Fluorinated apelin-13 mediates neuroprotective effects in multiple sclerosis models.

Multiple sclerosis (MS) is an autoimmune and neurodegenerative disease leading to demyelination and axonal loss. Current treatments are immunomodulatory or immunosuppressive drugs acting on the inflammatory component. However, these treatments do not adequately address the crucial aspect of neuroprotection. Recently, an association between an altered balance of adipokines and MS has been proposed as both a risk factor for developing MS and a chronic disease aggravating factor. Specifically, a decrease of apelin plasma levels in MS patients compared to controls correlates with the number of relapses and disease severity. Here we report a dramatic downregulation of apelin levels in the CNS of EAE mice which is also detected in MS patients brain samples compared to controls. Exploiting innovative design and synthesis techniques, we engineered a novel fluorinated apelin-13 peptide characterized by enhanced plasmatic stability compared to its native counterpart. With this peptide, we assessed the potential therapeutic benefits of apelin preventive supplementation in the EAE mouse model. We show that the fluorinated Apelin-13 peptide ameliorates EAE clinical score and preserves myelin content in the EAE MOG model recapitulating the progressive form of disease. These results combined with ex-vivo experiments in brain organotypic slices and in vitro studies in neurons and primary microglia and macrophages suggest that apelin has neuroprotective effects and influences the microglia/macrophages function.

Neuroprotective effect of apelin-13 and other apelin forms-a review.

Neurodegenerative diseases, which occur when neurons begin to deteriorate, affect millions of people worldwide. These age-related disorders are becoming more common partly because the elderly population has increased in recent years. While no treatments are accessible, every year an increasing number of therapeutic and supportive options become available. Various substances that may have neuroprotective effects are currently being researched. One of them is apelin. This review aims to illustrate the results of research on the neuroprotective effect of apelin amino acid oligopeptide which binds to the apelin receptor and exhibits neuroprotective effects in the central nervous system. The collected data indicate that apelin can protect the central nervous system against injury by several mechanisms. More studies are needed to thoroughly investigate the potential neuroprotective effects of this peptide in neurodegenerative diseases and various other types of brain damage.

A novel insight into the neuroprotective effects of cannabidiol: maintained apelin/dopamine synthesis, NRF2 signaling, and AKT/CREB/BDNF gene expressions.

Neuroinflammation is a process associated with degeneration and loss of neurons in different parts of the brain. The most important damage mechanisms in its formation are oxidative stress and inflammation. This study aimed to investigate the protective effects of cannabidiol (CBD) against neuroinflammation through various mechanisms. Thirty­two female rats were randomly divided into 4 groups as control, lipopolysaccharide (LPS), LPS + CBD and CBD groups. After six hours following LPS administration, rats were sacrificed, brain and cerebellum tissues were obtained. Tissues were stained with hematoxylin­eosin for histopathological analysis. Apelin and tyrosine hydroxylase synthesis were determined immunohistochemically. Total oxidant status and total antioxidant status levels were measured, and an oxidative stress index was calculated. Protein kinase B (AKT), brain-derived neurotrophic factor (BDNF), cyclic­AMP response element­binding protein (CREB) and nuclear factor erythroid 2­related factor 2 (NRF2) mRNA expression levels were also determined. In the LPS group, hyperemia, degeneration, loss of neurons and gliosis were seen in all three tissues. Additionally, Purkinje cell loss in the cerebellum, as well as neuronal loss in the cerebral cortex and hippocampus, were found throughout the LPS group. The expressions of AKT, BDNF, CREB and NRF2, apelin and tyrosine hydroxylase synthesis all decreased significantly. CBD treatment reversed these changes and ameliorated oxidative stress parameters. CBD showed protective effects against neuroinflammation via regulating AKT, CREB, BDNF expressions, NRF2 signaling, apelin and tyrosine hydroxylase synthesis.

The biological efficacy of Apelin against focal transient cerebral ischemia-reperfusion injury. A systematic review and meta-analysis of animal studies.

BACKGROUND: Apelin has been extensively studied, and emerging experimental evidence suggests that Apelin may have effects on stroke by reducing infarct volume and neurological deficits, inhibiting the apoptosis process and reducing brain water content. However, the credibility of the evidence is uncertain. Thus, we aimed to perform a systematic review and meta-analysis to evaluate preclinical studies that used Apelin for the treatment of transient focal cerebral ischemia. METHODS: Electronic bibliographic databases including PubMed, EMBASE, Scopus, and Google Scholar were searched for finding relevant studies from January 2000 to July 2023. The methodological quality and risk of bias scores for animal studies were calculated based on the CAMARADES and the SYRCLE's RoB tools, respectively. The effect sizes were assessed using Comprehensive Meta-Analysis (CMA) software. RESULTS: A total of twelve eligible studies were used for the systematic review and meta-analysis. The median scores of study quality and risk of bias were 7.5 out of 10, and 5 out of 10, respectively. Apelin treatment effectively decreased infarct volume (primary outcome) [Hedges' g = 2.72, 95 % CI (1.93, 3.51), p < 0.001], neurological deficit [Hedges' g = 1.76, 95 % CI (0.96, 2.55), p < 0.001], cleaved caspase 3 [Hedges' g = 2.16, 95 % CI (0.87, 3.44), p = 0.001], and apoptotic cell number [Hedges' g = 4.07, 95 % CI (1.25,6.89), p = 0.005] compared with the control group. According to subgroup analysis, more notable neuroprotective effects were observed with intravenous administration than with intracerebroventricular (ICV) administration. Moreover, we determined that effect size of infarct volume was markedly related to the species. The combined measurement of two studies demonstrated that Apelin could reduce BCL2 and TNF-α levels as well as brain water content compared with the control group. However, pooled measurement of two studies showed that no relevancy was discovered between CHOP and altering infarct volume. CONCLUSION: The present meta-analysis was conducted to assess preclinical studies related to Apelin treatment in rodent ischemic stroke. Apelin can exert promising neuroprotective effects by reducing infarct volume, neurological deficit, caspase 3, apoptotic cell number, TNF- α and brain water content and increasing BCL2. The current evidence supports the anti-apoptotic and anti-inflammatory properties of Apelin, but its effectiveness in decreasing CHOP level in animal models of ischemic stroke needs further elucidation. This study was registered within the International Prospective Register of Systematic Reviews (PROSPERO) as number CRD42023460926.

Protective effect of apelin-13 on ventilator-induced acute lung injury.

BACKGROUND: Mechanical Ventilation (MV) is an essential mechanism of life support in the clinic. It may also lead to ventilator-induced acute lung injury (VILI) due to local alveolar overstretching and/or repeated alveolar collapse. However, the pathogenesis of VILI is not completely understood, and its occurrence and development may be related to physiological processes such as the inflammatory response, oxidative stress, and apoptosis. Some studies have found that the the apelin/APJ axis is an endogenous antagonistic mechanism activated during acute respiratory distress syndrome(ARDS), that can counteract the injury response and prevent uncontrolled lung injury. To indicate that apelin-13 plays a protective role in VILI, an animal model of VILI was established in this study to explore whether apelin-13 can alleviate VILI in rats by inhibiting inflammation, apoptosis and oxidative stress. METHODS: SD rats were divided into four groups: control, high tidal volume, high tidal volume + normal saline and high tidal volume + apelin-13. After tracheotomy, the rats in control maintained spontaneous breathing, and the other rats were connected to the small animal ventilator for 4 h to establish the rat VILI model. The mRNA expression of apelin was measured by real-time quantitative polymerase chain reaction(qRT-PCR), immunofluorescence and Western blotting(WB) were used to detect the expression level of APJ, and WB was used to detect the expression of the apoptotic proteins Bax and bcl-2. The degree of lung injury was evaluated by pathological staining of lung tissue,W/D ratio, and BALF total protein concentration. The expression of inflammatory factors(IL-1ß, IL-6, TNF-α) in alveolar lavage fluid was measured using ELISA. The activities of MPO and cat and the content of MDA, an oxidative product, in lung tissue were measured to evaluate the degree of oxidative stress in the lung. RESULTS: After treatment with apelin-13, the apelin/APJ axis in the lung tissue of VILI model rats was activated, and the effect was further enhanced. The pathological damage of lung tissue was alleviated, the expression of the antiapoptotic protein Bcl-2 and the proapoptotic protein Bax was reversed, and the levels of the inflammatory cytokines IL-1ß, IL-6, TNF-α levels were all decreased. MPO activity and MDA content decreased, while CAT activity increased. CONCLUSION: The apelin/apj axis is activated in VILI. Overexpression of apelin-13 further plays a protective role in VILI, mainly by including reducing pathological damage, the inflammatory response, apoptosis and antioxidant stress in lung tissue, thus delaying the occurrence and development of VILI.

Apelin C-Terminal Fragments: Biological Properties and Therapeutic Potential.

Creation of bioactive molecules for treatment of cardiovascular diseases based on natural peptides is the focus of intensive experimental research. In the recent years, it has been established that C-terminal fragments of apelin, an endogenous ligand of the APJ receptor, reduce metabolic and functional disorders in experimental heart damage. The review presents literature data and generalized results of our own experiments on the effect of apelin-13, [Pyr]apelin-13, apelin-12, and their chemically modified analogues on the heart under normal and pathophysiological conditions in vitro and in vivo. It has been shown that the spectrum of action of apelin peptides on the damaged myocardium includes decrease in the death of cardiomyocytes from necrosis, reduction of damage to cardiomyocyte membranes, improvement in myocardial metabolic state, and decrease in formation of reactive oxygen species and lipid peroxidation products. The mechanisms of protective action of these peptides associated with activation of the APJ receptor and manifestation of antioxidant properties are discussed. The data presented in the review show promise of the molecular design of APJ receptor peptide agonists, which can serve as the basis for the development of cardioprotectors that affect the processes of free radical oxidation and metabolic adaptation.

Apelin/APJ system protects placental trophoblasts from hypoxia-induced oxidative stress through activating PI3K/Akt signaling pathway in preeclampsia.

BACKGROUND: Preeclampsia is a placentally induced syndrome with diverse clinical presentation that currently has no cure. Oxidative stress is a potent inducer of placental dysfunction. The apelin receptor (APJ) system is a pleiotropic pathway with a potential for therapeutic targeting in preeclampsia. This study examines the alteration of circulating apelin levels and placental APJ expression in preeclampsia and investigates whether apelin/APJ system can protect placental trophoblast from hypoxia-induced oxidative stress injury through PI3K/AKT signaling pathway. RESULTS: Our results confirmed that maternal apelin concentration was increased in women with preeclampsia, but APJ expression was reduced in the preeclamptic placentas. Apelin-13 treatment not only specifically attenuated CoCl2-induced superoxide production, but also prevented CoCl2-induced reduction of SOD activity and SOD1 expression. In addition, apelin-13 suppressed CoCl2-induced apoptosis by increasing the expression of bcl-2/bax ratio and by decreasing the expression of active caspase-3 in placental trophoblasts. Furthermore, we found that apelin-13 binding APJ activated the PI3K and AKT kinases and inhibition of PI3K kinase significantly blocked the anti-oxidative effects of apelin-13 in placental trophoblasts. CONCLUSIONS: Decrease of placental APJ expression is associated with oxidative stress-induced placental dysfunction in preeclampsia, and increased circulating apelin could be a moderately successful marker to differentiate subjects with preeclampsia from healthy pregnant women. Inhibition of superoxide production and caspase-3 cleavage, together with upregulation of SOD activity/expression and bcl-2/bax ratio, could be the potential molecular mechanisms by which apelin-13/APJ protects placental trophoblasts from oxidative stress injury.

Apelin-13 alleviates contrast-induced acute kidney injury by inhibiting endoplasmic reticulum stress.

Contrast-induced acute kidney injury (CI-AKI) is a severe complication associated with significant morbidity and mortality, and effective therapeutic strategies are still lacking. Apelin is an endogenous physiological regulator with antioxidative, anti-inflammatory and antiapoptotic properties. However, the role of apelin-13 in CI-AKI remains unclear. In our study, we found that the protein expression levels of apelin were significantly downregulated in rat kidney tissues and HK-2 cells during contrast media treatment. Moreover, we explored the protective effect of apelin-13 on renal tubule damage using in vitro and in vivo models of CI-AKI. Exogenous apelin-13 ameliorated endoplasmic reticulum stress, reactive oxygen species and apoptosis protein expression in contrast media-treated cells and rat kidney tissues. Mechanistically, the downregulation of endoplasmic reticulum stress contributed critically to the antiapoptotic effect of apelin-13. Collectively, our findings reveal the inherent mechanisms by which apelin-13 regulates CI-AKI and provide a prospective target for the prevention of CI-AKI.

Effects of Apelin on the fibrosis of retinal tissues and Müller cells in diabetes retinopathy through the JAK2/STAT3 signalling pathway.

Retinal fibrosis was a key characteristic of diabetes retinopathy (DR). Apelin was found to be a candidate for tissue fibrosis. Nevertheless, the role of Apelin in the Müller cells in DR remains unclear. This study identified the function and mechanism of Apelin in Müller cells and the fibrosis of retinal tissue. Western blot was carried out to detect the Apelin, GFAP, Collagen I, α-SMA, JAK2 and STAT3 protein levels. Masson staining was performed to display the histopathological changes in retinal tissue of diabetic mellitus (DM) rats. The immunofluorescence staining was conducted to evaluate the Apelin levels in the retinal tissue. The levels of GFAP, Collagen I and α-SMA in the retinal tissue of DM rats was visualised by the immunohistochemistry staining. The results showed that Apelin, GFAP, Collagen I andα-SMA expression was prominently elevated in the retinal tissue of DM rats and high glucose (HG)-exposed Müller cells. The results of Masson staining showed that the epiretinal fibrotic membrane was observed in DM rats. Apelin knockdown declined the GFAP, Collagen I andα-SMA levels. Besides, the protein levels of p-JAK2 and p-STAT3 were elevated in the HG-treated Müller cells, while Apelin knockdown declined them. FLLL32 treatment neutralised the role of Apelin. In conclusion, Apelin facilitated the fibrogenic activity of Müller cells through activating the JAK2/STAT3 signalling pathway, and thus inducing the retinal fibrosis in DR.

Hypophagia induced by intracerebroventricular injection of apelin-13 is mediated via CRF1/CRF2 and MC3/MC4 receptors in neonatal broiler chicken.

Previous studies have shown the role of apelin and its receptors in the regulation of food intake. In the present study, we investigate the mediating role of melanocortin, corticotropin, and neuropeptide Y systems in apelin-13- induced food intake in broilers. Eight trials were run in the current investigation to ascertain the relationships between the aforementioned systems and apelin-13 on food intake and behavioral changes after apelin-13 administration. In experiment 1, hens were given an intracerebroventricular administration of a solution for control in addition to apelin-13 (0.25, 0.5, and 1 µg). Astressin-B (a CRF1/CRF2 receptor antagonist, 30 µg), apelin-13 (1 µg), and administration of astressin-B and apelin-13 concurrently, were all injected into the birds in experiment 2. Experiments 3 through 8 were quite similar to experiment 2, with the exception of astressin2-B (CRF2 receptor antagonist, 30 µg), SHU9119 (MC3/MC4 receptor antagonist, 0.5 nmol), MCL0020 (MC4 receptor antagonist, 0.5 nmol), BIBP-3226 (NPY1 receptor antagonist, 1.25 nmol), BIIE 0246 (NPY2 receptor antagonist, 1.25 nmol), and CGP71683A (NPY5 receptor antagonist, 1.25 nmol) were injected instead of astressin-B. After then, total food consumption was monitored for 6 h. Apelin-13 injections of 0.5 and 1 µg decreased feeding (P < 0.05). The hypophagic effects of apelin were attenuated following the simultaneous administration of Astressin-B and Astressin2-B with apelin-13 (P > 0.05). Co-infusion of SHU9119 and apelin-13 reduced the appetite-decreasing effects of apelin-13 (P > 0.05). When MCL0020 and apelin-13 were injected at the same time, the hypophagia that apelin-13 induced was eliminated (P > 0.05). BIBP-3226, BIIE 0246, and CGP71683A had no effect on the hypophagia brought on by apelin-13 (P > 0.05). Also, apelin-13 significantly increased number of steps, jumps, exploratory food, pecks and standing time while decreased siting time (P < 0.05). These findings suggest that apelin-13-induced hypophagia in hens may involve the CRF1/CRF2 and MC3/MC4 receptors.

7ß-Hydroxycholesterol and 7-ketocholesterol: New oxidative stress biomarkers of sarcopenia inducing cytotoxic effects on myoblasts and myotubes.

Aging is a complex biological process which can be associated with skeletal muscle degradation leading to sarcopenia. The aim of this study consisted i) to determine the oxidative and inflammatory status of sarcopenic patients and ii) to clarify the impact of oxidative stress on myoblasts and myotubes. To this end, various biomarkers of inflammation (C-reactive protein (CRP), TNF-α, IL-6, IL-8, leukotriene B4 (LTB4)) and oxidative stress (malondialdehyde, conjugated dienes, carbonylated proteins and antioxidant enzymes: catalase, superoxide dismutase, glutathione peroxidase) as well as oxidized derivatives of cholesterol formed by cholesterol autoxidation (7-ketocholesterol, 7ß-hydroxycholesterol), were analyzed. Apelin, a myokine which contributes to muscle strength, was also quantified. To this end, a case-control study was conducted to evaluate the RedOx and inflammatory status in 45 elderly subjects (23 non-sarcopenic; 22 sarcopenic) from 65 years old and higher. SARCopenia-Formular (SARC-F) and Timed Up and Go (TUG) tests were used to distinguish between sarcopenic and non-sarcopenic subjects. By using red blood cells, plasma and/or serum, we observed in sarcopenic patients an increased activity of major antioxidant enzymes (superoxide dismutase, glutathione peroxidase, catalase) associated with lipid peroxidation and protein carbonylation (increased level of malondialdehyde, conjugated dienes and carbonylated proteins). Higher levels of 7-ketocholesterol and 7ß-hydroxycholesterol were also observed in the plasma of sarcopenic patients. Significant differences were only observed with 7ß-hydroxycholesterol. In sarcopenic patients comparatively to non-sarcopenic subjects, significant increase of CRP, LTB4 and apelin were observed whereas similar levels of TNF-α, IL-6 and IL-8 were found. The increased plasma level of 7-ketocholesterol and 7ß-hydroxycholesterol in sarcopenic patients led us to study the cytotoxic effect of these oxysterols on undifferentiated (myoblasts) and differentiated (myotubes) murine C2C12 cells. With the fluorescein diacetate and sulforhodamine 101 assays, an induction of cell death was observed both on undifferentiated and differentiated cells: the cytotoxic effects were less pronounced with 7-ketocholesterol. In addition, IL-6 secretion was never detected whatever the culture conditions, TNF-α secretion was significantly increased on undifferentiated and differentiated C2C12 cells treated with 7-ketocholesterol- and 7ß-hydroxycholesterol, and IL-8 secretion was increased on differentiated cells. 7-ketocholesterol- and 7ß-hydroxycholesterol-induced cell death was strongly attenuated by α-tocopherol and Pistacia lentiscus L. seed oil both on myoblasts and/or myotubes. TNF-α and/or IL-8 secretions were reduced by α-tocopherol and Pistacia lentiscus L. seed oil. Our data support the hypothesis that the enhancement of oxidative stress observed in sarcopenic patients could contribute, especially via 7ß-hydroxycholesterol, to skeletal muscle atrophy and inflammation via cytotoxic effects on myoblasts and myotubes. These data bring new elements to understand the pathophysiology of sarcopenia and open new perspectives for the treatment of this frequent age-related disease.

Apelin signalling in the periaqueductal grey matter alleviates capsaicin-evoked pulpal nocifensive behaviour and capsaicin-induced spatial learning and memory impairments in rat.

AIM: Pulpal pain is a common orofacial health issue that has been linked to cognitive impairment. Because of its prominent role in pain modulation and cognitive impairment, apelin (Apl) is regarded as a promising target for clinical pain management. The role of Apl in orofacial pain, however, is unknown. The purpose of this study was to determine the effects of intra-periaqueductal grey matter (PAG) administrations of Apl-13 on capsaicin-evoked pulpal nocifensive behaviour and capsaicin-induced spatial learning and memory impairments in rats. METHODOLOGY: Forty-nine male Wistar rats (200-250 g) were randomly divided into seven groups (n = 7 per group). The groups included: untreated intact, capsaicin (Caps) only, three Caps+Apl groups that received different dosages of intra-PAG injection of Apl-13 (1, 2 and 3 µg/rat) 20 min prior to capsaicin application, and two Apl+antagonist groups that received Apl receptor antagonist or naloxone (a µ opioid receptor) 20 min before Apl injection. Learning and memory were assessed using the Morris water maze test. One-way analysis of variance followed by Tukey post hoc tests was used for statistical analysis. RESULTS: Intra-PAG administration of Apl-13 significantly reduced the capsaicin-induced nocifensive behaviour (p < .01). This antinociception effect was inhibited by F13A and naloxone. Apl-13 inhibited nociception-induced learning and memory deficits (p < .01). The cognitive effects were also blocked by pre-treatment administration of F13A (3 µg/rat). CONCLUSIONS: These findings indicated that Apl-13, via Apl receptors (AR or APJ) and µ opioid receptors, alleviated capsaicin-induced dental nocifensive behaviour and protected against nociception-induced learning and memory impairments. As a result of our findings, Apl appears to be a promising analgesic option for further research in orofacial pain models and clinical trials.

The effects of apelin on IGF1/FSH-induced steroidogenesis, proliferation, Bax expression, and total antioxidant capacity in granulosa cells of buffalo ovarian follicles.

Apelin (APLN) was believed to be an adipokine secreted from adipose tissue. However, studies demonstrate that it is a pleiotropic peptide and has several effects on the female reproductive system. In this study, We examined the effects of different doses of IGF1 and FSH in the presence of APLN-13 on the production of progesterone in buffalo ovary granulosa cells. Furthermore, different doses of APLN isoforms (APLN-13 and APLN-17) were tested on proliferation, Bax protein expression, and antioxidant capacity in the same cells. Granulosa cells of buffalo ovaries were cultured in the presence of different doses of IGF1 and FSH with or without APLN-13 (10-9 M) to evaluate its effect on the secretion of progesterone tested by ELISA assay. The WST-1 method was used to survey the effect of APLN on granulosa cell proliferation and cytotoxicity. In addition, the antioxidant capacity of the cells in the presence of APLN was assessed using the FRAP method. mRNA and Bax protein levels were measured in granulosa cells treated with APLN using real-time PCR and western blot techniques. APLN-13 (10-9) stimulated the effect of IGF1 on the production of progesterone, and its levels were affected by APLN-13 dose-dependently. However, it did not significantly stimulate the effect of FSH on the secretion of progesterone. APLN-13 (all doses) and APLN-17 (10-8 and 10-9 M) improved the proliferation of granulosa cells. Moreover, preincubation of the cells for an hour by APLN receptor antagonist (ML221, 10 µM) did not significantly affect the proliferation of cells induced by APLN. Neither APLN-13 nor APLN-17 were not cytotoxic for the cells compared to the control treatment. APLN-13 at the doses of 10-6 and 10-8 M substantially up and down-regulated Bax protein expression; however, such effects were not observed when the cells were preincubated with ML221. In addition, APLN-17 did not influence the expression amount of Bax. Furthermore, both APLN-13 and -17 improved the total antioxidant capacity of the ovarian granulosa cells, but such effects were not seen when the cells were preincubated with ML221. According to these results, APLN enhanced the steroidogenesis induced by IGF1 but did not affect the steroidogenesis induced by FSH. APLN also enhanced the cell proliferation and antioxidant capacity of buffalo ovaries follicular granulosa cells; however, its effect on Bax expression was different.

Apelin triggers macrophage polarization to M2 type in head and neck cancer.

Cancer comes after cardiovascular diseases in terms of mortality rate in the world. Chemotherapy, radiotherapy and surgical interventions are the current cancer treatment. Recently, it has been observed that immunotherapeutic approaches provide a significant improvement when used along with these interventions. The mononuclear system mainly consists of macrophages that play an active role in the pathology of many diseases because of having high plasticity capacities. Previous research suggested that they can be used as an alternative to cancer treatment. Aim was to investigate the effect of apelin on macrophage polarization in the tumor microenvironment. Mouse macrophage cell line RAW264.7 cells and head and were chosen for this study. The apelin expression was knockdown in neck cell carcinoma cell line SCCL MT1 cells using shRNA technique. SCCL MT1 cells having normal or suppressed apelin expression were co-cultured with mouse macrophage RAW264.7 cells. The effect of co-culturing on the expression of inflammatory genes in RAW264.7 cells was investigated. Suppressed apelin expression in SCCL MT1 cells resulted in elevated pro-inflammatory response in co-cultured macrophages. Expression of the IL1ß, IL6, and TNFα genes significantly increased, however anti-inflammatory cytokine levels were significantly decreased. However, in the control group, a downregulation was determined in pro-inflammatory genes, while an increase was observed in anti-inflammatory genes. The protein levels of these cytokines in concordance with the RT-PCR analysis. As a result of this study, apelin released from cancer cells was found to affect macrophage polarization. These results indicated that the apelin peptide may cause the intense presence of M2-type macrophages in the tumor niche, and the therapeutic approaches targeting of apelin in cancer cells may have a potential role in macrophage polarization.

APELIN-13 AMELIORATES LPS-INDUCED ENDOTHELIAL-TO-MESENCHYMAL TRANSITION AND POST-ACUTE LUNG INJURY PULMONARY FIBROSIS BY SUPPRESSING TRANSFORMING GROWTH FACTOR-Β1 SIGNALING.

ABSTRACT: The pathophysiology of acute respiratory distress syndrome (ARDS) involves cytokine storms, alveolar-capillary barrier destruction, and fibrotic progression. Pulmonary interstitial fibrosis is an important factor affecting the prognosis of ARDS patients. Endothelial-to-mesenchymal transition (EndMT) plays an important role in the development of fibrotic diseases, and the occurrence of EndMT has been observed in experimental models of LPS-induced acute lung injury (ALI). Apelin is an endogenous active polypeptide that plays an important role in maintaining endothelial cell homeostasis and inhibiting fibrotic progression in various diseases. However, whether apelin attenuates EndMT in ALI and post-ALI pulmonary fibrosis remains unclear. We analyzed the serum levels of apelin-13 in patients with sepsis-associated ARDS to examine its possible clinical value. A murine model of LPS-induced pulmonary fibrosis and an LPS-challenged endothelial cell injury model were used to analyze the protective effect and underlying mechanism of apelin-13. Mice were treated with apelin-13 by i.p. injection, and human pulmonary microvascular endothelial cells were incubated with apelin-13 in vitro . We found that the circulating apelin-13 levels were significantly elevated in sepsis-associated ARDS patients compared with healthy controls. Our study also confirmed that LPS induced EndMT progression and pulmonary fibrosis, which were characterized by decreased CD31 expression and increased α-smooth muscle actin expression and collagen deposition. LPS also stimulated the production of transforming growth factor ß1 and activated the Smad signaling pathway. However, apelin-13 treatment significantly attenuated these changes. Our findings suggest that apelin-13 may be a novel biomarker in patients with sepsis-associated ARDS. These results demonstrate that apelin-13 ameliorates LPS-induced EndMT and post-ALI pulmonary fibrosis by suppressing transforming growth factor ß1 signaling.

The G protein-coupled receptor ligand apelin-13 ameliorates skeletal muscle atrophy induced by chronic kidney disease.

BACKGROUND: Targeting of the apelin-apelin receptor (Apj) system may serve as a useful therapeutic intervention for the management of chronic kidney disease (CKD)-induced skeletal muscle atrophy. We investigated the roles and efficacy of the apelin-Apj system in CKD-induced skeletal muscle atrophy. METHODS: The 5/6-nephrectomized mice were used as CKD models. AST-120, a charcoal adsorbent of uraemic toxins (8 w/w% in diet), or apelin (1 µmol/kg) was administered to CKD mice to investigate the mechanism and therapeutic potential of apelin on CKD-induced skeletal muscle atrophy. The effect of indoxyl sulfate, a uraemic toxin, or apelin on skeletal muscle atrophy was evaluated using mouse myoblast cells (C2C12 cells) in vitro. RESULTS: Skeletal muscle atrophy developed over time following nephrectomy at 12 weeks, as confirmed by a significant increase of atrogin-1 and myostatin mRNA expression in the gastrocnemius (GA) muscle and a decrease of lower limb skeletal muscle weight (P < 0.05, 0.01 and 0.05, respectively). Apelin expression in GA muscle was significantly decreased (P < 0.05) and elabela, another Apj endogenous ligand, tended to show a non-significant decrease at 12 weeks after nephrectomy. Administration of AST-120 inhibited the decline of muscle weight and increase of atrogin-1 and myostatin expression. Apelin and elabela expression was slightly improved by AST-120 administration but Apj expression was not, suggesting the involvement of uraemic toxins in endogenous Apj ligand expression. The administration of apelin at 1.0 µmol/kg for 4 weeks to CKD mice suppressed the increase of atrogin-1 and myostatin, increased apelin and Apj mRNA expression at 30 min after apelin administration and significantly ameliorated weight loss and a decrease of the cross-sectional area of hindlimb skeletal muscle. CONCLUSIONS: This study demonstrated for the first time the association of the Apj endogenous ligand-uraemic toxin axis with skeletal muscle atrophy in CKD and the utility of therapeutic targeting of the apelin-Apj system.

Potential role of the apelin-APJ pathway in sex-related differential cardiotoxicity induced by doxorubicin in mice.

Preclinical and clinical findings suggest sexual dimorphism in cardiotoxicity induced by a chemotherapeutic drug, doxorubicin (DOX). However, molecular alterations leading to sex-related differential vulnerability of heart to DOX toxicity are not fully explored. In the present study, RNA sequencing in hearts of B6C3F1 mice indicated more differentially expressed genes in males than females (224 vs. 19; ≥1.5-fold, False Discovery Rate [FDR] < 0.05) at 1 week after receiving 24 mg/kg total cumulative DOX dose that induced cardiac lesions only in males. Pathway analysis further revealed probable inactivation of cardiac apelin fibroblast signaling pathway (p = 0.00004) only in DOX-treated male mice that showed ≥1.25-fold downregulation in the transcript and protein levels of the apelin receptor, APJ. In hearts of DOX-treated females, the transcript levels of apelin (1.24-fold) and APJ (1.47-fold) were significantly (p < 0.05) increased compared to saline-treated controls. Sex-related differential DOX effect was also observed on molecular targets downstream of the apelin-APJ pathway in cardiac fibroblasts and cardiomyocytes. In cardiac fibroblasts, upregulation of Tgf-ß2, Ctgf, Sphk1, Serpine1, and Timp1 (fibrosis; FDR < 0.05) in DOX-treated males and upregulation of only Tgf-ß2 and Timp1 (p < 0.05) in females suggested a greater DOX toxicity in hearts of males than females. Additionally, Ryr2 and Serca2 (calcium handling; FDR < 0.05) were downregulated in conjunction with 1.35-fold upregulation of Casp12 (sarcoplasmic reticulum-mediated apoptosis; FDR < 0.05) in DOX-treated male mice. Drug effect on the transcript level of these genes was less severe in female hearts. Collectively, these data suggest a likely role of the apelin-APJ axis in sex-related differential DOX-induced cardiotoxicity in our mouse model.

Feedback Interaction Between Apelin and Endoplasmic Reticulum Stress in the Rat Myocardium.

ABSTRACT: Apelin is an endogenous active peptide, playing a crucial role in regulating cardiovascular homeostasis. This study aimed to investigate the interaction between apelin and endoplasmic reticulum stress (ERS). Tunicamycin (Tm) and dithiothreitol (DTT) were used to induce ERS in the ex vivo cultured myocardium of rats. Myocardial injury was determined by the activities of lactate dehydrogenase and creatine kinase-MB in the culture medium. The protein levels of an ERS-associated molecule, apelin, and its receptor angiotensin domain type 1 receptor-associated proteins (APJ) in the myocardium were determined by western blot analysis. The level of apelin in the culture medium was determined by enzyme immunoassay. Administration of Tm and DTT triggered ERS activation and myocardial injury, and led to a decrease in protein levels of apelin and APJ, in a dose-dependent manner. Integrated stress response inhibitor, an inhibitor of eukaryotic initiation factor 2α phosphorylation that is commonly used to prevent activation of protein kinase R-like ER kinase cascades, blocked ERS-induced myocardial injury and reduction of apelin and APJ levels. The ameliorative effect of integrated stress response inhibitor was partially inhibited by [Ala]-apelin-13, an antagonist of APJ. Furthermore, apelin treatment inhibited activation of the 3 branches of ERS induced by Tm and DTT in a dose-dependent manner, thereby preventing Tm-induced or DTT-induced myocardial injury. The negative feedback regulation between ERS activation and apelin/APJ suppression might play a critical role in myocardial injury. Restoration of apelin/APJ signaling provides a potential target for the treatment and prevention of ERS-associated tissue injury and diseases.

Apelin as a new therapeutic target for COVID-19 treatment.

BACKGROUND: Apelin is an endogenous neuropeptide that binds to the G-protein-coupled receptor (APJ) and participates in a variety of physiological processes in the heart, lungs and other peripheral organs. Intriguingly, [Pyr1]-Apelin-13, a highly potent pyroglutamic form of apelin, has the potential to bind to and be degraded by angiotensin-converting enzyme 2 (ACE2). ACE2 is known to operate as a viral receptor in the early stages of severe acute respiratory coronavirus (SARS-CoV-2) infection. AIM: This study aimed to determine if apelin protects against SARS-CoV-2 infection by inhibiting ACE2 binding to SARS-CoV-2 spike protein. DESIGN AND METHODS: To determine whether [Pyr1]-Apelin-13 inhibits ACE2 binding to the SARS-CoV-2 spike protein (S protein), we performed a cell-to-cell fusion assay using ACE2-expressing cells and S protein-expressing cells and a pseudovirus-based inhibition assay. We then analyzed publicly available transcriptome data while focusing on the beneficial effects of apelin on the lungs. RESULTS: We found that [Pyr1]-Apelin-13 inhibits cell-to-cell fusion mediated by ACE2 binding to the S protein. In this experiment, [Pyr1]-Apelin-13 protected human bronchial epithelial cells, infected with pseudo-typed lentivirus-producing S protein, against viral infection. In the presence of [Pyr1]-Apelin-13, the level of viral spike protein expression was also reduced in a concentration-dependent manner. Transcriptome analysis revealed that apelin may control inflammatory responses to viral infection by inhibiting the nuclear factor kappa B pathway. CONCLUSION: Apelin is a potential therapeutic candidate against SARS-CoV-2 infection.

Loss of thyroid gland circadian PER2 rhythmicity in aged mice and its potential association with thyroid cancer development.

Molecular clocks operate in peripheral tissues, including endocrine glands, and play important regulatory roles in this context. However, potential age-related changes in the expression rhythmicity of clock genes and the effects of these changes on the thyroid gland remain unknown. In the present study, we evaluated the expression rhythmicity of peripheral thyroid clock genes in aged mice using RNA-seq transcriptomic analysis in young (3.5-month) versus aged (20-month) mice. In addition, we determined the cellular effects of silencing of PER2, a major clock gene regulator, in human thyroid cell lines. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that differentially expressed genes (DEGs) in the thyroid glands of aged mice were involved in mitogen-activated protein kinase (MAPK) signaling, chemokine signaling, circadian entrainment, PI3K/AKT signaling, and Apelin signaling. The expression of circadian clock genes Arntl/Bmal1 was significantly downregulated in thyroid glands of aged mice, whereas the expression of genes involved in regulation of cell proliferation, migration, and tumorigenesis was upregulated. Peripheral thyroid clock genes, particularly Per mRNA and PER2 protein, were downregulated in the thyroid glands of aged mice, and circadian oscillation of these genes was declined. Knockdown of the circadian clock gene PER2 in human thyroid follicular cells induced AP-1 activity via JNK MAPK signaling activation, which increased cell proliferation. Furthermore, the aging-related loss of PER2 circadian oscillation activated the AP-1 transcription factor via the JNK MAPK pathway, which could contribute to thyroid hyperplasia, a common age-related condition.