[Effect of salvia miltiorrhiza combined with roxadustat on wound healing of full-thickness skin defects in diabetic rats and its mechanism].

Objective: To explore the effect of salvia miltiorrhiza combined with roxadustat on wound healing of full-thickness skin defects in diabetic rats and its mechanism. Methods: This study was an experimental study. Twenty male 8-week-old Sprague-Dawley rats were used to successfully establish diabetic model, then full-thickness skin defect wounds on their backs were made. The rats were divided into normal saline group, roxadustat alone group, salvia miltiorrhiza alone group, and roxadustat+salvia miltiorrhiza group according to the random number table, with 5 rats in each group. Immediately after injury, the rats in normal saline group were given 5 mL normal saline by gavage, the rats in roxadustat alone group were given 1.5 mg/mL roxadustat suspension by gavage at 25 mg/kg, the rats in salvia miltiorrhiza alone group were given 18 mg/mL salvia miltiorrhiza suspension by gavage at 300 mg/kg, and the rats in roxadustat+salvia miltiorrhiza group were given 19.5 mg/mL roxadustat and salvia miltiorrhiza suspension at roxadustat 25 mg/kg and salvia miltiorrhiza 300 mg/kg. All were administered once a day for 2 weeks. The wounds at 0 (immediately), 4, 8, and 12 d after injury were observed, and the wound healing rates at 4, 8, and 12 d after injury were calculated (n=5). At 14 d after injury, abdominal aortic blood was collected, and hemoglobin, red cell count, and white blood cell count were detected (n=5). The wound tissue was collected for hematoxylin-eosin staining to observe inflammatory infiltration, skin tissue structure, and neovascularization, for Masson staining to observe the proportion of collagen fiber (n=3), for Western blotting to detect the protein expression levels of vascular endothelial growth factor (VEGF), CD31, interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and IL-1ß (n=3), and for immunohistochemical staining to determine the protein expression levels of epidermal growth factor receptor (EGFR), hypoxia-inducible factor 1α (HIF-1α), and proliferating cell nuclear antigen (PCNA), with sample number of 3. Results: From 0 to 12 d after injury, the wound areas of rats in 4 groups were gradually decreased. At 4 d after injury, the wound healing rates of rats in salvia miltiorrhiza alone group and roxadustat+salvia miltiorrhiza group were significantly higher than those in normal saline group and roxadustat alone group (P<0.05). At 8 d after injury, the wound healing rates of rats in roxadustat alone group and salvia miltiorrhiza alone group were significantly higher than the rate in normal saline group (P<0.05), and the wound healing rate of rats in roxadustat+salvia miltiorrhiza group was significantly higher than the rates in the other 3 groups (with P values all <0.05). At 12 d after injury, the wound healing rates of rats in roxadustat alone group, salvia miltiorrhiza alone group, and roxadustat+salvia miltiorrhiza group were significantly higher than the rate in normal saline group (P<0.05). At 14 d after injury, there were no statistically significant differences in the hemoglobin or red blood cell count of rats in 4 groups (P<0.05). The white blood cell count of rats in roxadustat alone group, salvia miltiorrhiza alone group, and roxadustat+salvia miltiorrhiza group were respectively (24.3±1.2)×109/L, (26.3±2.4)×109/L, and (15.0±0.7)×109/L, which were significantly lower than (33.8±2.7)×109/L in normal saline group (P<0.05); the white blood cell count of rats in roxadustat+salvia miltiorrhiza group was significantly lower than that in roxadustat alone group and salvia miltiorrhiza alone group (with P values both <0.05). At 14 d after injury, a large number of inflammatory cell infiltration, disordered skin tissue structure, and few new blood vessels were observed in the wounds of rats in normal saline group; while a small amount of inflammatory cell infiltration, tight skin tissue structure, and rich neovascularization were observed in the wounds of rats in the other 3 groups. There were no statistically significant differences in the proportion of collagen fiber of wounds in rats among the 4 groups (P>0.05). At 14 d after injury, the protein expression levels of VEGF and CD31 in the wound tissue of rats in roxadustat alone group, salvia miltiorrhiza alone group, and roxadustat+salvia miltiorrhiza group were significantly higher than those in normal saline group (P<0.05), the protein expression level of CD31 in the wound tissue of rats in roxadustat+salvia miltiorrhiza group was significantly higher than the levels in roxadustat alone group and salvia miltiorrhiza alone group (with P values both <0.05). At 14 d after injury, the protein expression levels of IL-6, TNF-α, and IL-1ß in the wound tissue of rats in roxadustat alone group, salvia miltiorrhiza alone group, and roxadustat+salvia miltiorrhiza group were significantly lower than those in normal saline group (P<0.05); the protein expression levels of IL-6 and IL-1ß in the wound tissue of rats in roxadustat+salvia miltiorrhiza group were significantly lower than those in roxadustat alone group and salvia miltiorrhiza alone group (P<0.05); the protein expression level of TNF-α in the wound tissue of rats in roxadustat+salvia miltiorrhiza group was significantly lower than that in salvia miltiorrhiza alone group (P<0.05). At 14 d after injury, the protein expression level of EGFR in the wound tissue of rats in roxadustat+salvia miltiorrhiza group was significantly higher than the levels in the other 3 groups (with P values all <0.05); the protein expression levels of HIF-1α in the wound tissue of rats in roxadustat alone group and roxadustat+salvia miltiorrhiza group were significantly higher than the level in normal saline group (P<0.05), and the protein expression level of HIF-1α in the wound tissue of rats in roxadustat+salvia miltiorrhiza group was significantly higher than that in salvia miltiorrhiza alone group (P<0.05); there were no statistically significant differences in the protein expression level of PCNA in the wound tissue of rats in 4 groups (P>0.05). Conclusions: Roxadustat combined with salvia miltiorrhiza can promote the wound healing of full-thickness skin defects in diabetic rats by promoting blood vessel regeneration and reducing inflammatory response.

TMEM9 promotes lung adenocarcinoma progression via activating the MEK/ERK/STAT3 pathway to induce VEGF expression.

Abnormal Transmembrane protein 9 (TMEM9) expression has been identified in various human tumors. However, the prognostic potential and mechanistic role of TMEM9 in lung adenocarcinoma (LUAD) remain unclear. Here, we first found a significant upregulation of TMEM9 in LUAD tissues, and TMEM9 expression was positively correlated with microvessel density (MVD), T stage, and clinical stage. Survival analysis demonstrated TMEM9 was an independent indicator of poor prognosis in LUAD patients. In addition, downregulation of TMEM9 suppressed tumor growth and metastasis in vitro and in vivo models, and reduced HUVEC proliferation, migration, and tube formation in a cancer cell/HUVEC coculture model. Furthermore, TMEM9 upregulated VEGF expression, and VEGF-neutralizing antibodies reversed HUVEC angiogenesis and cancer cell migration ability caused by overexpression of TMEM9. In contrast, recombinant VEGF (rVEGF) abolished the inhibitory effect of TMEM9-knockdown LUAD cells on HUVEC angiogenesis and tumor cell migration. Moreover, we showed that TMEM9 upregulated VEGF expression by activating the mitogen-activated protein kinase/extracellular signal-regulated kinase/STAT3 (MEK/ERK/STAT3) pathway. Together, our study provides mechanistic insights into the role of TMEM9 in LUAD and highlights the potential of targeting the TMEM9/MEK/ERK/STAT3/VEGF pathway as a novel therapy for preventing LUAD progression.

Single-cell RNA sequencing reveals cellular and molecular landscape of fetal cystic hygroma.

BACKGROUND: The molecular mechanism of fetal cystic hygroma (CH) is still unclear, and no study has previously reported the transcriptome changes of single cells in CH. In this study, single-cell transcriptome sequencing (scRNA-seq) was used to investigate the characteristics of cell subsets in the lesion tissues of CH patients. METHODS: Lymphoid tissue collected from CH patients and control donors for scRNA-seq analysis. Differentially expressed gene enrichment in major cell subpopulations as well as cell-cell communication were analyzed. At the same time, the expression and interactions of important VEGF signaling pathway molecules were analyzed, and potential transcription factors that could bind to KDR (VEGFR2) were predicted. RESULTS: The results of scRNA-seq showed that fibroblasts accounted for the largest proportion in the lymphatic lesions of CH patients. There was a significant increase in the proportion of lymphatic endothelial cell subsets between the cases and controls. The VEGF signaling pathway is enriched in lymphatic endothelial cells and participates in the regulation of cell-cell communication between lymphatic endothelial cells and other cells. The key regulatory gene KDR in the VEGF signaling pathway is highly expressed in CH patients and interacts with other differentially expressed EDN1, TAGLN, and CLDN5 Finally, we found that STAT1 could bind to the KDR promoter region, which may play an important role in promoting KDR up-regulation. CONCLUSION: Our comprehensive delineation of the cellular composition in tumor tissues of CH patients using single-cell RNA-sequencing identified the enrichment of lymphatic endothelial cells in CH and highlighted the activation of the VEGF signaling pathway in lymphoid endothelial cells as a potential modulator. The molecular and cellular pathogenesis of fetal cystic hygroma (CH) remains largely unknown. This study examined the distribution and gene expression signature of each cell subpopulation and the possible role of VEGF signaling in lymphatic endothelial cells in regulating the progression of CH by single-cell transcriptome sequencing. The enrichment of lymphatic endothelial cells in CH and the activation of the VEGF signaling pathway in lymphatic endothelial cells provide some clues to the pathogenesis of CH from the perspective of cell subpopulations.

Interplay of precision therapeutics and MD study: Calocybe indica's potentials against cervical cancer and its interaction with VEGF via octadecanoic acid.

The evolving landscape of personalized medicine necessitates a shift from traditional therapeutic interventions towards precision-driven approaches. Embracing this paradigm, our research probes the therapeutic efficacy of the aqueous crude extract (ACE) of Calocybe indica in cervical cancer treatment, merging botanical insights with advanced molecular research. We observed that ACE exerts significant influences on nuclear morphology and cell cycle modulation, further inducing early apoptosis and showcasing prebiotic attributes. Characterization of ACE have identified several phytochemicals including significant presence of octadeconoic acid. Simultaneously, utilizing advanced Molecular Dynamics (MD) simulations, we deciphered the intricate molecular interactions between Vascular Endothelial Growth Factor (VEGF) and Octadecanoic acid to establish C.indica's role as an anticancer agent. Our study delineates Octadecanoic acid's potential as a robust binding partner for VEGF, with comprehensive analyses from RMSD and RMSF profiles highlighting the stability and adaptability of the protein-ligand interactions. Further in-depth thermodynamic explorations via MM-GBSA calculations reveal the binding landscape of the VEGF-Octadecanoic acid complex. Emerging therapeutic innovations, encompassing proteolysis-targeting chimeras (PROTACs) and avant-garde nanocarriers, are discussed in the context of their synergy with compounds like Calocybe indica P&C. This convergence underscores the profound therapeutic potential awaiting clinical exploration. This study offers a holistic perspective on the promising therapeutic avenues facilitated by C. indica against cervical cancer, intricately woven with advanced molecular interactions and the prospective integration of precision therapeutics in modern oncology.

Short Term Effect of Pre-Operative Anti-VEGF on Angiogenic and Fibrotic Profile of Fibrovascular Membranes of Proliferative Diabetic Retinopathy.

Purpose: Adjuvant, pre-operative intravitreal anti-vascular endothelial growth factor (anti-VEGF) injections have been used to reduce peri-operative bleeding in eyes undergoing pars-plana vitrectomy for complications of proliferative diabetic retinopathy (PDR). To address the concern over their potential off-target effects of progressive fibrous contraction, we sought to dissect the transcriptional changes in the surgically extracted fibrovascular membranes (FVMs). Methods: We analyzed surgically extracted FVMs from 10 eyes: 4 eyes pretreated with intravitreal bevacizumab (IVB) and 6 untreated eyes. FVMs were digested into single cells, mRNA was extracted from endothelial cell-enriched (microbead selection with CD31) and non-endothelial cell compartments, followed by RT-qPCR quantification. We then compared the relative expression of genes involved in angiogenesis, endothelial cell integrity, and myofibroblastic processes between treated and untreated FVMs. Results: Endothelial cells from IVB pretreated FVMs showed significant reduction of VEGFA, VEGF receptors (FLT1 and KDR), and angiopoietin 2 expression as well as increased vascular endothelial cadherin and endothelin, suggesting reduced angiogenesis and enhanced vascular integrity. The non-endothelial cell fraction showed decreased expression of VEGFA and fibronectin, without significant difference in the expression of other profibrotic factors. Conclusions: Our findings confirm that adjuvant pre-operative IVB decreased fibronectin and increase endothelin-1 expression without affecting other profibrotic gene expression, uncovering an important interaction between IVB and endothelin-1 that deserves further study.

[Mechanism of ultrafiltration extract of Angelicae Sinensis Radix and Hedysari Radix regulates HIF-1α signaling pathway mediated by renal hypoxia to ameliorate renal fibrosis in DKD rats].

This study explored the mechanism of the ultrafiltration extract of Angelicae Sinensis Radix and Hedysari Radix in ameliorating renal fibrosis in the rat model of diabetic kidney disease(DKD) based on the expression of hypoxia-inducible factor-1α(HIF-1α)/vascular endothelial growth factor(VEGF) and HIF-1α/platelet-derived growth factor(PDGF)/platelet-derived growth factor receptor(PDGFR) signaling pathways in the DKD rats. After 1 week of adaptive feeding, 50 male SPF-grade Wistar rats were randomized into a blank group(n=7) and a modeling group. After 24 h of fasting, the rats in the modeling group were subjected to intraperitoneal injection of streptozocin and fed with a high-sugar and high-fat diet to establish a DKD model. After modeling, the rats were randomly assigned into model(n=7), low-dose ultrafiltration extract(n=7), medium-dose ultrafiltration extract(n=7), irbesartan(n=8), and high-dose ultrafiltration extract(n=8) groups. After intervention by corresponding drugs for 12 weeks, the general conditions of the rats were observed. The body weights and blood glucose levels of the rats were measured weekly, and the 24 h urinary protein(24hUP) was measured at the 6th and 12th weeks of drug administration. After the last drug administration, the renal function indicators were determined. Masson staining was employed to observe the pathological changes of the renal tissue. The expression of prolyl hydroxylase domain 2(PHD2) and HIF-1α in the renal tissue was detected by immunohistochemistry(IHC). Real-time qPCR was employed to determine the mRNA levels of PHD2, VEGF, PDGF, and PDGFR in the renal tissue. Western blot was employed to determine the protein levels of HIF-1α, VEGF, PDGF, and PDGFR in the renal tissue. The results showed that compared with the model group, drug administration lowered the levels of glycosylated serum protein(GSP), aerum creatinine(Scr), and blood urea nitrogen(BUN) in a dose-dependent manner(P<0.05 or P<0.01) and mitigated the pathological changes in the renal tissue. Furthermore, drug administration up-regulated mRNA level of PHD2(P<0.05 or P<0.01), down-regulated the mRNA levels of VEGF, PDGF, and PDGFR(P<0.05 or P<0.01) and the protein levels of HIF-1α, VEGF, PDGF, and PDGFR(P<0.01) in the renal tissue, and increased the rate of PHD2-positive cells(P<0.01). In conclusion, the ultrafiltration extract of Angelicae Sinensis Radix and Hedysari Radix effectively alleviated the renal fibrosis in DKD rats by inhibiting the expression of key proteins in the HIF-1α signaling pathway mediated by renal hypoxia and reducing extracellular matrix(ECM) deposition.

DFMG decreases angiogenesis to uphold plaque stability by inhibiting the TLR4/VEGF pathway in mice.

The aim of this study was to elucidate the specific mechanism through which 7-difluoromethoxy-5,4'-dimethoxygenistein (DFMG) inhibits angiogenesis in atherosclerosis (AS) plaques, given its previously observed but poorly understood inhibitory effects. In vitro, a model using Human Umbilical Vein Endothelial (HUVEC-12) cells simulated the initial lesion in the atherosclerotic pathological process, specifically oxidative stress injury, by exposing cells to 30 µmol/L LPC. Additionally, an AS mouse model was developed in ApoE knockout mice through a 16-week period of high-fat feeding. DFMG demonstrated a reduction in tubule quantities in the tube formation assay and neovascularization induced by oxidative stress-damaged endothelial cells in the chicken embryo chorioallantoic membrane assay. Furthermore, DFMG decreased lipid levels in the blood of ApoE knockout mice with AS, along with a decrease in atherosclerotic plaques and neovascularizations in the aortic arch and descending aorta of AS animal models. DFMG treatment upregulated microRNA140 (miR-140) expression and suppressed VEGF secretion in HUVEC-12 cells. These effects were counteracted by Toll-like receptor 4 (TLR4) overexpression in HUVEC-12 cells subjected to oxidative injury or in a mouse model of AS. Dual-luciferase reporter assays demonstrated that miR-140 directly targeted TLR4. Immunohistochemical assay findings indicated a significant inverse relationship between miR-140 expression and TLR4 expression in ApoE knockout mice subjected to a high-fat diet. The study observed a close association between DFMG inhibitory effects on angiogenesis and plaque stability in AS, and the inhibition of the TLR4/NF-κB/VEGF signaling pathway, negatively regulated by miR-140.

Differential expression and clinical significance of IGF2BP3 in peritoneal dialysate of patients with varying duration of peritoneal dialysis.

This study aims to investigate the differential expression of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) in the peritoneal dialysate among patients with different durations of peritoneal dialysis and its association with the angiogenic marker vascular* endothelial growth factor (VEGF), the fibronectin (FN), and various clinical indicators. A cohort of 122 peritoneal dialysis patients was categorized into short-term (≤1 year, n = 33), mid-term (>1 and ≤5 years, n = 55), and long-term (>5 years, n = 34) groups based on dialysis duration. We utilized enzyme-linked immunosorbent assay (ELISA) and western blot assays to quantify the levels of IGF2BP3, VEGF, and FN in the dialysate. Our findings showed a progressive increase in IGF2BP3 levels with the duration of PD, with the long-term group exhibiting significantly higher levels than both the short-term and mid-term groups (p < 0.001). A positive correlation between IGF2BP3 and VEGF (r = 0.386, p = 0.013), as well as between IGF2BP3 and FN (r = 0.340, p = 0.030), was observed. IGF2BP3 levels also correlated positively with serum creatinine, calcium, and phosphorus levels. In vitro analysis further confirmed that IGF2BP3 expression is enhanced in human peritoneal mesothelial cells under high-glucose conditions (p < 0.05). The study highlights the potential of IGF2BP3 in PD effluent as a biomarker for monitoring PF progression, with its expression significantly correlated with the duration of PD (Pearson r = 0.897, p < 0.001). In conclusion, our results underscore a correlation between elevated IGF2BP3 levels and PD duration, suggesting the clinical significance of IGF2BP3 as a biomarker for PF progression.

Facilitation of neurological recovery in a complete spinal cord injury with NeuroAiD: case report.

INTRODUCTION: NeuroAiD (MLC601 & MLC901)'s neuroprotective capabilities include limiting exaggerated calcium influx, decreasing excitotoxicity, reducing oxidative stress, and preventing glutamate-induced cell death. It has also been shown to facilitate synaptogenesis, neurogenesis, and neuroplasticity. However, its clinical efficacy has primarily been studied in the context of brain injuries, particularly stroke. NeuroAiD's potential application in SCI remains largely untapped. CASE PRESENTATION: A 34-year-old male presented with C4 complete tetraplegia. Following surgical decompression and initial inpatient rehabilitation, he started consuming MLC901 two capsules three times daily at month 4 post injury for 6 months. He regained considerable neurological recovery following the supplementation. Apart from the improvement in the neurological level of injury, the patient exhibited motor recovery beyond the initial zone of partial preservation up to 24 months post injury. DISCUSSION: Our findings align with a recent animal study demonstrating MLC901's potential to downregulate Vascular Endothelial Growth Factor (VEGF), a molecule known to increase vascular permeability and exacerbate tissue edema and infarction. In another animal study involving stroke-affected mice, MLC901 demonstrates the ability to promote neurological recovery by regulating the expression of proteins mediating angiogenesis, such as hypoxic inducible factor 1α, erythropoietin, angiopoietins 1 and 2, as well as VEGF. The anecdotal findings from this case report offer preliminary insights into NeuroAiD's potential in facilitating recovery during post-acute and chronic phases of severe SCI, necessitating further exploration.

Exploring the Impact of Saccharin on Neovascular Age-Related Macular Degeneration: A Comprehensive Study in Patients and Mice.

Purpose: We aimed to determine the impact of artificial sweeteners (AS), especially saccharin, on the progression and treatment efficacy of patients with neovascular age-related macular degeneration (nAMD) under anti-vascular endothelial growth factor (anti-VEGF-A) treatment. Methods: In a cross-sectional study involving 46 patients with nAMD undergoing intravitreal anti-VEGF therapy, 6 AS metabolites were detected in peripheral blood using liquid chromatography - tandem mass spectrometry (LC-MS/MS). Disease features were statistically tested against these metabolite levels. Additionally, a murine choroidal neovascularization (CNV) model, induced by laser, was used to evaluate the effects of orally administered saccharin, assessing both imaging outcomes and gene expression patterns. Polymerase chain reaction (PCR) methods were used to evaluate functional expression of sweet taste receptors in a retinal pigment epithelium (RPE) cell line. Results: Saccharin levels in blood were significantly higher in patients with well-controlled CNV activity (P = 0.004) and those without subretinal hyper-reflective material (P = 0.015). In the murine model, saccharin-treated mice exhibited fewer leaking laser scars, lesser occurrence of bleeding, smaller fibrotic areas (P < 0.05), and a 40% decrease in mononuclear phagocyte accumulation (P = 0.06). Gene analysis indicated downregulation of inflammatory and VEGFR-1 response genes in the treated animals. Human RPE cells expressed taste receptor type 1 member 3 (TAS1R3) mRNA and reacted to saccharin stimulation with changes in mRNA expression. Conclusions: Saccharin appears to play a protective role in patients with nAMD undergoing intravitreal anti-VEGF treatment, aiding in better pathological lesion control and scar reduction. The murine study supports this observation, proposing saccharin's potential in mitigating pathological VEGFR-1-induced immune responses potentially via the RPE sensing saccharin in the blood stream.

Inhibition of IGF2BP1 attenuates the progression of endometriosis through PTBP1.

INTRODUCTION: Endometriosis (EMs), manifested by pain and infertility, is a chronic inflammatory disease. The precise pathophysiology of this disease remains uncertain. Insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1) and polypyrimidine tract-binding protein 1 (PTBP1) have both been found to regulate proliferation, apoptosis, and invasion. This study aimed to investigate the effects of IGF2BP1/PTBP1 in treating EMs. MATERIALS AND METHODS: qRT-PCR and western blotting were employed to quantify IGF2BP1 and PTBP1 expression in six patients with EMs (mean age 33.83 years). The correlation analysis, STRING database prediction, and RNA immunoprecipitation were utilized to identify the relationship between IGF2BP1 and PTBP1. Ectopic endometrial volume, weight, HE staining, and IGF2BP1 silencing were utilized to estimate the effects of IGF2BP1 in EMs model rats. qRT-PCR, CCK-8, 5-ethynyl-2'-deoxyuridine (EDU) labeling, Transwell assay, and flow cytometry were utilized to assess the effects of IGF2BP1/PTBP1 on the proliferation, migration, invasion, and apoptosis of ectopic endometrial stromal cells (eESCs). Furthermore, western blotting was employed to evaluate expressions of PCNA, VEGF, and E-cadherin in EMs rats and eESCs. RESULTS: The mRNA and protein levels of IGF2BP1 and PTBP1 in the ectopic and eutopic endometrium of EMs patients were significantly increased. RNA immunoprecipitation revealed a close interaction of IGF2BP1 with PTBP1. Additionally, the endometrial volume, weight, and histopathologic scores in rats were significantly reduced after IGF2BP1 silencing. IGF2BP1 silencing also decreased the expression of PCNA and VEGF, and increased E-cadherin expression in endometrial tissues of EMs rats. Moreover, IGF2BP1 silencing inhibited proliferation, migration, and invasion and promoted apoptosis through PTBP1 in eESCs. CONCLUSIONS: IGF2BP1 exhibits potential beneficial properties in the management of EMs by interacting with PTBP1, thereby highlighting IGF2BP1 as a promising therapeutic target for EMs.

Endomucin selectively regulates vascular endothelial growth factor receptor-2 endocytosis through its interaction with AP2.

The endothelial glycocalyx, located at the luminal surface of the endothelium, plays an important role in the regulation of leukocyte adhesion, vascular permeability, and vascular homeostasis. Endomucin (EMCN), a component of the endothelial glycocalyx, is a mucin-like transmembrane glycoprotein selectively expressed by venous and capillary endothelium. We have previously shown that knockdown of EMCN impairs retinal vascular development in vivo and vascular endothelial growth factor 165 isoform (VEGF165)-induced cell migration, proliferation, and tube formation by human retinal endothelial cells in vitro and that EMCN is essential for VEGF165-stimulated clathrin-mediated endocytosis and signaling of VEGF receptor 2 (VEGFR2). Clathrin-mediated endocytosis is an essential step in receptor signaling and is of paramount importance for a number of receptors for growth factors involved in angiogenesis. In this study, we further investigated the molecular mechanism underlying EMCN's involvement in the regulation of VEGF-induced endocytosis. In addition, we examined the specificity of EMCN's role in angiogenesis-related cell surface receptor tyrosine kinase endocytosis and signaling. We identified that EMCN interacts with AP2 complex, which is essential for clathrin-mediated endocytosis. Lack of EMCN did not affect clathrin recruitment to the AP2 complex following VEGF stimulation, but it is necessary for the interaction between VEGFR2 and the AP2 complex during endocytosis. EMCN does not inhibit VEGFR1 and FGFR1 internalization or their downstream activities since EMCN interacts with VEGFR2 but not VEGFR1 or FGFR1. Additionally, EMCN also regulates VEGF121-induced VEGFR2 phosphorylation and internalization.

Eucommia granules activate Wnt/ß-catenin pathway, and improve oxidative stress, inflammation, and endothelial injury in preeclampsia rats.

PURPOSE: Pre-eclampsia (PE) is a pregnancy-related complication. Eucommia is effective in the treatment of hypertensive disorders in pregnancy, but the specific effects and possible mechanisms of Eucommia granules (EG) in PE remain unknown. The aim of this study was to investigate the effects and possible mechanisms of EG in PE rats. METHODS: Pregnant Sprague Dawley rats were divided into five groups (n = 6): the control group, the model group, the low-dose group, the medium-dose group, and the high-dose group of EG. The PE model was established by subcutaneous injection of levonitroarginine methyl ester. Saline was given to the blank and model groups, and the Eucommia granules were given by gavage to the remaining groups. Blood pressure and urinary protein were detected. The body length and weight of the pups and the weight of the placenta were recorded. Superoxide dismutase (SOD) activity and levels of malondialdehyde (MDA), placental growth factor (PIGF), and soluble vascular endothelial growth factor receptor-1 (sFIt-1) were measured in the placenta. Pathological changes were observed by hematoxylin-eosin staining. Wnt/ß-catenin pathway-related protein expression was detected using Western blot. RESULTS: Compared with the model group, the PE rats treated with EG had lower blood pressure and urinary protein. The length and weight of the pups and placental weight were increased. Inflammation and necrosis in the placental tissue was improved. SOD level increased, MDA content and sFIt-1/PIGF ratio decreased, and Wnt/ß-catenin pathway-related protein expression level increased. Moreover, the results of EG on PE rats increased with higher doses of EG. CONCLUSIONS: EG may activate the Wnt/ß-catenin pathway and inhibit oxidative stress, inflammation, and vascular endothelial injury in PE rats, thereby improving the perinatal prognosis of preeclamptic rats. EG may inhibit oxidative stress, inflammation, and vascular endothelial injury through activation of the Wnt/ß-catenin pathway in preeclampsia rats, thereby improving perinatal outcomes in PE rats.

Autocrine VEGF drives neural stem cell proximity to the adult hippocampus vascular niche.

The vasculature is a key component of adult brain neural stem cell (NSC) niches. In the adult mammalian hippocampus, NSCs reside in close contact with a dense capillary network. How this niche is maintained is unclear. We recently found that adult hippocampal NSCs express VEGF, a soluble factor with chemoattractive properties for vascular endothelia. Here, we show that global and NSC-specific VEGF loss led to dissociation of NSCs and their intermediate progenitor daughter cells from local vasculature. Surprisingly, though, we found no changes in local vascular density. Instead, we found that NSC-derived VEGF supports maintenance of gene expression programs in NSCs and their progeny related to cell migration and adhesion. In vitro assays revealed that blockade of VEGF receptor 2 impaired NSC motility and adhesion. Our findings suggest that NSCs maintain their own proximity to vasculature via self-stimulated VEGF signaling that supports their motility towards and/or adhesion to local blood vessels.

Deciphering microglial activation and neuronal apoptosis post­traumatic brain injury: The role of TYROBP in inflammation regulation networks.

Traumatic Brain Injury (TBI) represents a significant public health challenge. Recovery from brain injury necessitates the collaborative efforts of various resident neural cells, predominantly microglia. The present study analyzed rat and mouse RNA expression micro­arrays, high­throughput RNA sequencing and single­cell sequencing data sourced from public databases. To construct an inflammation regulation network around TYRO protein tyrosine kinase­binding protein (TYROBP), to evaluate the role of TYROBP in cell death after TBI. These findings indicate that following TBI, neurons predominantly communicate with one another through the CXC chemokine ligand (CXCL) and CC chemokine ligand (CCL) signaling pathways, employing a paracrine mechanism to activate microglia. These activated microglia intensify the pathological progression of brain injury by releasing factors such as tumor necrosis factor α (TNF­α), vascular endothelial growth factor and transforming growth factor ß via the NF­κB pathway. Cells co­culture experiments demonstrated that neurons, impaired by mechanical injury, interact with microglia through non­contact mechanisms. Activated microglia secrete cytokines, including TNF­α, CXCL­8 and CCL2, which trigger an inflammatory response and facilitate neuronal apoptosis. TYROBP gene knockout in microglia was demonstrated to reduce this interaction and reduce neuronal cell apoptosis rates.

MiR-34a ameliorates arterial blood flow in rats with lower limb arteriosclerosis obliterans via Sirt1 signaling pathway.

In this study, we investigated the impact of microRNA-34a (miR-34a) on lower limb arteriosclerosis obliterans in rats through the Sirtuin 1 (Sirt1) signaling pathway. Thirty-six Sprague-Dawley rats were divided into normal, model, and miR-34a mimics groups. Rats in the normal group were raised normally, while the model group underwent lower limb arteriosclerosis obliterans induction and received saline injections. The miR-34a mimics group also underwent arteriosclerosis obliterans modeling but received miR-34a mimics injections. Immunohistochemistry revealed significantly increased vascular endothelial growth factor (VEGF) expression in both model and miR-34a mimics groups compared to the normal group, with the miR-34a mimics group showing higher levels. Western blotting indicated elevated Sirt1 protein expression in both non-normal groups, with the miR-34a mimics group exhibiting significantly higher levels. Quantitative polymerase chain reaction (qPCR) demonstrated higher levels of miR-34a, VEGF mRNA, and Sirt1 mRNA in the model group compared to the normal group, but significantly lower levels than the miR-34a mimics group. Enzyme-linked immunosorbent assay (ELISA) showed increased VEGF content in the model group compared to the normal group but decreased compared to the miR-34a mimics group. Hemorrheological detection revealed a reduced PU index in both non-normal groups compared to the normal group, with a significant increase in the miR-34a mimics group compared to the model group. Overall, miR-34a upregulation enhanced VEGF expression in rat blood vessels, ameliorating arterial blood flow in lower limb arteriosclerosis obliterans through the Sirt1 signaling pathway.

Overexpression of RAD54L attenuates osteoarthritis by suppressing the HIF-1α/VEGF signaling pathway: Bioinformatics analysis and experimental validation.

Osteoarthritis (OA) is a widespread chronic, progressive, degenerative joint disease that causes pain and disability. Current treatments for OA have limited effectiveness and new biomarkers need to be identified. Bioinformatics analysis was conducted to explore differentially expressed genes and DNA repair/recombination protein 54 L (RAD54L) was selected. We firstly overexpressed RAD54L in interleukin-1ß (IL-1ß)-induced human articular chondrocytes or in OA rats to investigate its effect on OA. Chondrocyte viability and apoptotic rate were measured by Cell Counting Kit-8 and flow cytometry, respectively. Then we evaluated OA severity in vivo by Hematoxylin-eosin staining and Osteoarthritis Research Society International standards. The expression of inflammatory mediators was tested by enzyme-linked immunosorbent assay. Finally, western blot was performed to determine the relative expression level of hypoxia-inducible factors 1α (HIF-1α) and vascular endothelial growth factor (VEGF). Overexpression of RAD54L promoted cell viability and attenuated apoptosis in IL-1ß-induced human chondrocytes. A lower Osteoarthritis Research Society International score and a remarkable alleviation of chondrocyte disordering and infiltration of inflammatory cells were found in cartilage tissues of OA rats after overexpressing RAD54L. The inflammatory response induced by OA was decreased by RAD54L overexpression in vitro and in vivo. In addition, RAD54L overexpression decreased the relative expression level of HIF-1α and VEGF. Overexpression of RAD54L could attenuate OA by suppressing the HIF-1α/VEGF signaling pathway, indicating that RAD54L may be a potential treatment target for OA.

Proteomic studies of VEGFR2 in human placentas reveal protein associations with preeclampsia, diabetes, gravidity, and labor.

VEGFR2 (Vascular endothelial growth factor receptor 2) is a central regulator of placental angiogenesis. The study of the VEGFR2 proteome of chorionic villi at term revealed its partners MDMX (Double minute 4 protein) and PICALM (Phosphatidylinositol-binding clathrin assembly protein). Subsequently, the oxytocin receptor (OT-R) and vasopressin V1aR receptor were detected in MDMX and PICALM immunoprecipitations. Immunogold electron microscopy showed VEGFR2 on endothelial cell (EC) nuclei, mitochondria, and Hofbauer cells (HC), tissue-resident macrophages of the placenta. MDMX, PICALM, and V1aR were located on EC plasma membranes, nuclei, and HC nuclei. Unexpectedly, PICALM and OT-R were detected on EC projections into the fetal lumen and OT-R on 20-150 nm clusters therein, prompting the hypothesis that placental exosomes transport OT-R to the fetus and across the blood-brain barrier. Insights on gestational complications were gained by univariable and multivariable regression analyses associating preeclampsia with lower MDMX protein levels in membrane extracts of chorionic villi, and lower MDMX, PICALM, OT-R, and V1aR with spontaneous vaginal deliveries compared to cesarean deliveries before the onset of labor. We found select associations between higher MDMX, PICALM, OT-R protein levels and either gravidity, diabetes, BMI, maternal age, or neonatal weight, and correlations only between PICALM-OT-R (p < 2.7 × 10-8), PICALM-V1aR (p < 0.006), and OT-R-V1aR (p < 0.001). These results offer for exploration new partnerships in metabolic networks, tissue-resident immunity, and labor, notably for HC that predominantly express MDMX.

Central subfield thickness of diabetic macular edema: Correlation with the aqueous humor proteome.

Purpose: Diabetic macular edema (DME) is a sight-threatening complication of diabetes. Consequently, studying the proteome of DME may provide novel insights into underlying molecular mechanisms. Methods: In this study, aqueous humor samples from eyes with treatment-naïve clinically significant DME (n = 13) and age-matched controls (n = 11) were compared with label-free liquid chromatography-tandem mass spectrometry. Additional aqueous humor samples from eyes with treatment-naïve DME (n = 15) and controls (n = 8) were obtained for validation by enzyme-linked immunosorbent assay (ELISA). Best-corrected visual acuity (BCVA) was evaluated, and the severity of DME was measured as central subfield thickness (CST) employing optical coherence tomography. Control samples were obtained before cataract surgery. Significantly changed proteins were identified using a permutation-based calculation, with a false discovery rate of 0.05. A human donor eye with DME and a control eye were used for immunofluorescence. Results: A total of 101 proteins were differentially expressed in the DME. Regulated proteins were involved in complement activation, glycolysis, extracellular matrix interaction, and cholesterol metabolism. The highest-fold change was observed for the fibrinogen alpha chain (fold change = 17.8). Complement components C2, C5, and C8, fibronectin, and hepatocyte growth factor-like protein were increased in DME and correlated with best-corrected visual acuity (BCVA). Ceruloplasmin and complement component C8 correlated with central subfield thickness (CST). Hemopexin, plasma kallikrein, monocyte differentiation antigen CD14 (CD14), and lipopolysaccharide-binding protein (LBP) were upregulated in the DME. LBP was correlated with vascular endothelial growth factor. The increased level of LBP in DME was confirmed using ELISA. The proteins involved in desmosomal integrity, including desmocollin-1 and desmoglein-1, were downregulated in DME and correlated negatively with CST. Immunofluorescence confirmed the extravasation of fibrinogen at the retinal level in the DME. Conclusion: Elevated levels of pro-inflammatory proteins, including the complement components LBP and CD14, were observed in DME. DME was associated with the loss of basal membrane proteins, compromised desmosomal integrity, and perturbation of glycolysis.

CircSNX6 promotes proliferation, metastasis, and angiogenesis in hepatocellular carcinoma via miR-383-5p/VEGFA signaling pathway.

The role of circular RNA (circRNAs) in hepatocellular carcinoma (HCC) has been extensively studied. Previous research has highlighted the regulatory role of circSNX6 in HCC cells and tissues. However, the precise mechanism underlying HCC progression still requires comprehensive investigation. The study initially utilized quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to assess circSNX6 expression levels in HCC cell lines and tissues. Subsequently, the stability of circRNA was evaluated through Ribonuclease R and actinomycin D treatment assays. The impact of circSNX6 knockdown on proliferation, migration, invasion, and angiogenesis abilities was determined using various assays including colony formation, Transwell culture system, tube formation assay, and cell counting kit (CCK)-8 assays. Additionally, RNA immunoprecipitation chip and dual-luciferase reporter assays were employed to investigate the interactions between circSNX6 and miR-383-5p. Finally, an HCC xenograft tumor model in mice was established to assess the in vivo expression of circSNX6 and its functional role in HCC. Our findings revealed an elevated circSNX6 expression in HCC tissues, which was correlated with poor patient prognosis. Knockdown of circSNX6 suppressed HCC cell growth, invasion, metastasis, and angiogenesis. The downregulation of miR-383-5p, a target of circSNX6, significantly attenuated the tumor-suppressive effects induced by circSNX6 knockdown. Moreover, circSNX6 was found to modulate VEGFA expression by targeting miR-383-5p. The inhibition of HCC cell proliferation by miR-383-5p could be partially reversed by overexpressing VEGFA. Silencing circSNX6 also suppressed tumor formation and the metastasis of HCC cells in a mouse model. In summary, our findings suggest that circSNX6 promotes cell proliferation, metastasis, and angiogenesis in HCC by regulating the miR-383-5p/VEGFA pathway.