Expression of wingless type (WNT) genes and their antagonists at mRNA levels in equine endometrium during the estrous cycle and early pregnancy

WNT signaling pathway plays important roles in reproductive events. Aims were to (1) determine presence of WNT genes and their antagonists in equine endometrium; and (2) to evaluate their expression profiles during early pregnancy. Endometrial biopsies were obtained from mares on day of ovulation (d0, n=4) and on days of 14 (P14, n=4), 18 (P18, n=4), 22 (P22, n=4) of early pregnancy. Biopsies were also collected from cyclic mares during late diestrus (LD, on day of 13.5-14, n=4) and after luteolysis in estrus phase (AL, on day of 17.5-18, n=4) of the cycle. PCR was used to detect expression of genes studied and then relative expression levels were quantified using real-time PCR analysis. A mixed model was fitted on the normalized data and least significant difference test (α=0.05) was employed. Eleven WNT genes (WNT2, WNT2B, WNT4, WNT5A, WNT5B, WNT7A, WNT8A, WNT9B, WNT10B, WNT11 and WNT16) and their antagonists (SFRP1, SFRP2, SFRP5, DKK1, DKK2 and WIF-1) were detected in equine endometrium. Compared to d0, WNT2, WNT5B, WNT7A and SFRP1 expressions were downregulated by the pregnancy while DKK1 was upregulated. WNT5A, WNT11 and WIF-1 were upregulated on P14 and P18, but WNT2B increased only on P14. When LD and P14 were compared, level of WNT8A decreased on P14 while increase in WNT4 level on P14 was slightly significant (P<0.06). Levels of WNT7A and SFRP1 decreased while DKK1 and WIF-1 increased by the pregnancy on P18 compared to AL. Moreover, WNT2B, WNT5A, WNT9B, WNT10B, WNT11, WNT16 DKK1 and WIF-1 were upregulated on LD compared to AL whereas WNT4, WNT7A, SFRP1 were downregulated. In conclusion, the results demonstrate that WNT genes and their antagonists appear to be regulated during early pregnancy in equine endometrium possibly due to embryonic factors and/or maternal progesterone.     

冠状动脉粥样硬化性心脏病患者血清SFRP5、FGF21、IGF-I水平与血脂和冠状动脉病变严重程度的相关性研究

目的:探讨冠状动脉粥样硬化性心脏病(CHD)患者血清分泌型卷曲蛋白5(SFRP5)、成纤维细胞生长因子21(FGF21)、胰岛素样生长因子-1(IGF-I)水平与血脂和冠状动脉病变严重程度的相关性。方法:选择2018年6月至2021年6月我院收治的109例CHD患者(CHD组),根据冠心病类型分为稳定型心绞痛组(SAP组,32例),不稳定型心绞痛组(UA组,42例)、急性心肌梗死组(AMI组,35例),根据Gensini积分分为轻度病变组(≤20分,42例)、中度病变组(21~40分,44例)和重度病变组(>40分,23例),另选择同期在我院行冠脉造影检查结果为正常的53例患者为对照组。检测并比较各组血清SFRP5、FGF21、IGF-I、血脂水平,分析血清SFRP5、FGF21、IGF-I与血脂和Gensini积分的相关性。结果:不同类型、不同冠脉病变程度CHD患者的血清SFRP5、FGF21、IGF-I、HDL-C水平均低于对照组,血清TC、TG、LDL-C水平均高于对照组,差异有统计学意义(P<0.05);血清SFRP5、FGF21、IGF-I水平的差异比较中,UA组低于SAP组,AMI组又低于UA组,中度病变组低于轻度病变组,重度病变组又低于中度病变组,差异均有统计学意义(P<0.05);血清TC、LDL-C水平的差异比较中,UA组高于SAP组,AMI组又高于UA组,中度病变组高于轻度病变组,重度病变组又高于中度病变组,差异均有统计学意义(P<0.05);而SAP组、UA组、AMI组之间两两比较以及轻度病变组、中度病变组、重度病变组之间两两比较的血清TG、HDL-C水平差异无统计学意义(P>0.05)。Pearson相关性分析结果显示:血清SFRP5、FGF21、IGF-I水平与TC、LDL-C、Gensini积分呈负相关(P<0.05)。结论:CHD患者血清SFRP5、FGF21、IGF-I水平均降低,且与血脂水平增高以及CHD病变程度加重均有关。     

Insulin resistance,lipotoxicity and endothelial dysfunction

The number of people with the insulin-resistant conditions of type 2 diabetes mellitus (T2DM) and obesity has reached epidemic proportions worldwide. Eighty percent of people with T2DM will die from the complications of cardiovascular atherosclerosis. Insulin resistance is characterised by endothelial dysfunction, which is a pivotal step in the initiation/progression of atherosclerosis. A hallmark of endothelial dysfunction is an unfavourable imbalance between the bioavailability of the antiatherosclerotic signalling molecule nitric oxide (NO) and proatherosclerotic reactive oxygen species. In this review we discuss the mechanisms linking insulin resistance to endothelial dysfunction, with a particular emphasis on a potential role for a toxic effect of free fatty acids on endothelial cell homeostasis.     

The role of WNT5A and Ror2 in peritoneal membrane injury

Patients on peritoneal dialysis are at risk of developing peritoneal fibrosis and angiogenesis, which can lead to dysfunction of the peritoneal membrane. Recent evidence has identified cross-talk between transforming growth factor beta (TGFB) and the WNT/β-catenin pathway to induce fibrosis and angiogenesis. Limited evidence exists describing the role of non-canonical WNT signalling in peritoneal membrane injury. Non-canonical WNT5A is suggested to have different effects depending on the receptor environment. WNT5A has been implicated in antagonizing canonical WNT/β-catenin signalling in the presence of receptor tyrosine kinase-like orphan receptor (Ror2). We co-expressed TGFB and WNT5A using adenovirus and examined its role in the development of peritoneal fibrosis and angiogenesis. Treatment of mouse peritoneum with AdWNT5A decreased the submesothelial thickening and angiogenesis induced by AdTGFB. WNT5A appeared to block WNT/β-catenin signalling by inhibiting phosphorylation of glycogen synthase kinase 3 beta (GSK3B) and reducing levels of total β-catenin and target proteins. To examine the function of Ror2, we silenced Ror2 in a human mesothelial cell line. We treated cells with AdWNT5A and observed a significant increase in fibronectin compared with AdWNT5A alone. We also analysed fibronectin and vascular endothelial growth factor (VEGF) in a TGFB model of mesothelial cell injury. Both fibronectin and VEGF were significantly increased in response to Ror2 silencing when cells were exposed to TGFB. Our results suggest that WNT5A inhibits peritoneal injury and this is associated with a decrease in WNT/β-catenin signalling. In human mesothelial cells, Ror2 is involved in regulating levels of fibronectin and VEGF.     

WNT pathways in the neonatal ovine uterus: potential specification of endometrial gland morphogenesis by SFRP2

Endometrial glands are critical for uterine function and develop between birth (Postnatal Day [P] 0) and P56 in the neonatal ewe. Endometrial gland morphogenesis or adenogenesis involves the site-specific budding differentiation of the glandular epithelium from the luminal epithelium followed by their coiling/branching development within the stroma of the intercaruncular areas of the endometrium. To determine whether WNT signaling regulates endometrial adenogenesis, the WNT signaling system was studied in the neonatal ovine uterus. WNT5A, WNT7A, and WNT11 were expressed in the uterine epithelia, whereas WNT2B was in the stroma. The WNT receptors FZD2 and FZD6 and coreceptor LRP6 were detected in all uterine cells, and FZD6 was particularly abundant in the endometrial epithelia. Secreted FZD-related protein-2 (SFRP2), a WNT antagonist, was not detected in the P0 uterus, but was abundant in the aglandular caruncular areas of the endometrium between P7 and P56. Exposure of ewes to estrogens during critical developmental periods inhibits or retards endometrial adenogenesis. Estrogen-induced disruption of endometrial adenogenesis was associated with reduction or ablation of WNT2B, WNT7A, and WNT11, and with an increase in WNT2 and SFRP2 mRNA, depending on exposure period. Collectively, results implicate the canonical and noncanonical WNT pathways in regulation of postnatal ovine uterine development and endometrial adenogenesis. Expression of SFRP2 in aglandular caruncular areas may inhibit the WNT signaling pathway, thereby concentrating WNT signaling and restricting endometrial adenogenesis in the intercaruncular areas of the uterus. Further, estrogen-induced inhibition of adenogenesis may be mediated by a reduction in WNT signaling caused by aberrant induction of SFRP2 and loss of several critical WNTs.     

脂联素基因单核苷酸多态性与Ⅱ型糖尿病合并肥胖及胰岛素抵抗相关联

脂联素是近年新发现的脂肪组织特异性的细胞因子,其mRNA是脂肪组织中含量最丰富的基因转录产物,该因子可通过多种途径影响个体对胰岛素的敏感性。脂联素基因多态性与肥胖、胰岛素抵抗和2型糖尿病密切相关,而与冠心病相关性研究的报道较少。本研究以中国汉族人群1,098例为对象,其中304例冠心病(CHD)患者,389例糖尿病患者(T2DM),及405例性别年龄相匹配的正常对照,采用PCR-RFLP技术对脂联素基因-4522C/T进行基因分型,并分别对血脂水平、胰岛素抵抗、体重指数等临床数据进行分析比较。研究结果显示,脂联素基因-4522C/T各基因型及等位基因在CHD组与对照组、T2DM组与对照组中的分布差异无显著性;经分组分析发现,T2DM合并肥胖患者BMI≥25kg/m2TT基因型及T等位基因明显多于对照组,差异有显著性,P=0.014和P=0.034;TT基因型T2DM患者胰岛素抵抗指数(HOMA-IR)显著高于携带有C等位基因的T2DM患者,P=0.0069。本研究提示脂联素基因-4522C/T与中国汉族人群T2DM合并肥胖的发生及T2DM患者胰岛素抵抗相关,是引发糖尿病患者肥胖和胰岛素抵抗的重要候选基因,而与冠心病的发生无关联。     

The role of melatonin in the treatment of type 2 diabetes mellitus and Alzheimer's disease

In type 2 diabetes mellitus (T2DM) and its related disorders like obesity, the abnormal protein processing, oxidative stress and proinflammatory cytokines will drive the activation of inflammatory pathways, leading to low-grade chronic inflammation and insulin resistance (IR) in the periphery and impaired neuronal insulin signaling in the brain. Studies have shown that such inflammation and impaired insulin signaling contribute to the development of Alzheimer''s disease (AD). Therefore, new therapeutic strategies are needed for the treatment of T2DM and T2DM-linked AD. Melatonin is primarily known for its circadian role which conveys message of darkness and induces night-state physiological functions. Besides rhythm-related effects, melatonin has anti-inflammatory and antioxidant properties. Melatonin levels are downregulated in metabolic disorders with IR, and activation of melatonin signaling delays disease progression. The aim of this Review is to highlight the therapeutic potentials of melatonin in preventing the acceleration of AD in T2DM individuals through its therapeutic mechanisms, including antioxidative effects, anti-inflammatory effects, restoring mitochondrial function and insulin sensitivity.     

Epigenetic loss of putative tumor suppressor SFRP3 correlates with poor prognosis of lung adenocarcinoma patients

Secreted frizzled related protein 3 (SFRP3) contains a cysteine-rich domain (CRD) that shares homology with Frizzled CRD and regulates WNT signaling. Independent studies showed epigenetic silencing of SFRP3 in melanoma and hepatocellular carcinoma. Moreover, a tumor suppressive function of SFRP3 was shown in androgen-independent prostate and gastric cancer cells. The current study is the first to investigate SFRP3 expression and its potential clinical impact on non-small cell lung carcinoma (NSCLC). WNT signaling components present on NSCLC subtypes were preliminary elucidated by expression data of The Cancer Genome Atlas (TCGA). We identified a distinct expression signature of relevant WNT signaling components that differ between adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). Of interest, canonical WNT signaling is predominant in LUAD samples and non-canonical WNT signaling is predominant in LUSC. In line, high SFRP3 expression resulted in beneficial clinical outcome for LUAD but not for LUSC patients. Furthermore, SFRP3 mRNA expression was significantly decreased in NSCLC tissue compared to normal lung samples. TCGA data verified the reduction of SFRP3 in LUAD and LUSC patients. Moreover, DNA hypermethylation of SFRP3 was evaluated in the TCGA methylation dataset resulting in epigenetic inactivation of SFRP3 expression in LUAD, but not in LUSC, and was validated by pyrosequencing of our NSCLC tissue cohort and in vitro demethylation experiments. Immunohistochemistry confirmed SFRP3 protein downregulation in primary NSCLC and indicated abundant expression in normal lung tissue. Two adenocarcinoma gain-of-function models were used to analyze the functional impact of SFRP3 on cell proliferation and regulation of CyclinD1 expression in vitro. Our results indicate that SFRP3 acts as a novel putative tumor suppressor gene in adenocarcinoma of the lung possibly regulating canonical WNT signaling.     

Cloning and Expression of SFRP5 in Tibetan Chicken and its Relationship with IMF Deposition

Secreted frizzled related protein 5 (SFRP5), an anti-inflammatory adipokine, is relevant to the adipocyte differentiation. In order to clarify its role in regulating intramuscular fat (IMF) deposition in Tibetan chicken, the full-length sequence of the Tibetan chicken SFRP5 gene was cloned. The relative expression of SFRP5 gene was detected using quantitative RT-PCR in various tissues of 154 days old Tibetan chicken, as well as in breast muscle, thigh muscle, and adipose tissue at different growth stages. The results showed that SFRP5 gene was expressed in all examined tissues but highly enriched in adipose tissue. Temporal expression profile showed that the expression of SFRP5 was gradually decreased in breast muscle, but was fluctuated in thigh muscle and adipose tissue with the growth of Tibetan chicken. Furthermore, correlation analysis demonstrated that the expression of SFRP5 in breast muscle, thigh muscle and adipose tissue was correlated with IMF content at different levels. The results indicated that Tibetan chicken SFRP5 is involved in IMF deposition.     

Beta-arrestin and casein kinase 1/2 define distinct branches of non-canonical WNT signalling pathways

Recent advances in understanding beta-catenin-independent WNT (non-canonical) signalling suggest an increasing complexity, raising the question of how individual non-canonical pathways are induced and regulated. Here, we examine whether intracellular signalling components such as beta-arrestin (beta-arr) and casein kinases 1 and 2 (CK1 and CK2) can contribute to determining signalling specificity in beta-catenin-independent WNT signalling to the small GTPase RAC-1. Our findings indicate that beta-arr is sufficient and required for WNT/RAC-1 signalling, and that casein kinases act as a switch that prevents the activation of RAC-1 and promotes other non-canonical WNT pathways through the phosphorylation of dishevelled (DVL, xDSH in Xenopus). Thus, our results indicate that the balance between beta-arr and CK1/2 determines whether WNT/RAC-1 or other non-canonical WNT pathways are activated.     

Benzo(a)pyrene inhibits endometrial cell apoptosis in early pregnant mice via the WNT5A pathway

Benzo(a)pyrene (BaP) is an endocrine-disrupting pollutant present in various aspects of daily life, and studies have demonstrated that BaP exerts reproductive toxicity. We previously showed that BaP damages endometrial morphology and decreases the number of implantation sites in early pregnant mice, but the mechanisms underlying these effects remain unclear. The endometrial function is crucial for implantation, which is associated with endometrial cell apoptosis. In this study, we focused on the effect of BaP on endometrial cell apoptosis and the role of WNT signaling during this process. Pregnant mice were gavaged with corn oil (control group) or 0.2 mg·kg⁻¹·day ⁻¹ BaP (treatment group) from Days 1 to 6 of pregnancy. BaP impaired endometrial function by decreasing the expression of HOXA10 and BMP2, two markers of receptivity and decidualization. WNT5A and β-catenin were activated in the BaP group. BaP affected the expression of apoptosis-related proteins and inhibited the apoptosis of endometrial stromal cells. In vitro, human endometrial stromal cells (HESCs) were treated with different concentrations of BaP (dimethyl sulfoxide (DMSO); 5, 10 µM). WNT5A and β-catenin were also upregulated in the BaP treatment group. HESC apoptosis was restrained by BaP. Inhibiting WNT5A by SFRP5 partially restored the effect of BaP on apoptosis. In summary, these results suggested that BaP exposure during early pregnancy activates WNT5A/β-catenin signaling pathway, which inhibits the endometrial cell apoptosis and potentially destroys endometrial function.     

SHH-N upregulates Sfrp2 to mediate its competitive interaction with WNT1 and WNT4 in the somitic mesoderm

Dorsoventral polarity of the somitic mesoderm is established by competitive signals originating from adjacent tissues. The ventrally located notochord provides the ventralizing signals to specify the sclerotome, while the dorsally located surface ectoderm and dorsal neural tube provide the dorsalizing signals to specify the dermomyotome. Noggin and SHH-N have been implicated as the ventralizing signals produced by the notochord. Members of the WNT family of proteins, on the other hand, have been implicated as the dorsalizing signals derived from the ectoderm and dorsal neural tube. When presomitic explants are confronted with cells secreting SHH-N and WNT1 simultaneously, competition to specify the sclerotome and dermomyotome domains within the naive mesoderm can be observed. Here, using these explant cultures, we provide evidence that SHH-N competes with WNT1, not only by upregulating its own receptor Ptc1, but also by upregulating Sfrp2 (Secreted frizzled-related protein 2), which encodes a potential WNT antagonist. Among the four known Sfrps, Sfrp2 is the only member expressed in the sclerotome and upregulated by SHH-N recombinant protein. We further show that SFRP2-expressing cells can reduce the dermomyotome-inducing activity of WNT1 and WNT4, but not that of WNT3a. Together, our results support the model that SHH-N at least in part employs SFRP2 to reduce WNT1/4 activity in the somitic mesoderm.     

Effect of dapagliflozin on diabetic patients with cardiovascular disease via MAPK signalling pathway

Clinical studies have shown that dapagliflozin can reduce cardiovascular outcome in patients with type 2 diabetes mellitus (T2DM), but the exact mechanism is unclear. In this study, we used the molecular docking and network pharmacology methods to explore the potential mechanism of dapagliflozin on T2DM complicated with cardiovascular diseases (CVD). Dapagliflozin's potential targets were predicted via the Swiss Target Prediction platform. The pathogenic targets of T2DM and CVD were screened by the Online Mendelian Inheritance in Man (OMIM) and Gene Cards databases. The common targets of dapagliflozin, T2DM and CVD were used to establish a protein-protein interaction (PPI) network; the potential protein functional modules in the PPI network were found out by MCODE. Metascape tool was used for Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analysis. A potential protein functional module with the best score was obtained from the PPI network and 9 targets in the protein functional module all showed good binding properties when docking with dapagliflozin. The results of KEGG pathway enrichment analysis showed that the underlying mechanism mainly involved AGE-RAGE signalling pathway in diabetic complications, TNF signalling pathway and MAPK signalling pathway. Significantly, the MAPK signalling pathway was considered as the key pathway. In conclusion, we speculated that dapagliflozin played a therapeutic role in T2DM complicated with CVD mainly through MAPK signalling pathway. This study preliminarily reveals the possible mechanism of dapagliflozin in the treatment of T2DM complicated with CVD and provides a theoretical basis for future clinical research.     

Secreted frizzled related protein 1 is a paracrine modulator of epithelial branching morphogenesis, proliferation, and secretory gene expression in the prostate

Previous in vitro studies identified secreted frizzled related protein 1 (SFRP1) as a candidate pro-proliferative signal during prostatic development and cancer progression. This study determined the in vivo roles of SFRP1 in the prostate using expression studies in mice and by creating loss- and gain-of-function mouse genetic models. Expression studies using an Sfrp1^lacZ knock-in allele showed that Sfrp1 is expressed in the developing mesenchyme/stroma of the prostate. Nevertheless, Sfrp1 null prostates exhibited multiple prostatic developmental defects in the epithelium including reduced branching morphogenesis, delayed proliferation, and increased expression of genes encoding prostate-specific secretory proteins. Interestingly, over-expression of SFRP1 in the adult prostates of transgenic mice yielded opposite effects including prolonged epithelial proliferation and decreased expression of genes encoding secretory proteins. These data demonstrated a previously unrecognized role for Sfrp1 as a stromal-to-epithelial paracrine modulator of epithelial growth, branching morphogenesis, and epithelial gene expression. To clarify the mechanism of SFRP1 action in the prostate, the response of WNT signaling pathways to SFRP1 was examined. Forced expression of SFRP1 in prostatic epithelial cells did not alter canonical WNT/β-catenin signaling or the activation of CamKII. However, forced expression of SFRP1 led to sustained activation of JNK, and inhibition of JNK activity blocked the SFRP1-induced proliferation of prostatic epithelial cells, suggesting that SFRP1 acts through the non-canonical WNT/JNK pathway in the prostate.     

Optimization,validation, and identification of two reliable antibodies for immunodetection of WNT5A

WNT5A is a secreted, noncanonical WNT signaling protein that has been reported to promote progression of several types of cancer, including oral squamous cell carcinoma. Many WNT5A antibodies are available commercially for immunohistochemistry (IHC) and western blot analysis. Validation of the primary antibodies, however, is often neglected. We characterized antibodies for detecting WNT5A by IHC and western blot analysis. We evaluated one polyclonal and three monoclonal commercially available WNT5A antibodies. After optimization of the IHC assay, all four antibodies showed cytoplasmic WNT5A expression in tissue samples; in contrast, only one antibody detected WNT5A in western blots. A pre-absorption test with recombinant WNT5A showed that AF645 and 3A4 antibodies specifically detected WNT5A in different assays. We suggest that the monoclonal 3A4 antibody is the most appropriate for use with IHC, while the polyclonal AF645 antibody is the best for western blot analysis.     

WNT‐3A and WNT‐5A counteract lipopolysaccharide‐induced pro‐inflammatory changes in mouse primary microglia

Surveying microglia, the resident macrophage‐like cells in the central nervous system, continuously screen their surroundings to sense imbalance in tissue homeostasis. Their activity is tightly regulated in both a pro‐ and anti‐inflammatory manner. We have previously shown that the lipoglycoproteins WNT‐3A and WNT‐5A drive pro‐inflammatory transformation in primary mouse microglia cells, arguing that WNTs have a role in the modulation of the central nervous system immune response. In this study, we address the effects of recombinant WNT‐3A and WNT‐5A on lipopolysaccharide (LPS)‐activated mouse primary microglia to investigate the putative anti‐inflammatory modulation of microglia by WNTs. While both WNT‐3A and WNT‐5A alone induce an up‐regulation of cyclooxygenase 2 (COX2), a generic pro‐inflammatory microglia marker, LPS exceeds these effects dramatically. However, combination of LPS and WNTs results in a dose‐dependent decrease in LPS‐induced cyclooxygenase 2 protein and mRNA expression. In conclusion, our data suggest that WNTs have a dual and context‐dependent effect on microglia acting in a homeostatic pro‐ and anti‐inflammatory manner.     

A systematic review and meta-analysis of the relationship between lipoprotein lipase Asn291Ser variant and diseases

This systematic review attempted to summarize the associations between the Asn291Ser variant in the lipoprotein lipase (LPL) gene and dyslipidemia, the risk of type 2 diabetes mellitus (T2DM), and coronary heart disease (CHD). In addition, the relationships between the Asn291Ser variant and other metabolic diseases such as obesity and high blood pressure were also investigated in this systematic review. We systematically reviewed the literature by means of a meta-analysis. Twenty-one articles, including 19,246 white subjects, were selected for this meta-analysis. The summary standardized mean difference (SMD) of plasma triglyceride (TG) for carriers compared with noncarriers of the Asn291Ser variant was 3.23 (P < 0.00001). The summary SMD of plasma HDL-cholsterol (HDL-C) for carriers compared with noncarriers of the Asn291Ser variant was -3.42 (P < 0.0001). The summary SMD of the association of the Asn291Ser variant with plasma TG increased with increasing age and weight gain. Significant interactions between the LPL Asn291Ser variant and fasting glucose, T2DM, and CHD were seen (P = 0.02, 0.04, and 0.01, respectively). No significant interactions were seen between the LPL Asn291Ser variant and body mass index, waist-hip ratio, and blood pressure (P > 0.05). This meta-analysis indicates that the Asn291Ser variant in the LPL gene is a risk factor for dyslipidemia, characterized by hypertriglyceridemia and low HDL-C levels. And the Asn291Ser variant in the LPL gene predisposes to more severe dyslipidemia with increasing age and weight gain. Also, this meta-analysis shows that the LPL Asn291Ser variant is associated with CHD and T2DM.