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The intact tendon-bone interface (TBI) consists of four histological layers-tendon, fibrocartilage, calcified fibrocartilage, and bone-that gradually merge into each other, making complete structural restoration after injury challenging. Osteoporosis poses a significant risk for rotator cuff tears (RCTs) and re-tears after arthroscopic rotator cuff repair (ARCR). Activating Leptin receptor (Lepr) mediated Stat3 signaling transduction facilitates the transcription of Runx2 and Sox9, respectively, and promotes osteogenesis and chondrogenesis. Sixty-five female Sprague Dawley rats were used. Animal models-ovariectomy (OVX) and rotator cuff tear and repair (RC)-were employed to simulate typical tendon-bone healing and TBI reconstruction under deficient bone-forming capability. And, grip strength, transcriptome, ELISA, histochemistry, and qPCR were performed to reveal the distinct functional recovery between RC and OVX + RC rats, as well as pathophysiologic exhibition in the TBI at 2-week and 8-week. RC rats exhibited better functional recovery during the proliferative phase of TBI reconstruction, i.e., 2-week, compared to OVX + RC rats, while both RC and OVX + RC rats showed a lower grip strength in the upper limbs during the remodeling phase, i.e., 8-week. In RCTs, where adipogenesis was suppressed in RCT healing, the osteoblast-derived Leptin (Lep) and Angiopoietin like 4 (Angptl4), the Lepr ligands, facilitate osteogenesis and chondrogenesis, resulting in an obvious mineralized band in the reconstructed TBI and a transit cartilage band during the proliferative phase in RC rats. In osteoporosis-comorbid RCTs, where osteogenesis was suppressed while adipogenesis was activated, the adipocyte-derived Lep and Angptl4, particularly Angptl4, facilitated Stat3 phosphorylation and nucleus transfer, Sox9 transcription, and chondrogenesis, which was observed in OVX + RC rats and led to excessive cartilage regeneration. This study demonstrated the role of Lep and Angptl4 in TBI reconstruction, via activating Lepr-mediated Stat3-Sox9 and Stat3-Runx2 signaling pathways, differentially regulating osteogenesis and chondrogenesis, and leading to the distinct clinical outcomes post-ARCR in RCTs and osteoporosis-comorbid RCTs. This study provides fundamental support for increasing Angptl4 in situ for chronogenesis in RCTs and lowering Angptl4 to Lep ratio for osteogenesis in RCTs with osteoporosis comorbidity. Show less

Triglyceride-rich lipoproteins carry lipids in the bloodstream, where the fatty acid moieties are liberated by lipoprotein lipase (LPL) and taken up by peripheral tissues such as brown adipose tissue (BAT) and white adipose tissue (WAT), whereas the remaining cholesterol-rich remnant particles are cleared mainly by the liver. Elevated triglyceride (TG) levels and prolonged circulation of cholesterol-rich remnants are risk factors for cardiovascular diseases. Acute cold exposure decreases postprandial TG levels and is a potential therapeutic approach to treat hypertriglyceridemia. However, how acute cold exposure regulates TG metabolism remains incompletely understood. In the current study, we found that acute cold exposure simultaneously increases postprandial very-low-density lipoprotein production and TG clearance, with the latter playing a dominant role and resulting in decreased TG levels. Acute cold exposure increases LPL activity and TG uptake in BAT, while suppressing LPL activity and TG uptake in WAT. Mechanistically, acute cold exposure increases BAT LPL activity through transcriptional upregulation of Lpl and posttranscriptional regulation via inhibiting the hepatic insulin-ANGPTL8-ANGPTL3 axis, while suppressing WAT LPL activity through upregulation of ANGPTL4. Angptl8 knockout mice have dramatically decreased levels of circulating TG. In the absence of ANGPTL8, acute cold exposure increases rather than decreases circulating TG levels. Thus, our study reveals multilayered regulation of acute cold response and postprandial TG metabolism, highlighting the key functions of ANGPTL3, 4, and 8 in response to acute cold exposure. Show less

Hypoxia-inducible factor-1 alpha (HIF-1α) plays a key role in the cellular response to hypoxia, which plays a crucial role in the induction of abnormal angiogenesis and metastasis. Understanding the mechanism for the regulation of angiogenesis by HIF-1α-regulating miRNA will contribute to developing the strategy to prevent metastasis. We conducted a functional screening for HIF-1α-inhibiting miRNAs by evaluating the effects of miRNA mimics on HIF-1α expression and identified miR-5586-5p as an angiogenesis inhibitor through a mechanistic study. Angiogenic activity was assessed by tube formation assays using HUVEC cells exposed to conditioned media from miRNA-transfected breast cancer cells. In vivo activity of miR-5586-5p was examined through intratumoral injection of miRNA in orthotopic xenograft mice established by injecting MDA-MB-231 cells into the mammary fat pads of BALB/c nu/nu mice. The expression of the critical proangiogenic factors vascular endothelial growth factor A (VEGFA) and angiopoietin-like protein 4 (ANGPTL4) was inhibited by miR-5586-5p. Migration and tube formation of human umbilical vein endothelial cells were reduced in the conditioned medium prepared from miR-5586-5p-transfected cells. miR-5586-5p also suppressed the expression of heparin-binding EGF-like growth factor (HBEGF) and a disintegrin and metalloprotease 17 (ADAM17), which play a role in hypoxic signaling to induce the expression of VEGFA and ANGPTL4. HIF-1α, HBEGF, and ADAM17 were verified as the direct targets of miR-5586-5p responsible for the angiogenesis-suppressing function of miR-5586-5p. Expression levels of miR-5586-5p were lower in tumor tissues than in neighboring normal tissues of breast cancer patients. The expression of miR-5586-5p was inversely correlated to those of HIF-1α, HBEGF, ADAM17, VEGFA, and ANGPTL4. Angiogenesis and subsequent tumor growth were suppressed by intratumoral injection of miR-5586-5p in orthotopic MDA-MB-231 xenografts in mice. A potent tumor-suppressive function of miR-5586-5p applicable for the development of a novel cancer treatment strategy is herein described. Show less

Renal fibrosis is a common pathological process in various chronic kidney diseases. The accumulation of senescent renal tubular epithelial cells (TECs) in renal tissues plays an important role in the development of renal fibrosis. Eliminating senescent TECs has been proven to effectively reduce renal fibrosis. Procyanidin C1 (PCC1) plays a senolytic role by specifically eliminating senescent cells and extending its overall lifespan. However, whether PCC1 can alleviate unilateral ureteral obstruction (UUO)-induced renal fibrosis and the associated therapeutic mechanisms remains unclear. Here, we observed a marked increase in senescent TECs within obstructed human renal tissue and demonstrated the positive correlation between the accumulation of senescent TECs and renal fibrosis in UUO-induced renal fibrosis in mice. We found that PCC1 reduced the number of senescent TECs, restored the regenerative phenotype in kidneys with reduced fibrosis, and improved tubular repair after UUO-induced injury. In vitro, PCC1 effectively cleared senescent HK2 cells by inducing apoptosis via ANGPTL4/NOX4 signaling. Incubation with culture medium from senescent HK2 cells promoted fibroblast activation, whereas PCC1 impeded profibrotic effects by downregulating senescence-associated secretory phenotype (SASP) factors from senescent HK2 cells. Therefore, PCC1 alleviated interstitial renal fibrosis not only by clearing senescent TECs and improving tubular repair but also by indirectly attenuating myofibroblast activation by reducing the level of SASP. In summary, PCC1 may be a novel therapeutic senolytic agent for treating renal fibrosis. Show less

Coronary artery disease (CAD) and cancer are 2 leading global causes of mortality, with shared modifiable risk factors, yet the genetic and molecular mechanisms underlying their comorbidity remain poorly understood. We performed a genome-wide pleiotropy analysis to identify shared genetic mechanisms across CAD and 4 common cancers that share modifiable risk factors with CAD (breast, colorectal, lung, prostate). Using genome-wide pleiotropy and colocalization analysis, we identified 60 colocalized susceptibility loci shared by CAD and site-specific cancer, of which 43 are novel, including loci at Our findings highlight shared and opposing genetic loci between CAD and cancer and provide insight into molecular intermediates mediating joint disease risk. Importantly, they indicate potential drug repurposing opportunities for dual CAD and cancer prevention while highlighting possible adverse and divergent effects of existing medications across both conditions. Show less

Cell immortalization corresponds to a biologically relevant clinical feature that allows cells to acquire a high proliferative potential during carcinogenesis. In multiple cancer types, Protein Kinase D3 (PKD3) has often been reported as a dysregulated oncogenic kinase that promotes cell proliferation. Using mouse embryonic fibroblasts (MEFs), in a spontaneous immortalization model, PKD3 has been demonstrated as a critical regulator of cell proliferation after immortalization. However, the mechanisms by which PKD3 regulates proliferation in immortalized MEFs require further elucidation. Using a previously validated Show less

Senescence is significantly associated with cancer promotion. This study aimed to characterize senescent cells at the single-cell level in nasopharyngeal carcinoma (NPC) and elucidate the phenotype of tumorigenic senescent cell clusters. The composition of NPC based on the single-cell sequencing dataset GSE150430 from clinical specimens of 15 treatment-naïve patients and one patient with chronic nasopharyngitis were investigated. Using single-cell transcriptomics, we identified the major types of senescent cells in NPC and determined that senescent epithelial C3 cells and SPP1+ macrophages were associated with tumor progression, and expressed unique arrays of pro-tumor surface proteins and senescence-associated secretory phenotype (SASP) factors. SASP is endowed with inflammatory cytokines and chemokines, which involve in the process of 'inflammatory ageing' and tumor progression. Epithelial cell cluster C3 upregulated epithelial-mesenchymal transition (EMT)-related genes associated with tumor metastasis. SPP1+ macrophages displayed a distinct secretome dominated by pro-inflammatory cytokines such as CCL2, CCL8, and IL-6, and were more enriched in glycolytic pathways compared with other subpopulations of macrophages. In particular, the senescent cell population showed higher and stronger intercellular communication compared with the non-senescent cell population. Furthermore, C3 interacted with SPP1+ macrophages through ANGPTL4-SD2. Our findings reveal the important role of senescent cells in the development of NPC, highlighting potential therapeutic pathways and cancer prevention strategies. Show less

Respiratory distress syndrome threatens neonates' life. This study probed the predictive value of lung ultrasound scores combined with serum angiopoietin-like protein 4 (ANGPTL4) levels on neonatal respiratory distress syndrome (NRDS) severity and prognosis. The NRDS group (n = 115) and control group (n = 30) were established. In both groups, lung ultrasound scores and serum ANGPTL4 levels, lung ultrasound scores and serum ANGPTL4 levels of newborns with NRDS of different severity, the risk factors affecting the poor prognosis of NRDS neonates, and the value of serum ANGPTL4 levels combined with lung ultrasound scores in determining the severity and prognosis of newborns with NRDS were analyzed. The NRDS groups had higher lung ultrasound scores and serum ANGPTL4 levels, and lower Apgar scores than the control group; lung ultrasound scores and serum ANGPTL4 levels were higher in the moderate and severe groups than in the mild group, and those were higher in the severe group than in the moderate group (all Lung ultrasound scores and serum ANGPTL4 levels are closely related to the severity and prognosis of NRDS neonates, and the combination of the two improves the assessed value of the severity and prognosis of NRDS neonates. The study provided a reference for the disease severity assessment of NRDS and the prediction of its prognosis. Show less

Social interaction with others is essential to life. Although social isolation and loneliness have been implicated as increased risks of cardiometabolic and cardiovascular diseases and all-cause mortality, the cellular and molecular mechanisms by which social connection maintains cardiometabolic and cardiovascular health remain largely unresolved. To investigate how social connection protects against cardiometabolic and cardiovascular diseases, atherosclerosis-prone, high-fat diet-fed These results identify a novel brain-liver axis that links sociality to hepatic lipid metabolism, thus proposing a potential therapeutic strategy for loneliness-associated atherosclerosis progression. Show less

Movement of lipoprotein lipase (LPL) from myocytes or adipocytes to the capillary lumen is essential for intravascular lipolysis and plasma triglyceride homeostasis-low LPL activity in the capillary lumen causes hypertriglyceridemia. The trans-endothelial transport of LPL depends on ionic interactions with GPIHBP1's intrinsically disordered N-terminal tail, which harbors two acidic clusters at positions 5-12 and 19-30. This polyanionic tail provides a molecular switch that controls LPL detachment from heparan sulfate proteoglycans (HSPGs) by competitive displacement. When the acidic tail was neutralized in gene-edited mice, LPL remained trapped in the sub-endothelial spaces triggering hypertriglyceridemia. Due to its disordered state, the crystal structure of LPL•GPIHBP1 provided no information on these electrostatic interactions between LPL and GPIHBP1 acidic tail. In the current study, we positioned the acidic tail on LPL using zero-length crosslinking. Acidic residues at positions 19-30 in GPIHBP1 mapped to Lys Show less

The early detection of high-risk individuals is crucial to delay and reduce the incidence of type 2 diabetes. In this study, we aimed to explore the performance of a novel subgroup-specific biomarker strategy in the prediction of incident diabetes. In the Taiwan Lifestyle Cohort Study, adult subjects without diabetes were included and followed for the incidence of diabetes in 2006-2019. The biomarkers measured included blood secretogranin III (SCG3), vascular adhesion protein-1 (VAP-1), fibrinogen-like protein 1 (FGL1), angiopoietin-like protein 6 (ANGPTL6), and angiopoietin-like protein 4 (ANGPTL4). Among the 1,287 subjects, 12.2% developed diabetes during a 6 year follow-up. Blood VAP-1 was significantly associated with incident diabetes in the overall population (HR = 0.724, P < 0.05), participants under 65 years old (HR = 0.685, P < 0.05), those with a BMI of ≥24 kg/m Gender- and BMI-specific biomarker strategy can improve the prediction of incident diabetes. A subgroup-specific biomarker strategy is a novel approach in the prediction of incident diabetes. Show less

DHAV-3 is one of the main causative agents of duck viral hepatitis (DVH), an acute and highly lethal infectious disease in duck industry. However, the understanding of the pathogenesis of this virus in ducklings is limited. To dissect the molecular characteristics associated with pathobiology of ducklings to DHAV-3, we applied single-cell RNA-sequencing approach to profile the transcriptome of 1.4 million cells from 14 livers of DHAV-3 susceptible (S) and resistant (R) ducklings during viral infection and 4 uninfected healthy controls. We found that infected S ducks exhibited the activation of type I and II interferon pathways with elevated expression of interferon-stimulated genes (ISGs) compared to infected R ducks and healthy controls. DHAV-3 promoted proinflammatory phenotype and inhibited the cell apoptosis pathway of Kupffer cells of S ducks. Furthermore, we observed the elevated expression of host factor PLAC8 in S ducks and validated its ability to facilitate the infection of DHAV-3. We identified significant dysregulation of various genes in complement and coagulation cascades in hepatocytes2 exclusive to S ducks, together with over-secretion of ANGPTL4 from endothelial cells in S ducks which is confirmed to promote cellular migration, suggesting etiology of coagulopathic complications in ducks with severe DVH. Collectively, this study provides a rich resource for understanding the inflammatory immune signatures and cell communications underlying the pathogenesis of DHAV-3 infection, which may accelerate the development of better diagnostic methods and strategies for controlling this disease. Show less

ANGPTL3/4/8 (angiopoietin-like proteins 3, 4, and 8) are important regulators of LPL (lipoprotein lipase). ANGPTL8 forms complexes with ANGPTL3 and ANGPTL4. ANGPTL4/8 complex formation converts ANGPTL4 from a furin substrate to a plasmin substrate, and both cleavages generate similar C-terminal domain-containing (CD)-ANGPTL4 fragments. Whereas several studies have investigated associations of free ANGPTL proteins with cardiovascular risk, there are no data describing associations of the complexes and CD-ANGPTL4 with outcomes or describing the effects of the complexes on LPL bound to GPIHBP1 (glycosylphosphatidylinositol HDL-binding protein 1). Recombinant protein assays were used to study ANGPTL protein and complex effects on GPIHBP1-LPL activity. ANGPTL3/8, ANGPTL3, ANGPTL4/8, and CD-ANGPTL4 were measured with dedicated immunoassays in 2394 LURIC (Ludwigshafen Risk and Cardiovascular Health) study participants undergoing coronary angiography and 6188 getABI study (German Epidemiological Trial on Ankle Brachial Index) participants undergoing ankle brachial index measurement. There was a follow-up for cardiovascular death with a median (interquartile range) duration of 9.80 (8.75-10.40) years in the LURIC study and 7.06 (7.00-7.14) years in the getABI study. ANGPTL3/8 potently inhibited GPIHBP1-LPL activity and showed positive associations with LDL-C (low-density lipoprotein cholesterol) and triglycerides (both ANGPTL3/8 potently inhibited GPIHBP1-LPL enzymatic activity, consistent with its positive association with serum lipids. However, ANGPTL3/8, LDL-C, and triglyceride levels were not associated with cardiovascular death in the LURIC and getABI cohorts. In contrast, concentrations of ANGPTL4/8 and particularly CD-ANGPTL4 were positively associated with inflammation, the prevalence of diabetes, and cardiovascular mortality. Show less

Angiopoietin-like 4 (ANGPTL4), a key protein involved in lipoprotein metabolism, has diverse effects. There is an association between Angptl4 and diabetic kidney disease; however, this association has not been well investigated. We show that both podocyte- and tubule-specific ANGPTL4 are crucial fibrogenic molecules in diabetes. Diabetes accelerates the fibrogenic phenotype in control mice but not in ANGPTL4 mutant mice. The protective effect observed in ANGPTL4 mutant mice is correlated with a reduction in stimulator of interferon genes pathway activation, expression of pro-inflammatory cytokines, reduced epithelial-to-mesenchymal transition and endothelial-to-mesenchymal transition, lessened mitochondrial damage, and increased fatty acid oxidation. Mechanistically, we demonstrate that podocyte- or tubule-secreted Show less

Senescence is recognized as a principal risk factor for cardiovascular diseases, with a significant association between the senescence of cardiomyocytes and inferior cardiac function. Furthermore, type 2 diabetes exacerbates this aging process. Sodium-glucose co-transporter 2 inhibitor (SGLT2i) has well-established cardiovascular benefits and, in recent years, has been posited to possess anti-aging properties. However, there are no reported data on their improvement of cardiomyocytes function through the alleviation of aging. Consequently, our study aims to investigate the mechanism by which SGLT2i exerts anti-aging and protective effects at the cardiac level through its action on the FOXO1-ANGPTL4 pathway. To elucidate the underlying functions and mechanisms, we established both in vivo and in vitro disease models, utilizing mice with diabetic cardiomyopathy (DCM) induced by type 2 diabetes mellitus (T2DM) through high-fat diet combined with streptozotocin (STZ) administration, and AC16 human cardiomyocyte cell subjected to stimulation with high glucose (HG) and palmitic acid (PA). These models were employed to assess the changes in the senescence phenotype of cardiomyocytes and cardiac function following treatment with SGLT2i. Concurrently, we identified ANGPTL4, a key factor contributing to senescence in DCM, using RNA sequencing (RNA-seq) technology and bioinformatics methods. We further clarified ANGPTL4 role in promoting pathological aging of cardiomyocytes induced by hyperglycemia and hyperlipidemia through knockdown and overexpression of the factor, as well as analyzed the impact of SGLT2i intervention on ANGPTL4 expression. Additionally, we utilized chromatin immunoprecipitation followed by quantitative real-time PCR (ChIP-qPCR) to confirm that FOXO1 is essential for the transcriptional activation of ANGPTL4. The therapeutic intervention with SGLT2i alleviated the senescence phenotype in cardiomyocytes of the DCM mouse model constructed by high-fat feeding combined with STZ, as well as in the AC16 model stimulated by HG and PA, while also improving cardiac function in DCM mice. We observed that the knockdown of ANGPTL4, a key senescence-promoting factor in DCM identified through RNA-seq technology and bioinformatics, mitigated the senescence of cardiomyocytes, whereas overexpression of ANGPTL4 exacerbated it. Moreover, SGLT2i improved the senescence phenotype by suppressing the overexpression of ANGPTL4. In fact, we discovered that SGLT2i exert their effects by regulating the upstream transcription factor FOXO1 of ANGPTL4. Under conditions of hyperglycemia and hyperlipidemia, compared to the control group without FOXO1, the overexpression of FOXO1 in conjunction with SGLT2i intervention significantly reduced both ANGPTL4 mRNA and protein levels. This suggests that the FOXO1-ANGPTL4 axis may be a potential target for the cardioprotective effects of SGLT2i. Collectively, our study demonstrates that SGLT2i ameliorate the pathological aging of cardiomyocytes induced by a high glucose and high fat metabolic milieu by regulating the interaction between FOXO1 and ANGPTL4, thereby suppressing the transcriptional synthesis of the latter, and consequently restoring cardiac function. Show less

Angiopoietin-like protein 8 (Angptl8), expressed in the liver and adipocytes, forms a complex with Angptl3 or Angptl4, which regulates lipoprotein lipase and triglyceride metabolism. However, the precise functions of adipocyte Angptl8 remain elusive. Here we report that adipocyte-specific inducible Angptl8-knockout (AT-A8-KO) male mice on normal diet showed minor phenotypic changes, but after a high-fat high fructose (HFHF) diet, exhibited decreased body weight gain and glycemia, elevated rectal temperature and early dark phase energy expenditure compared to the Cre controls. AT-A8-KO mice also displayed improved glucose tolerance, a trend for better insulin sensitivity, improved insulin-stimulated glucose uptake in adipose tissues, and reduced visceral adipose tissue crown-like structures, plasma MCP-1 and leptin levels. The results indicate the importance of adipose Angptl8 in the context of nutri-stress and obesity, as its deletion in mice promotes a metabolically healthy obese phenotype by slightly ameliorating obesity, improving glucose and energy homeostasis, and mitigating inflammation. Show less

Glycolysis plays a major role in progression of idiopathic pulmonary fibrosis (IPF). Here, we aim to explore the predictive signature based on glycolysis-related genes for predicting the prognosis and identified a potential therapeutic target for IPF. Gene expression data of bronchoalveolar lavage (BAL) cells and clinical information were downloaded from the Gene Expression Omnibus database. Bioinformatic analysis was then performed to identify differentially expressed genes (DEGs). Lasso multivariate cox analysis and multivariate Cox regression were used to establish a gene signature. The prediction model was evaluated using the time-dependent receiver operating characteristic (ROC) curve and validated using an external independent dataset. The expression of these key genes in cellular level analyzed from Single Cell Expression Atlas. Cell Counting Kit-8 assay, immunofluorescence, wound healing and plasmid transfection were performed. A total of 4 gene (ANGPTL4, ME2, TPBG and IER3), which were associated with the prognosis of IPF patients, were selected to establish our signature. The prediction model was an independent prognostic indicator for IPF patients. ANGPTL4 was significantly upregulated in pleural mesothelial cells (PMCs). In vitro assay showed that ANGPTL4 promoted PMCs proliferation and migration. Knockdown of ANGPTL4 can inhibit mesothelial-mesenchymal transition by suppressed glycolysis-associated gene, such as PGM1, GPI, PGK1, LDHA, ALDOA, ENO1 and TPI1. Our research established a glycolysis-associated gene signature that holds potential to assist clinicians in the personalized management of IPF. Furthermore, we identified that ANGPTL4 mediates mesothelial-mesenchymal transition, suggesting its viability as a therapeutic target for IPF treatment. Show less

The most common cause of death in patients with peripheral artery disease (PAD) are major adverse cardiovascular events (MACEs), including myocardial infarction (MI) and stroke. However, data on biomarkers that could be used to help predict MACEs in patients with PAD to guide clinical decision making is limited. Angiogenesis-related proteins have been demonstrated to play an important role in systemic atherosclerosis and may act as prognostic biomarkers for MACEs in patients with PAD. In this study, we evaluated a large panel of angiogenesis-related proteins and identified specific biomarkers associated with MACEs in patients with PAD. We conducted a prognostic study using a prospectively recruited cohort of 406 patients (254 with PAD and 152 without PAD). Plasma concentrations of 22 circulating angiogenesis-related proteins were measured at baseline, and the cohort was followed for 2 years. The primary outcome was 2-year MACEs (composite of MI, stroke, or death). Plasma protein concentrations were compared between PAD patients with and without 2-year MACEs using Mann-Whitney U tests. Differentially expressed proteins were further investigated in terms of their prognostic potential. Specifically, Cox proportional hazards analysis was performed to determine the independent association between differentially expressed proteins and 2-year MACEs, controlling for all baseline demographic and clinical characteristics, including existing coronary artery disease and cerebrovascular disease. Kaplan-Meier analysis was conducted to assess 2-year freedom from MACEs in patients with low vs. high levels of the differentially expressed proteins based on median plasma concentrations. The mean age of the cohort was 68.8 (SD 11.1), and 134 (33%) patients were female. Two-year MACEs occurred in 63 (16%) individuals. The following proteins were significantly elevated in PAD patients with 2-year MACEs compared to those without 2-year MACEs: endostatin (69.15 [SD 58.15] vs. 51.34 [SD 29.07] pg/mL, Among a panel of 22 angiogenesis-related proteins, endostatin, ANGPTL4, and ANGPTL3 were identified to be independently and specifically associated with 2-year MACEs in patients with PAD. Measurement of plasma concentrations of these proteins can support MACE risk stratification in patients with PAD, thereby informing clinical decisions on multidisciplinary referrals to cardiologists, neurologists, and vascular medicine specialists and guiding aggressiveness of medical treatment, thereby improving cardiovascular outcomes in patients with PAD. Show less

Fetal growth restriction (FGR) is a common complication of pregnancy, which seriously endangers fetal health and still lacks effective therapeutic targets. Clostridium difficile (C. difficile) is associated with fetal birth weight, and its membrane vesicles (MVs) are pathogenic vectors. However, the role of C. difficile and its MVs in FGR remains unclear. Here we found that supplementation with C. difficile altered the characteristics of gut microbiota and reduced the birth weight in mice. Interestingly, C. difficile MVs entered placenta, inhibited trophoblast motility, and induced fetal weight loss in mice. Mechanistically, C. difficile MVs activated the PPAR pathway via enhancing the transcriptional activity of PPARγ promoter, consequently inhibiting trophoblast motility. Moreover, PPARγ expression was significantly elevated in FGR placenta, and negatively correlated with fetal birth weight. Together, our findings reveal the significance of C. difficile and its MVs in FGR, providing new insights into the mechanisms of FGR development. Show less

To elucidate the unidentified roles of a selective peroxisome proliferator-activated receptor α (PPARα) agonist, pemafibrate (Pema), on the pathogenesis of retinal ischemic diseases (RID)s, the pharmacological effects of Pema on the retinal pigment epithelium (RPE), which is involved in the pathogenesis of RID, were compared with the pharmacological effects of the non-fibrate PPARα agonist GW7647 (GW). For this purpose, the human RPE cell line ARPE19 that was untreated (NT) or treated with Pema or GW was subjected to Seahorse cellular metabolic analysis and RNA sequencing analysis. Real-time cellular metabolic function analysis revealed that pharmacological effects of the PPARα agonist actions on essential metabolic functions in RPE cells were substantially different between Pema-treated cells and GW-treated cells. RNA sequencing analysis revealed the following differentially expressed genes (DEGs): (1) NT vs. Pema-treated cells, 37 substantially upregulated and 72 substantially downregulated DEGs; (2) NT vs. GW-treated cells, 32 substantially upregulated and 54 substantially downregulated DEGs; and (3) Pema vs. GW, 67 substantially upregulated and 51 markedly downregulated DEGs. Gene ontology (GO) analysis and ingenuity pathway analysis (IPA) showed several overlaps or differences in biological functions and pathways estimated by the DEGs between NT and Pema-treated cells and between NT and GW-treated cells, presumably due to common PPARα agonist actions or unspecific off-target effects to each. For further estimation, overlaps of DEGs among different pairs of comparisons (NT vs. Pema, NT vs. GW, and Pema vs. GW) were listed up. Angiopoietin-like 4 (ANGPTL4), which has been shown to cause deterioration of RID, was the only DEG identified as a common significantly upregulated DEG in all three pairs of comparisons, suggesting that ANGPTL4 was upregulated by the PPARα agonist action but that its levels were substantially lower in Pema-treated cells than in GW-treated cells. In qPCR analysis, such lower efficacy for upregulation of the mRNA expression of ANGPTL4 by Pema than by GW was confirmed, in addition to substantial upregulation of the mRNA expression of HIF1α by both agonists. However, different Pema and GW-induced effects on mRNA expression of HIF1α (Pema, no change; GW, significantly downregulated) and mRNA expression of ANGPTL4 (Pema, significantly upregulated; GW, significantly downregulated) were observed in HepG2 cells, a human hepatocyte cell line. The results of this study suggest that actions of the PPARα agonists Pema and GW are significantly organ-specific and that lower upregulation of mRNA expression of the DR-worsening factor ANGPTL4 by Pema than by GW in ARPE19 cells may minimize the risk for development of RID. Show less

Retinopathy is a common complication of diabetes mellitus and the leading cause of visual impairment. Danggui Buxue decoction (RRP) has been used as a traditional drug for the treatment of diabetic nephropathy for many years. The aim of this study was to investigate the effects of RRP on hypoxia-induced retinal Müller cell injury. A model of retinal Müller cell damage was created using high glucose levels (25 mmol/L) and/or exposure to low oxygen conditions (1% O2). RRP was given to rats by continuous gavage for 7 days to obtain drug-containing serum. After sterilization, the serum was added to the culture medium at a ratio of 10%. Cell viability, apoptosis, and cell proliferation were assessed using the CCK-8 kit, Annexin V-FITC/propidium iodide apoptosis kit, and EdU kit. The mRNA levels of angiogenesis factors (ANGPTL4, VEGF) and inflammatory factors (IL-1B, ICAM-1) were detected by RT-qPCR. Western blot analysis was employed to assess the levels of proteins related to the ATF4/CHOP pathway. Following hypoxia for 48 h and 72 h, there was a significant decrease in cell viability and proliferation, as well as a notable increase in apoptosis compared to the control group (21% O2). However, high glucose stimulation had no significant effect, and high glucose combined with hypoxia had no further damage to cells. After 48 h of exposure to low oxygen levels, the mRNA expression levels of ANGPTL4, VEGF, IL-1B, and ICAM-1 in retinal Müller cells were significantly higher than in the control group (21% O2). RRP treatment significantly alleviated the increase of cell apoptosis and the upregulation of IL-1B and-1 in retinal Müller cells induced by hypoxia. RRP has the potential to reduce the suppression of the ATF4/CHOP pathway in hypoxia-induced retinal Müller cells, and it significantly alleviates cell apoptosis through regulating inflammatory factors and the ATF4/CHOP pathway. Show less

The global burden of renal diseases is increasingly severe, underscoring the need for in-depth exploration of the molecular mechanisms underlying renal disease progression and the development of potential novel biomarkers or therapeutic targets. Angiopoietin-like protein 4 (ANGPTL4) is a multifunctional cytokine involved in the regulation of key biological processes, such as glucose and lipid metabolism, inflammation, vascular permeability, and angiogenesis, all of which play crucial roles in the pathogenesis of kidney diseases. Over the past 2 decades, ANGPTL4 has been regarded as playing a pivotal role in the progression of various kidney diseases, prompting significant interest from the scientific community regarding its potential clinical utility in renal disorders. This review synthesizes the available literature, provides a concise overview of the molecular biological effects of ANGPTL4, and highlights its relationship with multiple renal diseases and recent research advancements. These findings underscore the important gaps that warrant further investigation to develop novel targets for the prediction or treatment of various renal diseases. Show less

This study investigates the impact of perioperative tourniquet on skeletal muscle cells during total knee arthroplasty (TKA) and its effects on the gene expression of apoptotic, inflammatory, and angiogenic pathways. The randomized controlled trial included 44 patients undergoing TKA. The patients were randomized to undergo surgery with (n = 23) or without (n = 21) tourniquet. The tourniquet was inflated before skin incision and deflated before wound closure in the tourniquet group. Biopsies from the lateral vastus muscle were obtained from both groups before wound closure and 8 weeks after surgery. The messenger ribonucleic acid (mRNA) expression and protein levels of angiopoietin-like 4 (ANGPTL4), Hypoxia-inducible Factor 1α, and Vascular Endothelial Growth Factor Alpha (VEGF-A) in the biopsies were examined by reverse transcription-quantitative polymerase chain reaction and tissue microarray, respectively. Differences in mean values (ΔC Show less

This study aimed to investigate the possible mechanisms by which ANGPTL4 is involved in the pathogenesis of choroidal neovascularization (CNV) and subretinal fibrosis. Differentially expressed genes in retinal pigmented epithelium (RPE)-choroid-sclera complex tissues from nAMD patients and control individuals were identified via the GEO database, followed by GO and KEGG analyses. A Venn diagram was used to identify EndMT-related DEGs. A logistic regression model was constructed to screen for prognostic genes. Laser-induced CNV mouse models were established and validated with FFA and OCTA. The expression of ANGPTL4 and EndMT-related markers in the RPE-choroid-sclera complex was measured via RT‒qPCR and Western blotting. TGF-β2-induced HUVECs were used as EndMT cell models, and specific siRNAs targeting ANGPTL4 (si-ANGPTL4) were designed and screened. The effects of ANGPTL4 knockdown on the migration and invasion of HUVECs were also examined. Laser-induced CNV mouse models were constructed, and an intravitreal injection of cholesterol-modified si-ANGPTL4 was used to knock down ANGPTL4. FFA, OCTA and immunofluorescence staining were used to observe CNV formation and subretinal fibrosis, and the expression of ANGPTL4 and EndMT-related markers was determined. ANGPTL4 expression was significantly increased in mice with CNV and colocalized with IB4. In TGF-β2-induced EndMT, ANGPTL4 was also upregulated, and its knockdown led to the inhibition of EndMT and cell migration and invasion, while its overexpression promoted the EndMT process. ANGPTL4 knockdown reduced the formation of CNV and subretinal fibrosis in mice with CNV by suppressing EndMT. ANGPTL4 may promote CNV and subretinal fibrosis through EndMT, suggesting that ANGPTL4 may be a novel potential target for nAMD therapy. Show less

Apolipoprotein E (APOE) ε4 allele is the strongest genetic risk factor for late onset Alzheimer's disease (AD). This case-cohort study used targeted plasma biomarkers and large-scale proteomics to examine the biological mechanisms that allow some APOEε4 carriers to maintain normal cognitive functioning in older adulthood. APOEε4 carriers and APOEε3 homozygotes enrolled in the Women's Health Initiative Memory Study (WHIMS) from 1996 to 1999 were classified as resilient if they remained cognitively unimpaired beyond age 80, and as non-resilient if they developed cognitive impairment before or at age 80. AD pathology (Aß A total of 1610 participants were included (baseline age: 71.3 [3.8 SD] years; all White; 42% APOEε4 carriers). Compared to resilient APOEε4 carriers, non-resilient APOEε4 carriers had lower Aß We identified and replicated a plasma proteomic signature associated with cognitive resilience among APOEε4 carriers. These proteins implicate specific immune processes in the preservation of cognitive status despite elevated genetic risk for AD. Future studies in diverse cohorts will be needed to assess the generalizability of these results. Show less

Increasing studies have shown degeneration of nucleus pulposus cells (NPCs) as an critical part of the progression of intervertebral disc degeneration (IVDD). However, there are relatively few studies on single-cell transcriptome contrasts in human degenerated NPCs. Moreover, differences in Wnt/Ca We performed bioinformatics analysis using our previously published findings to construct single cell expression profiles of normal and degenerated nucleus pulposus. Then, in-depth differential analysis was used to characterize the expression of Wnt/Ca The obtained cell data were clustered into five different chondrocytes clusters, which chondrocyte 4 and chondrocyte 5 mainly accounted for a high proportion in degenerated nucleus pulposus tissues, but rarely in normal nucleus pulposus tissues. Genes associated within the Wnt/Ca Single-cell RNA sequencing revealed differential expression of Wnt/Ca Show less