VEGF Signaling Pathway Research Articles

FGFR3 mutated (FGFR3mut+) urothelial carcinoma of bladder (UCB) or upper tract (UTUC): A comparative genomic landscape study.

540 Background: Activating DNA sequence genomic alterations (GA) in the FGFR3 gene including short variant (SV) mutations and kinase domain activating gene rearrangements/fusions (RE-FUS) are known drivers and relevant precision treatment targets for pts with UC. THOR trial showed a significant overall survival benefit with erdafitinib (FGFR inhibitor) vs (taxane or vinflunine) in patients (pts) with advanced UCB or UTUC. PROOF-302 trial showed higher frequency of FGFR3 GA in UTUC (30%) vs UCB (13%). We sought to explore differences in frequency of FGFR3 alterations and the genomic landscapes of FGFR3-altered UCB and UTUC. Methods: 10,402 UCB and 2,325 UTUC (combined ureter and renal pelvis) underwent hybrid capture-based comprehensive genomic profiling (CGP) using a hybrid capture-based to assess all classes of GA and measure MSI status, TMB level, genomic ancestry, genomic signature, germline mutations and HRD score. PD-L1 was determined by IHC (Dako 22C3 using the TPS system. Results were compared using the Fisher Exact method with the Benjamini-Hochberg adjustment. Results: FGFR3mut+ status was significantly more frequent in UTUC than in UBC (24.9% vs 17.7%; p<.0001) (Table). UCB pts were more often male than UTUC (P=.0003). Age (71-72 yrs), EUR ancestry (85%) and GA/tumor (8.9-9.0) were similar. The number of GA/case and the frequency of EUR ancestry were similar. Targetable GA in ERBB2 and PIK3CA were more frequent in FGFR3mut+ UBC vs FGFR3mut+ UTUC. GA in PTEN, TSC1 and MTAP currently associated with clinical trials were similar in UBC and UTUC. The KEGG ERBB and VEGF signaling pathways were more frequently identified in UCB (p=0.036) and the MMR pathway more frequently identified in UTUC (p=0.014). The HRD signature was similar in both groups (2.3-3.1%). Anti-PD(L)1 putative biomarkers included a significantly higher frequency of MSI-high status in UTUC vs UCB but no significant difference in TMB or PD-L1 expression levels. Conclusions: Although histologically similar, the genomic landscape of FGFR3mut+ UTUC has notable differences with FGFR3mut+ UCB. Limitations include lack of clinical data annotation. The findings may impact of clinical trial designs for UCB and UTUC, including evaluating combinations of anti-FGFR3 with other agents. [Table: see text]

Study on chemical components of Hypericum himalaicum and mechanism of anti-inflammatory based on network pharmacology and molecular docking technology

The chemical constituents of ethyl acetate from Hypericum himalaicum were isolated by silica gel column chromatography, gel column chromatography, and high-performance liquid chromatography. The structure of the isolated compounds was identified by modern spectral techniques(NMR, MS, IR, and UV), and the potential anti-inflammatory targets and action pathways were analyzed and predicted by network pharmacology and molecular docking methods.Ten compounds were isolated from H. himalaicum and identified as 5,9,11-trihydroxy-3,3-dimethyl-3H,8H-benzo[6,7][1,4]dioxepino[2,3-f]chromen-8-one(1), betulinic acid(2), demethyltorosaflavone C(3), kaempferol(4), quercetin(5), hyperwightin B(6), toxyloxanthone B(7), 1,7-dihydroxy-xanthone(8), emodin(9), and 1,7-dihydroxy-4-methoxy-xanthone(10). Among them, compound 1 was a new compound, and compounds 2-10 were isolated from H. himalaicum for the first time. Network pharmacology screened 60 key anti-inflammatory targets. By acting on TNF, AKT1, CASP3, and other key targets, involving PI3K-AKT signaling pathway, IL-17 signaling pathway, VEGF signaling pathway, MAPK signaling pathway, and other signaling pathways, and phosphorylation, cell migration and movement, protein tyrosine kinase, and other biological processes were regulated to achieve anti-inflammatory effects. The results of molecular docking show that the above components have good binding properties with the core targets.

Expression of PTGS2 along with genes regulating VEGF signalling pathway and association with high-risk factors in locally advanced oral squamous cell carcinoma.

PTGS2 encodes cyclooxygenase-2 (COX-2), which catalyses the committed step in prostaglandin synthesis. Various invivo and invitro data suggest that COX-2 mediates the VEGF signalling pathway. In silico analysis performed in TCGA, PanCancer Atlas for head and neck cancers, demonstrated significant expression and co-expression of PTGS2 and genes that regulate VEGF signalling. This study was designed to elucidate the expression pattern of PTGS2 and genes regulating VEGF signalling in patients with locally advanced oral squamous cell carcinoma (OSCC). Tumour and normal tissue samples were collected from patients with locally advanced OSCC. RNA was isolated from tissue samples, followed by cDNA synthesis. The cDNA was used for gene expression analysis (RT-PCR) using target-specific primers. The results obtained were compared with the in silico gene expression of the target genes in the TCGA datasets. Co-expression analysis was performed to establish an association between PTGS2 and VEGF signalling genes. Tumour and normal tissue samples were collected from 24 OSCC patients. Significant upregulation of PTGS2 expression was observed. Furthermore, VEGFA, KDR, CXCR1 and CXCR2 were significantly upregulated in tumour samples compared with paired normal samples, except for VEGFB, whose expression was not statistically significant. A similar expression pattern was observed in silico, except for CXCR2 which was highly expressed in the normal samples. Co-expression analysis showed a significant positive correlation between PTGS2 and VEGF signalling genes, except for VEGFB which showed a negative correlation. PTGS2 and VEGF signalling genes are upregulated in OSCC, which has a profound impact on clinical outcomes.

Selective induction of human renal interstitial progenitor-like cell lineages from iPSCs reveals development of mesangial and EPO-producing cells

Recent regenerative studies using human pluripotent stem cells (hPSCs) have developed multiple kidney-lineage cells and organoids. However, to further form functional segments of the kidney, interactions of epithelial and interstitial cells are required. Here we describe a selective differentiation of renal interstitial progenitor-like cells (IPLCs) from human induced pluripotent stem cells (hiPSCs) by modifying our previous induction method for nephron progenitor cells (NPCs) and analyzing mouse embryonic interstitial progenitor cell (IPC) development. Our IPLCs combined with hiPSC-derived NPCs and nephric duct cells form nephrogenic niche- and mesangium-like structures invitro. Furthermore, we successfully induce hiPSC-derived IPLCs to differentiate into mesangial and erythropoietin-producing cell lineages invitro by screening differentiation-inducing factors and confirm that p38 MAPK, hypoxia, and VEGF signaling pathways are involved in the differentiation of mesangial-lineage cells. These findings indicate that our IPC-lineage induction method contributes to kidney regeneration and developmental research.

THBS1 promotes angiogenesis and accelerates ESCC malignant progression by the HIF-1/VEGF signaling pathway.

Previously, we demonstrated that the expression of THBS1 is increased in esophageal squamous cell carcinoma (ESCC) tissues and is correlated with lymph node metastasis and poor prognosis, indicating that THBS1 might be a candidate oncogene in ESCC. In this study, we future studied the specific role of THBS1 in ESCC and its molecular mechanism. Silencing THBS1 expression resulted in inhibition of cell migration and cell invasion of ESCC cells, the decrease of colony formation and proliferation. Tube formation of human umbilical vein endothelial cells (HUVECs) in vitro was decreased when cultured with conditioned medium from THBS1-silenced cells. The expression of CD31, a marker for blood vessel endothelial cells, was decreased in tumor tissues derived from THBS1-silenced tumors in vivo. Silencing THBS1 leaded the decreased of hypoxia-inducible factor-1α (HIF-1α), HIF-1β, and VEGFA protein. The expression of p-ERK and p-AKT were declined in HUVECs following incubation with conditioned medium from THBS1-silenced ESCC cells compared conditioned medium from control cells. Furthermore, the treatment with bevacizumab boosted the decrease of the p-ERK and p-AKT levels in HUVECs incubated with the conditioned medium from THBS1-silenced ESCC cells. THBS1 silencing combined with bevacizumab blocked VEGF, inhibited to the tube formation, colony formation and migration of HUVECs, which were superior to that of bevacizumab alone. We presumed that THBS1 can enhance HIF-1/VEGF signaling and subsequently induce angiogenesis by activating the AKT and ERK pathways in HUVECs, resulting in bevacizumab resistance. THBS1 would be a potential target in tumor antiangiogenesis therapies.

VEGF and EGFR signaling pathways are involved in the baicalein attenuation of OVA-induced airway inflammation and airway remodeling in mice

BackgroundAlthough Traditional Chinese Medicine (TCM) has been used for treating asthma for centuries, the understanding of its mechanism of action is still limited. Thus, the purpose of this study was to explore the possible therapeutic effects, and underlying mechanism of baicalein in the treatment of asthma.MethodsFreely availabled atabases (e.g. OMIM, TTD, Genecards, BATMAN-TCM, STITCH 5.0, SEA, SwissTargetPrediction) and software (e.g. Ligplot 2.2.5 and PyMoL) were used for disease drug target prediction and molecular docking by network pharmacology. The efficacy and mechanism of action of baicalein in the treatment of asthma were validated using an ovalbumin (OVA)-induced asthma mouse model and molecular biology techniques.ResultsA total of 1655 asthma-related genes and 161 baicalein-related targets were identified from public databases. Utilizing common databases and software for network pharmacology and molecular docking analysis, seven potential target proteins for the therapeutic effects of baicalein on asthma were selected, including v-akt murine thymoma viral oncogene homolog 1 (AKT1), vascular endothelial growth factor A (VEGFA), epidermal growth factor receptor (EGFR), proto-oncogene tyrosine-protein kinase Src (SRC), mitogen-activated protein kinase 3 (MAPK3), matrix metallopeptidase 9 (MMP9), and MAPK1. In vivo, baicalein treatment via intraperitoneal injection at a dose of 50 mg/kg significantly reduced airway inflammation, collagen deposition, smooth muscle thickness, lung interleukin (IL)-4 and IL-13 levels, peripheral blood immunoglobulin (Ig)E levels, as well as the count and ratio of eosinophils in bronchoalveolar lavage fluid (BALF) in an OVA-induced asthma mouse model. Further validation by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting analysis revealed that the VEGF and EGFR signaling pathways involving VEGFA, MAPK1, MAPK3, and EGFR were inhibited by baicalein in the asthma mouse model.ConclusionBaicalein attenuates airway inflammation and airway remodeling through inhibition of VEGF and EGFR signaling pathways in an OVA-induced asthma mouse model. This will provide a new basis for the development of baicalein as a treatment for asthma and highlights the potential of network pharmacology and molecular docking in drug discovery and development.

Analysis of the differentially expressed genes in the combs and testes of Qingyuan partridge roosters at different developmental stages

BackgroundThe sexual maturity of chickens is an important economic trait, and the breeding of precocious and delayed puberty roosters is an important selection strategy for broilers. The comb serves as an important secondary sexual characteristic of roosters and determines their sexual precocity. Moreover, comb development is closely associated with gonad development in roosters. However, the underlying molecular mechanism regulating the sexual maturity of roosters has not yet been fully explored.ResultsIn order to identify the genes related to precocious puberty in Qingyuan partridge roosters, and based on the synchrony of testis and combs development, combined with histological observation and RNA-seq method, the developmental status and gene expression profile of combs and testis were obtained. The results showed that during the early growth and development period (77 days of age), the development of combs and testis was significant in the high comb (H) group versus the low comb (L) group (p < 0.05); however, the morphological characteristic of the comb and testicular tissues converged during the late growth and development period (112 days of age) in the H and L groups. Based on these results, RNA-sequencing analysis was performed on the comb and testis tissues of the 77 and 112 days old Qingyuan Partridge roosters with different comb height traits. GO and KEGG analysis enrichment analysis showed that the differentially expressed genes were primarily enriched in MAPK signaling, VEGF signaling, and retinol metabolism pathways. Moreover, weighted correlation network analysis and module co-expression network analysis identified WNT6, AMH, IHH, STT3A, PEX16, KPNA7, CATHL2, ROR2, PAMR1, WISP2, IL17REL, NDRG4, CYP26B1, and CRHBP as the key genes associated with the regulation of precocity and delayed puberty in Qingyuan Partridge roosters.ConclusionsIn summary, we identified the key regulatory genes of sexual precocity in roosters, which provide a theoretical basis for understanding the developmental differences between precocious and delayed puberty in roosters.

Network pharmacology and experimental verification: Unraveling Zhiwei Fuwei Pills's role and mechanism in angiogenesis of precancerous lesions of gastric cancer.

Precancerous lesions of gastric cancer (PLGC) are key pathological stages in the transformation of gastric "inflammation-cancer", and timely and effective intervention at this stage is of great importance in the prevention and treatment of gastric cancer. Zhiwei Fuwei Pills (ZWFW), as a traditional Chinese medicine formulation, has been proven to have good clinical efficacy in the treatment of PLGC, but its specific mechanism of action has not been fully explained. Thus, this study validated the efficacy and explored the potential mechanisms of ZWFW in treating PLGC by integrating network pharmacology analyses and experimental verification. The TCMSP database was used to obtain the active ingredients of ZWFW and their corresponding targets, and the GeneCards database was used to retrieve PLGC-related targets. The intersecting targets between ZWFW and PLGC were obtained through mapping, and protein-protein interaction (PPI) networks and "drug-active ingredient-target" networks were constructed by using Cytoscape software. The DAVID database was used for GO functional enrichment analysis and KEGG pathway enrichment analysis. AutoDockTools software was used for molecular docking of key active ingredients and key targets. In order to verify the analysis results of network pharmacology, TEM and H&E were used to observe the effects of different dosage groups of ZWFW on gastric mucosal microvasculature in PLGC rats. Subsequently, the ELISA, IF, IHC, RT-PCR and western blot were used to detected the expression levels of relevant targets in the tissues, so as to verify the potential mechanism of ZWFW in intervening PLGC. After the screening, 258 effective active ingredients and 325 targets were obtained, and 1294 disease-related targets were determined, resulting in 139 intersection targets through mapping. The KEGG enrichment results showed that PI3K/Akt and HIF-1 signaling pathway might play important roles in the treatment mechanism of PLGC. The molecular docking results showed that active ingredients of ZWFW all had a strong affinity and stable structure with key targets, including AKT1 and VEGF. In vivo experiments confirmed that ZWFW could improve gastric mucosal microvascular abnormalities in PLGC, effectively intervene in gastric mucosal pathological grading. Meanwhile, compared with the model group, this formulation could reduce the expression levels of PI3K, Akt, mTOR, HIF-1α, and VEGF in gastric mucosa, showing a dose-effect relationship. ZWFW can intervene in the neovascularization and pathological evolution of PLGC, and this mechanism of action may be achieved by inhibiting abnormal activation of the PI3K/Akt/mTOR/HIF-1α/VEGF signaling pathway.

Exploring the Mechanism of Si-miao-yong-an Decoction in the Treatment of Coronary Heart Disease based on Network Pharmacology and Experimental Verification.

To investigate the active ingredients and the mechanisms of Si-miaoyong- an Decoction (SMYA) in the treatment of coronary heart disease (CHD) by using network pharmacology, molecular docking technology, and in vitro validation. Through the Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP), Uniprot database, GeneCards database, and DAVID database, we explored the core compounds, core targets and signal pathways of the effective compounds of SMYA in the treatment of CHD. Molecular docking technology was applied to evaluate the interactions between active compounds and key targets. The hypoxia-reoxygenation H9C2 cell model was applied to carry out in vitro verification experiments. A total of 109 active ingredients and 242 potential targets were screened from SMYA. A total of 1491 CHD-related targets were retrieved through the Gene- Cards database and 155 overlapping CHD-related SMYA targets were obtained. PPI network topology analysis indicated that the core targets of SMYA in the treatment of CHD include interleukin- 6 (IL-6), tumor suppressor gene (TP53), tumor necrosis factor (TNF), vascular endothelial growth factor A (VEGFA), phosphorylated protein kinase (AKT1) and mitogen-activated protein kinase (MAPK). KEGG enrichment analysis demonstrated that SMYA could regulate Pathways in cancer, phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signaling pathway, hypoxiainducible factor-1(HIF-1) signaling pathway, VEGF signaling pathway, etc. Results: Molecular docking showed that quercetin had a significant binding activity with VEGFA and AKT1. In vitro studies verified that quercetin, the major effective component of SMYA, has a protective effect on the cell injury model of cardiomyocytes, partially by up-regulating expressions of phosphorylated AKT1 and VEGFA. SMYA has multiple components and treats CHD by acting on multiple targets. Quercetin is one of its key ingredients and may protect against CHD by regulating AKT/VEGFA pathway.

PLA2G2D fosters angiogenesis in non-small cell lung cancer through aerobic glycolysis

Non-small cell lung cancer (NSCLC) stands prominent among the prevailing and formidable oncological entities. The immune and metabolic-related molecule Phospholipase A2, group IID (PLA2G2D) exerts promotional effects on tumor progression. However, its involvement in cancer angiogenesis remains elusive. Therefore, this investigation delved into the functional significance of PLA2G2D concerning angiogenesis in NSCLC. This study analyzed the expression and enriched pathways of PLA2G2D in NSCLC tissues through bioinformatics analysis, and measured the expression of PLA2G2D in NSCLC cells using qRT-PCR and western blot (WB). Subsequently, the viability and angiogenic potential of NSCLC cells were assessed employing CCK-8 and angiogenesis assays, respectively. The expression profile of angiogenic factors was analyzed through WB. Finally, the expression of glycolysis pathway-related genes, extracellular acidification rate and oxygen consumption rate, and the levels of pyruvate, lactate, citrate, and malate were analyzed in NSCLC cells using qRT-PCR, Seahorse XF 96, and related kits. Bioinformatics analysis revealed the upregulation of PLA2G2D in NSCLC tissues and its association with VEGF and glycolysis signaling pathways. Molecular and cellular experiments demonstrated that upregulated PLA2G2D promoted the viability, angiogenic ability, and glycolysis pathway of NSCLC cells. Rescue assays revealed that the effects of high expression of PLA2G2D on the viability, angiogenic ability, and glycolysis of NSCLC cells were weakened after the addition of the glycolysis inhibitor 2-DG. In summary, PLA2G2D plays a key role in NSCLC angiogenesis through aerobic glycolysis, displaying great potential as a target for anti-angiogenesis therapy.

STX4 as a potential biomarker for predicting prognosis and guiding clinical treatment decisions in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) represents a frequent subtype of kidney cancer, with the prognosis remaining poor for individuals with metastatic disease. Given its resistance to both radiation and chemotherapy, targeted therapies and immunotherapies have emerged as critical for effective ccRCC treatment. Within this context, the SNARE protein STX4, which is associated with malignant cancer cell migration, provides a promising focus. The underlying mechanism, however, requires further illumination. Furthermore, the influence of STX4 on the ccRCC tumor microenvironment remains to be determined. In our research, we utilized multiple databases and immunohistochemical staining to confirm differential STX4 expression and its prognostic implications. We evaluated the potential tumor-promoting function of STX4 in ccRCC cell lines through molecular studies. Additionally, we conducted functional enrichment analysis to delve deeper into the underlying mechanisms and performed immune infiltration and drug sensitivity analyses to assess the potential of STX4 as a prognostic biomarker and therapeutic target. Our study reveals that STX4 contributes to cancer progression by enhancing AKT expression and stimulating the activation of VEGF signaling pathways. Additionally, STX4 further fosters CD8+ T-cell infiltration and diminishes the percentage of CAFs and M2-TAMs. Our findings suggest that patients presenting higher STX4 levels may exhibit enhanced responsiveness to immunotherapy and higher sensitivity to the medications axitinib and everolimus. Finally, we propose STX4 expression assessment as a novel approach to predict patient response to respective immunotherapies and targeted treatments, hence potentially improving patient outcomes.

Study on the Mechanism of Opuntia Dillenii Polysaccharide against Cadmium-Induced Liver Injury Based on Network Pharmacology and Oxidative Stress Pathway

This study aims to investigate the mechanism of action of Opuntia dillenii polysaccharides (ODPs) in protecting against cadmium-induced liver injury from the perspectives of network pharmacology and oxidative stress pathway. Using network pharmacology methods, we analyzed the interactions between ODPs and target molecules, and predicted potential mechanisms of action. Additionally, we focused on the oxidative stress pathway, which plays a crucial role in cadmium-induced liver injury. The results showed that the active components of ODPs were glucuronic acid, galacturonic acid, rhamnose, fucose, glucose, galactose, mannose, fructose, xylose and arabinose in order. According to the analysis of PPI network and the “compound - liver injury target-pathway” network, 69 ODPs anti-cadmium-induced liver injury targets were selected, including AKT1, CASP3, MAPK8, JAK2, MAPK14, IL2, MMP9, IGF1R, NOS2, etc. Enrichment analysis showed that the main signaling pathways involved were VEGF signaling pathway, cancer signaling pathway, PI3K-AKT signaling pathway, Rap1 signaling pathway, Ras signaling pathway, chemokine signaling pathway, sphingolipid signaling pathway, toll-like receptor signaling pathway, tumor necrosis factor signaling pathway, and MAPK signaling pathway. Network pharmacological analysis showed that cactus polysaccharide could significantly reduce the level of Caspase-3 and MMP-9 in liver, increase the level of IL-2, and effectively inhibit the occurrence of liver cell apoptosis and inflammation caused by cadmium. Studies on the mechanism of action based on traditional oxidative stress indexes show that ODPs has good antioxidant capacity, can significantly inhibit the activity of SOD and GSH-PX caused by cadmium, reduce the content of lipid peroxidation product MDA, and restore the content of antioxidant enzymes in liver to normal level. This study can provide a scientific basis for the in-depth development and application of ODPs, and also provide a potential scientific and technological basis for the development of drugs or health food for the treatment of cadmium-induced liver injury.

Phenobarbital ameliorates hyperglycemia-induced angiogenesis in diabetic nephropathy-possible intervention at the HIF-1α/VEGF axis

Hyperglycemia contributes to a cascade of inflammatory responses in kidneys that result in the development of renal hypoxia and angiogenesis with subsequent chronic renal failure. As the hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) axis is a key pathway for neovascularization, the inhibition of this axis is a target for renal angiogenesis therapy. We speculate that Phenobarbital (PB) which has a potential to reduce vascularization in clinical settings might have an influence on the development of angiogenesis in diabetic kidney. The aim of the study was to explore the effects of PB on the HIF-1α and VEGF expression and angiogenesis in renal tissue of rats with hyperglycemia and diabetic nephropathy. Sixty-four male Wistar rats were devided into 4 groups: control group received a single intraperitoneal saline injection; PB group received 0.05% PB orally in drinking water; diabetic group received a single intra-peritoneal STZ (65 mg/kg) injection; PB-STZ group received 0.05% PB orally two weeks before STZ administration. At the end of the experiment period (8 weeks), the kidneys were removed and used for biochemical analyses. Serum glucose, urea and creatinine levels, IL-6 levels in kidney homogenate and changes in HIF-1α and VEGF gene expression were estimated. Hematoxylin-eosin staining was performed for histopathological exa­mination. The results obtained showed that both HIF-1α and VEGF gene expression and IL6 level in diabetic rat group were significantly elevated compared to that in control group, whereas in PB and PB-STZ groups, these indices were significantly down-regulated compared to the diabetic group. Abundant glomerular congestion and mesangial proliferation were detected in diabetic rat renal tissues. However, in PB-treated diabetic group, newly formed vessels were significantly decreased. These findings confirmed that phenobarbital, affecting the HIF-1α/VEGF signaling pathway, ameliorates angiogenesis after hyperglycemic kidney injury. Keywords: angiogenesis, HIF-1α, hyperglycemia, IL6, kidney injury, VEGF

Abstract B123: Mechanism of action of ivonescimab (AK112/SMT112): a first-in-class tetravalent Fc-silent bispecific antibody with dual blockade of PD-1 and VEGF that promotes cooperative biological effects

Abstract Background: Combination treatments using anti-PD-1/PD-L1 antibodies with other VEGF antagonists have shown enhanced clinical antitumor activities1. The expression of PD-1 and VEGF are found to be frequently upregulated and co-expressed in solid tumors. Importantly, VEGF promotes tumor angiogenesis and suppresses antitumor immune response2,3. Consequently, we characterized the mechanism-of-action of a novel first-in-class anti-PD-1/VEGF bispecific antibody, ivonescimab, designed to simultaneously inhibit PD-1-mediated immunosuppression and block tumor angiogenesis in the tumor microenvironment. Methods: Binding activity of ivonescimab to PD-1/VEGF were assessed by ELISA. Blockade of PD-1/VEGF signaling pathways was determined in luciferase reporter cell assays. Ivonescimab-VEGF complex formation was detected by SEC-HPLC. Cooperative binding of ivonescimab-VEGF complex to PD-1 or ivonescimab-PD-1 complex to VEGF was measured by Octet BLI. The enhanced PD-1 blockade bioactivity of ivonescimab with VEGF was evaluated in hPBMC and engineered cell-line co-culture/luciferase-reporter cell assays. Antitumor activity of ivonescimab was investigated in hPBMC-humanized SCID/Beige mice implanted with HCC827 (mEGFR lung adenocarcinoma) or U87MG (glioblastoma). Immuno-safety was assessed by FcγR binding, ADCC, ADCP assays, and reported clinical irAEs. Results: Ivonescimab displayed strong binding activity to human PD-1 and VEGF alone or simultaneously, effectively blocking interactions with ligands and the downstream signaling effects. In presence of VEGF, ivonescimab forms soluble complexes with VEGF dimers, leading to over 10-fold enhanced binding affinity (KD) of ivonescimab to PD-1. The avidity increase was consistent with reduced cell surface PD-1 expression on human T-cell lines, increased potency on blockade of PD-1/PD-L1 signaling and subsequent enhanced T cell activation in-vitro. Likewise, PD-1 binding enhanced ivonescimab binding to VEGF which was associated with enhanced VEGF-signaling blockade. Also, ivonescimab treatment demonstrated statistically significant dose-dependent antitumor response in hPBMC-humanized murine HCC827 and U87MG tumor models. Additionally, ivonescimab enhanced antitumor response in combination with anti-CD47 (AK117) or anti-CD73 (AK119) in mouse models. Finally, ivonescimab contains Fc-silencing mutations abrogating FcγRI/IIIa binding and showed significantly reduced ADCC, ADCP activities and cytokine release in-vitro. Clinically, this is consistent with the safety profile in Phase 1/2 studies of ivonescimab in advanced solid tumors4,5. Conclusions: Ivonescimab is a novel tetravalent anti-PD-1/VEGF bispecific antibody displaying unique cooperative binding to each of its intended targets consistent with increased in-vitro functional bioactivities compared with bevacizumab or PD-1 inhibitors alone. Importantly, the Fc-null IgG1 design resulted in reduced FcgR interactions and minimal ADCC, ADCP activities consistent with its clinical immunosafety profile. Ivonescimab is currently in Ph3 NSCLC trials in the US and EU. Citation Format: Tingting Zhong, Zhaoliang Huang, Xinghua Pang, Chunshan Jin, Xinrong He, Jose G Montoya, Betty Y Chang, Michelle Xia, Baiyong Li, Jing Min. Mechanism of action of ivonescimab (AK112/SMT112): a first-in-class tetravalent Fc-silent bispecific antibody with dual blockade of PD-1 and VEGF that promotes cooperative biological effects [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B123.

Inhibiting MiR-320a promotes myocardial apoptosis in myocardial infarction rats through activating VEGF pathway.

To detect the expressions of vascular endothelial growth factor (VEGF) and micro ribonucleic acid (miR)-320a in myocardial cells of rats with myocardial infarction (MI), and to study the detailed mechanism of the role of miR-320a in myocardial apoptosis in MI rats. The Sprague-Dawley rat model of MI was established, and the rats were randomly divided into a control group (n=8), recombinant adeno-associated virus (rAAV)-miR-320a group (n=8) and rAAV-miR-320a TuDs group (n=8). The corresponding rAAV (1×1011 virion-like particles) was intravenously injected. At 2 weeks after the injection of rAAV, all rats were euthanatized, and the organs were collected, frozen in liquid nitrogen and stored at -80°C for further experiments. The total RNA and total protein were extracted from heart tissues, and the expression levels of rAAV-miR-320a and rAAV-miR-320aTuDs in heart tissues were determined via reverse transcription-polymerase chain reaction (RT-PCR). Moreover, RT-PCR and Western blotting were performed to detect the mRNA and protein expressions in heart tissues, respectively. At the same time, myocardial apoptosis was evaluated through flow cytometry. After treatment with miR-320a TuDs, the mRNA and protein expressions of VEGF in heart tissues in MI were significantly increased (P<0.05). The results of flow cytometry showed that miR-320a TuDs intervention could promote myocardial apoptosis in MI (P<0.05). In addition, the results of Western blotting revealed that miR-320a TuDs could facilitate the activation of the VEGF signaling pathway in heart tissues in MI (P<0.05). Inhibiting miR-320a can promote myocardial apoptosis through activating the VEGF signaling pathway in myocardial cells in MI.

Comparative Transcriptome Analysis Reveals Sexually Dimorphic Gene Expression in the Gonads of Brachymystax tsinlingensis Li

Brachymystax tsinlingensis Li is an endangered cold-water salmonid fish native to China. This study aimed to identify sex-related genes and biological pathways via gonadal transcriptome sequencing of B. tsinlingensis Li. A total of 167,904 unigenes were identified with an average length of 836 bp and an N50 of 1452 bp, of which 84,977 (50.61%) unigenes were successfully annotated in six major databases. Comparative transcriptome analysis identified 22,864 differentially expressed genes (DEGs), of which 17,231 were up-regulated (male-biased genes, mDEGs) and 5633 were down-regulated (female-biased genes, fDEGs). Several DEGs associated with gonadal development were found through Gene Ontology enrichment analysis, such as ccnb1, zp3, bmp15, dmrt1, and psmc3ip. Signaling pathways related to gonadal development were found to be enriched through analysis using the Kyoto Encyclopedia of Genes and Genomes Pathway database, such as genes involves in base excision repair, the notch signaling pathway, neuroactive ligand-receptor interaction, the VEGF signaling pathway, and the estrogen signaling pathway. In addition, mRNA expression levels of 19 DEGs were determined to validate the reliability of the transcriptomic data by quantitative real-time polymerase chain reaction. These results revealed genes and signaling pathways potentially involved in gonadal development in B. tsinlingensis Li and provided basic molecular data for future research on reproductive regulation and breeding of B. tsinlingensis Li.

Discovery of β-sitosterol's effects on molecular changes in rat diabetic wounds and its impact on angiogenesis and macrophages

Diabetes care, particularly for diabetic foot ulcers (DFUs)-related complications, increases treatment costs substantially. Failure to provide timely and appropriate treatment for severe DFUs significantly increases amputation risk. Neovascularization and macrophage polarization play an important role in diabetic wound healing during different stages of the wound repair process. Therefore, a new treatment method that promotes neovascularization and macrophage polarization may accelerate diabetic wound healing. β-sitosterol possesses anti-inflammatory, lipid-lowering, and antidiabetic properties. However, its therapeutic potential in diabetic wound healing remains underexplored. This study evaluated the healing effects of β-sitosterol on diabetic ulcer wounds in rats. We found that β-sitosterol can promote angiogenesis, alternatively activated macrophages (M2 macrophage) proliferation, and collagen synthesis in diabetic wounds. Transcriptomics analysis and proteomics analysis revealed that MAPK, mTOR and VEGF signaling pathways were enriched in β-sitosterol-treated wounds. Molecular docking revealed Ndufb5 maybe the target of β-sitosterol-treated wounds. Our findings confirm the significant diabetic wound healing effects of β-sitosterol in a rat model. β-sitosterol treatment to diabetic wounds accelerates wound healing through promoting M2 macrophage proliferation and angiogenesis. Interestingly, we also found that the process of M2 macrophage proliferation accompanies angiogenesis. Thus, β-sitosterol may be a promising therapeutic approach to enhance diabetic wound healing and reduce amputation in diabetes.

Identification of a 2-phenylthiazole derivative acetylcholinesterase modulator with invitro antitumor activity in breast cancer cells.

Acetylcholinesterase (AchE) is a serine hydrolase with classical function to degrade acetylcholine and terminate neurotransmission. While "nonclassical" functions of AchE were involved in cell growth, death, invasion, etc. The expression and activity of AchE is changed in tumors, suggesting AChE inhibitors (AchEIs) may serve as potential antitumor drugs. In this study, the antitumor activity of a series of 2-phenylthiazole derivatives originally designed and synthesized as AchEIs were investigated. One compound named A6, was screened out with superior antitumor efficacy, especially against breast cancer MCF-7 cells. A6 significantly disrupted the amino acid metabolism and inhibited migration of MCF-7. In addition, A6 induced apoptosis of MCF-7 cells. To clarify how A6 affected on MCF-7 cells, RNA-seq analysis was conducted to evaluate the whole genome effect of A6 on gene expression. A total of 153 genes were increased, and the expression of 81 genes was decreased. GO and KEGG enrichment analysis showed A6 treatment mainly disrupted sterol/cholesterol pathway, Ras signaling pathway, VEGF signaling pathway, etc. Moreover, bioinformatic analysis and cell viability test showed A6 plays anticancer role by regulating Best1 and HIST1H2BJ. These results indicate that AchEI A6 could be a potential antitumor agent for breast cancer patients and could help the development of novel therapies.

Metagenomic Sequencing Analysis of the Effects of Acetylcysteine on the Pig Gut Microbiome

The gut microbiota is engaged in multiple interactions affecting host health, and gut dysbiosis can lead to many diseases. However, the effects of acetylcysteine (NAC) on the gut microbiome composition in pigs using metagenomic sequencing have not been reported. In this study, we used metagenome sequencing to study the effects of NAC on the pig gut microbiome. Sequencing results showed that microbial diversity was changed after NAC treatment. Antibiotic Resistance Genes Database (ARDB) analysis demonstrated that the main genes modified were macb, tsnr, norm, bl2be-per, vansb and pbp1b in the NAC group. Our data showed that NAC could affect microbial distribution at the phylum, gene and species levels. At the species level, NAC significantly increased the abundances of Megasphaera, Lactobacillus reuteri and Megasphaeraelsdenii and reduced the abundances of Phascolarctobacterium succinatutens, Prevotellacopri and Selenomonasbovis compared with the control group. In addition, Gene Ontology (GO) analysis revealed that in the NAC group, cellular process, metabolic process and single-organism process were the dominant terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that RNA transport, MAPK signaling pathway, cell cycle, glycosylphosphatidylinositol (GPI)-anchor biosynthesis and VEGF signaling pathway were the dominant signaling pathways in the NAC group. In conclusion, our results suggest that NAC may modify the piglet gut microbiome composition and these findings might provide a new strategy for maintaining animal and human health in the future.

HDAC1 suppresses radiotherapy sensitivity in cervical cancer via regulating HIF-1α/VEGF signaling pathway.

Cervical cancer (CC) is the fourth most common cancer amongst females worldwide. Histone deacetylase (HDAC) 1 plays a vital role in several tumors. Nevertheless, its potential and mechanism in radiotherapy sensitivity underlying CC remains obscure. Hence, the objective of this research was to probe the potential of HDAC1 in CC radiotherapy sensitivity and its mechanism of action. The expression HDACs and survival analysis of HDAC1 were investigated based on the GEPIA database. Immunohistochemical staining was implemented to detect HDAC1 and Ki-67 expression in tumor tissues. RT-qPCR and Western blot were conducted to assess HDAC1, HIF-1α, VEGFA, along with VEGFR expressions in CC cells and tumor tissues. Cell viability, apoptosis, invasion, migration, along with cell cycle were analyzed by functional assays. Tumor-bearing nude mice model was established, and the tumor weight and volume were determined. HDAC1 was high-expressed in the tumor tissue and CC cells. In vitro, overexpression of HDAC1 suppressed radiotherapy sensitivity in C33A cells, while knockdown of HDAC1 promoted radiotherapy sensitivity in SiHa cells. In vivo, we found that HDAC1 silence hindered tumor growth and cell proliferation and promoted tumor cell apoptosis in nude mice after radiotherapy. In addition, we found that HDAC1 impacted radiotherapy sensitivity by modulating the HIF-1α/VEGF signaling pathway. In conclusion, HDAC1 suppressed the radiotherapy sensitivity of CC via regulating HIF-1α/VEGF signaling pathway, suggesting that HDAC1 may act as a crucial participant in regulating CC radiosensitivity, which may provide a novel method for treating CC.