCR5 regulates Aß1-42-induced learning and memory deficits in mice.

C-C chemokine receptor 5 (CCR5) is a chemokine receptor involved in immune responses and a co-receptor for HIV infection. Recently, CCR5 has also been reported to play a role in synaptic plasticity, learning and memory, and cognitive deficits associated with normal aging, traumatic brain injury (TBI), and HIV-associated neurocognitive disorder (HAND). In contrast, the role of CCR5 in cognitive deficits associated with other disorders, including Alzheimer's disease (AD), is much less understood. Studies have reported an increase in expression of CCR5 or its ligands in both AD patients and AD rodent models, suggesting a correlation between AD and CCR5 expression. However, whether blocking CCR5 in specific brain regions, such as the hippocampus, could improve memory deficits in AD mouse models is unknown. To study the potential causal role of CCR5 in cognitive deficits in AD, we injected soluble Aß1-42 or a control (Aß42-1) oligomers in the dorsal CA1 region of the hippocampus and found that Aß1-42 injection resulted in severe memory impairment in the object place recognition (OPR) and novel object recognition (NOR) tests. Aß1-42 injection caused an increase in Ccr5, Ccl3, and Ccl4 in the dorsal hippocampus, and the expression levels of CCR5 and its ligands remained elevated at 2 weeks after Aß1-42 injection. Knocking down Ccr5 in the CA1 region of dorsal hippocampus reversed the increase in microglia number and size in dorsal CA1 and rescued memory deficits. These results indicate that CCR5 plays an important role in modulating Aß1-42-induced learning and memory deficits, and suggest that CCR5 antagonists may serve as a potential treatment to improve cognitive deficits associated with AD.

Ophthalmic drug effects on the amyloidogenesis of a transforming growth factor ß-induced protein (TGFBIp) peptide fragment.

Drugs that can treat one disease may either be detrimental or beneficial toward another due to possible cross-interactions. Therefore, care in choosing a suitable drug for patients with multiple diseases is crucial in successful patient management. This study explores several currently available ophthalmic drugs used to treat common ocular diseases to understand how they can affect the amyloidogenesis of a transforming growth factor ß-induced protein (TGFBIp) peptide fragment found in abundance in the corneal protein aggregation deposits of lattice corneal dystrophy (LCD) patients. Results from this study provided supporting evidence that some drugs intended to treat other diseases can enhance or inhibit fibrillar aggregation of TGFBIp peptide, which may have potential implication of affecting the disease progression of LCD by either worsening or ameliorating it. Comparisons of the different properties of ophthalmic compounds explored in this study may also provide some guidance for future design of drugs geared toward the treatment of LCD.

Exogenous Angiotensin-(1-7) Provides Protection Against Inflammatory Bone Resorption and Osteoclastogenesis by Inhibition of TNF-α Expression in Macrophages.

Renin-angiotensin-aldosterone system plays a crucial role in the regulation of blood pressure and fluid homeostasis. It is reported to be involved in mediating osteoclastogenesis and bone loss in diseases of inflammatory bone resorption such as osteoporosis. Angiotensin-(1-7), a product of Angiotensin I and II (Ang I, II), is cleaved by Angiotensin-converting enzyme 2 and then binds to Mas receptor to counteract inflammatory effects produced by Ang II. However, the mechanism by which Ang-(1-7) reduces bone resorption remains unclear. Therefore, we aim to elucidate the effects of Ang-(1-7) on lipopolysaccharide (LPS)-induced osteoclastogenesis. In vivo, mice were supracalvarial injected with Ang-(1-7) or LPS ± Ang-(1-7) subcutaneously. Bone resorption and osteoclast formation were compared using micro-computed tomography, tartrate-resistant acid phosphatase (TRAP) stain, and real-time PCR. We found that Ang-(1-7) attenuated tumor necrosis factor (TNF)-α, TRAP, and Cathepsin K expression from calvaria and decreased osteoclast number along with bone resorption at the suture mesenchyme. In vitro, RANKL/TNF-α ± Ang-(1-7) was added to cultures of bone marrow-derived macrophages (BMMs) and osteoclast formation was measured via TRAP staining. The effect of Ang-(1-7) on LPS-induced osteoblasts RANKL expression and peritoneal macrophages TNF-α expression was also investigated. The effect of Ang-(1-7) on the MAPK and NF-κB pathway was studied by Western blotting. As a result, Ang-(1-7) reduced LPS-stimulated macrophages TNF-α expression and inhibited the MAPK and NF-κB pathway activation. However, Ang-(1-7) did not affect osteoclastogenesis induced by RANKL/TNF-α nor reduce osteoblasts RANKL expression in vitro. In conclusion, Ang-(1-7) alleviated LPS-induced osteoclastogenesis and bone resorption in vivo via inhibiting TNF-α expression in macrophages.

A single dose of angiotensin-(1-7) resolves eosinophilic inflammation and protects the lungs from a secondary inflammatory challenge.

OBJECTIVE: Angiotensin-(1-7) [Ang-(1-7)] is a pro-resolving mediator. It is not known whether the pro-resolving effects of Ang-(1-7) are sustained and protect the lung from a subsequent inflammatory challenge. This study sought to investigate the impact of treatment in face of a second allergic or lipopolysaccharide (LPS) challenge. METHODS: Mice, sensitized and challenged with ovalbumin (OVA), received a single Ang-(1-7) dose at the peak of eosinophilic inflammation, 24 h after the final OVA challenge. Subsequently, mice were euthanized at 48, 72, 96, and 120 h following the OVA challenge, and cellular infiltrate, inflammatory mediators, lung histopathology, and macrophage-mediated efferocytic activity were evaluated. The secondary inflammatory stimulus (OVA or LPS) was administered 120 h after the last OVA challenge, and subsequent inflammatory analyses were performed. RESULTS: Treatment with Ang-(1-7) resulted in elevated levels of IL-10, CD4+Foxp3+, Mres in the lungs and enhanced macrophage-mediated efferocytic capacity. Moreover, in allergic mice treated with Ang-(1-7) and then subjected to a secondary OVA challenge, inflammation was also reduced. Similarly, in mice exposed to LPS, Ang-(1-7) effectively prevented the lung inflammation. CONCLUSION: A single dose of Ang-(1-7) resolves lung inflammation and protect the lung from a subsequent inflammatory challenge highlighting its potential therapeutic for individuals with asthma.

Ac2-26 Reduced Lung Injury After Cardiopulmonary Bypass via the AKT1/GSK3ß/eNOS Pathway.

INTRODUCTION: Cardiopulmonary bypass (CPB) leads to severe inflammation and lung injury. Our previous study showed that Ac2-26 (an active n-terminal peptide of Annexin A1) can reduce acute lung injury. The aim of this study was to evaluate the effect of Ac2-26 on lung injury in CPB rats. METHODS: Forty rats were randomly divided into the sham, CPB, Ac, Ac/serine/threonine kinase 1 (AKT1), and Ac/ glycogen synthase kinase (GSK)-3ß groups. The rats in the sham group only received anesthesia, intubation, and cannulation. The rats in the other 4 groups received the standard CPB procedure. The rats in the CPB, Ac, Ac/AKT1, and Ac/GSK3ß groups were immediately injected with saline, Ac2-26 (1 mg/kg), Ac2-26 combined with short hairpin RNA (AKT1), or Ac2-26 combined with a GSK3ß inhibitor after CPB. At 12 h after the end of CPB, the PaO2/ fraction of inspired oxygen ratio, wet/dry weight ratio and protein content in the bronchoalveolar lavage fluid (BALF) were recorded. The numbers of macrophages and neutrophils in the BALF and blood were determined. Cytokine levels in the blood and BALF were investigated. Lung tissue histology and apoptosis were estimated. The expression of nuclear factor kappa- B, AKT1, GSK3ß, endothelial nitric oxide synthase and apoptosis-related proteins was analyzed. The survival of all the rats was recorded. RESULTS: Compared with the rats in the sham group, all the parameters examined worsened in the rats that received CPB. Compared with those in the CPB group, Ac2-26 significantly improved pulmonary capillary permeability, reduced cytokine levels, and decreased histological scores and apoptosis. The protective effect of Ac2-26 on lung injury was significantly reversed by AKT1 short hairpin RNA or a GSK3ß inhibitor. CONCLUSIONS: Ac2-26 significantly reduced lung injury and inflammation after CPB. The protective effect of Ac2-26 mainly depended on the AKT1/GSK3ß/endothelial nitric oxide synthase pathway.

Angiotensin-(1-7) Treatment Early in Life Prevents Cardiac Hypertrophy in Adult Hypertensive Rats.

ABSTRACT: Angiotensin (Ang)-(1-7) is a cardioprotective peptide of the renin-angiotensin system. Prepuberty has been considered as a later susceptible window of development, and stressful factors in this life phase can induce chronic diseases in adulthood. We aimed to investigate whether the treatment with Ang-(1-7) during the prepuberty could attenuate the development of hypertension and cardiac injury in adult spontaneously hypertensive rats (SHRs). SHRs were treated with Ang-(1-7) (24 µg/kg/h) from age 4 to 7 weeks. Systolic blood pressure was measured by tail-cuff plethysmography up to 17th week. Thereafter, echocardiography was performed, and the rats were euthanized for the collection of tissues and blood. Ang-(1-7) did not change the systolic blood pressure but reduced the septal and posterior wall thickness, and cardiomyocyte hypertrophy and fibrosis in SHR. In addition, Ang-(1-7) reduced the gene expression of atrial natriuretic peptide and brain natriuretic peptide, increased the metalloproteinase 9 expression, and reduced the extracellular signal-regulated kinases 1/2 phosphorylation. Ang-(1-7) also prevented the reduction of Mas receptor but did not change the protein expression of angiotensin-converting enzyme, angiotensin-converting enzyme 2, AT1, and AT2. The treatment with Ang-(1-7) decreased the malondialdehyde (MDA) levels and increased superoxide dismutase-1 and catalase activities and protein expression of catalase. Our findings demonstrate that the treatment of SHR with Ang-(1-7) for 3 weeks early in life promotes beneficial effects in the heart later in life, even without altering blood pressure, through mechanisms involving the reduction of oxidative stress and ERK1/2 phosphorylation. In addition, this study supports the prepuberty as an important programming window.

Synthesis and mechanistic study of Aß42 C-terminus domain derived tetrapeptides that inhibit Alzheimer's Aß-aggregation-induced neurotoxicity.

Amyloid plaque formation in the brain is mainly responsible for the onset of Alzheimer's disease (AD). Structure-based peptides have gained importance in recent years, and rational design of the peptide sequences for the prevention of Aß-aggregation and related toxicity is imperative. In this study, we investigate the structural modification of tetrapeptides derived from the hydrophobic C-terminal region of Aß42 "VVIA-NH2" and its retro-sequence "AIVV-NH2." A preliminary screening of synthesized peptides through an MTT cell viability assay followed by a ThT fluorescence assay revealed a peptide 13 (Ala-Ile-Aib-Val-NH2) that showed protection against Aß-aggregation and associated neurotoxicity. The presence of the α-helix inducer "Aib" in peptide 13 manifested the conformational transition from cross-ß-sheets to α-helical content in Aß42. The absence of fibrils in electron microscopic analysis suggested the inhibitory potential of peptide 13. The HRMS, DLS, and ANS studies further confirmed the inhibitory activity of 13, and no cytotoxicity was observed. The structure-based peptide described herein is a promising amyloid-ß inhibitor and provides a new lead for the development of AD therapeutics.