Prime Editing of Vascular Endothelial Growth Factor Receptor 2 Attenuates Angiogenesis In Vitro.

Vascular endothelial growth factor receptor (VEGFR)-2 is a key switch for angiogenesis, which is observed in various human diseases. In this study, a novel system for advanced prime editing (PE), termed PE6h, is developed, consisting of dual lentiviral vectors: (1) a clustered regularly interspaced palindromic repeat-associated protein 9 (H840A) nickase fused with reverse transcriptase and an enhanced PE guide RNA and (2) a dominant negative (DN) MutL homolog 1 gene with nicking guide RNA. PE6h was used to edit VEGFR2 (c.18315T>A, 50.8%) to generate a premature stop codon (TAG from AAG), resulting in the production of DN-VEGFR2 (787 aa) in human retinal microvascular endothelial cells (HRECs). DN-VEGFR2 impeded VEGF-induced phosphorylation of VEGFR2, Akt, and extracellular signal-regulated kinase-1/2 and tube formation in PE6h-edited HRECs in vitro. Overall, our results highlight the potential of PE6h to inhibit angiogenesis in vivo.

Endothelial cell heterogeneity in colorectal cancer: tip cells drive angiogenesis.

This study aims to uncover the heterogeneity of endothelial cells (ECs) in colorectal cancer (CRC) and their crucial role in angiogenesis, with a special focus on tip cells. Using single-cell RNA sequencing to profile ECs, our data suggests that CRC ECs predominantly exhibit enhanced angiogenesis and decreased antigen presentation, a shift in phenotype largely steered by tip cells. We also observed that an increase in the density and proportion of tip cells correlates with CRC occurrence, progression, and poorer patient prognosis. Furthermore, we identified endothelial cell-specific molecule 1 (ESM1), specifically expressed in tip cells, sustains a VEGFA-KDR-ESM1 positive feedback loop, promoting angiogenesis and CRC proliferation and migration. We also found the enrichment of KDR in tip cells and spotlight a unique long-tail effect in VEGFA expression: while VEGFA is primarily expressed by epithelial cells, the highest level of VEGFA expression is found in individual myeloid cells. Moreover, we observed that effective PD-1 blockade immunotherapy significantly reduced tip cells, disrupting the VEGFA-KDR-ESM1 positive feedback loop in the process. Our investigation into the heterogeneity of ECs in CRC at a single-cell level offers important insights that may contribute to the development of more effective immunotherapies targeting tip cells in CRC.

Therapeutic efficacy of ECs Foxp1 targeting Hif1α-Hk2 glycolysis signal to restrict angiogenesis.

Endothelial cells (ECs) rely on glycolysis for energy production to maintain vascular homeostasis and the normalization of hyperglycolysis in tumor vessels has recently gained attention as a therapeutic target. We analyzed the TCGA database and found reduced Foxp1 expression in lung carcinoma. Immunostaining demonstrated reduced expression more restricted at tumor vascular ECs. Therefore, we investigated the function and mechanisms of Foxp1 in EC metabolism for tumor angiogenesis required for tumor growth. EC-Foxp1 deletion mice exhibited a significant increase of tumor and retinal developmental angiogenesis and Hif1α was identified as Foxp1 target gene, and Hk2 as Hif1α target gene. The Foxp1-Hif1α-Hk2 pathway in ECs is important in the regulation of glycolytic metabolism to govern tumor angiogenesis. Finally, we used genetic deletion of EC-Hif1α and RGD-peptide nanoparticles EC target delivery of Hif1α/Hk2-siRNAs to knockdown gene expression which reduced the tumor EC hyperglycolysis state and restricted angiogenesis for tumor growth. This study advances our understanding of EC metabolism for tumor angiogenesis, and meanwhile provides evidence for future therapeutic intervention of hyperglycolysis in tumor ECs for suppression of tumor growth.

YAP/TAZ Signaling Enhances Angiogenesis of Retinal Microvascular Endothelial Cells in a High-Glucose Environment.

PURPOSE: Diabetic retinopathy (DR) is a major cause of irreversible blindness in the working-age population. Neovascularization is an important hallmark of advanced DR. There is evidence that Yes-associated protein (YAP)/transcriptional co-activator with a PDZ binding domain (TAZ) plays an important role in angiogenesis and that its activity is regulated by vascular endothelial growth factor (VEGF). Therefore, the aim of this study was to investigate the effect of YAP/TAZ-VEGF crosstalk on the angiogenic capacity of human retinal microvascular endothelial cells (hRECs) in a high-glucose environment. METHODS: The expression of YAP and TAZ of hRECs under normal conditions, hypertonic conditions and high glucose were observed. YAP overexpression (OE-YAP), YAP silencing (sh-YAP), VEGF overexpression (OE-VEGF) and VEGF silencing (sh-VEGF) plasmids were constructed. Cell counting kit-8 assay was performed to detect cells proliferation ability, transwell assay to detect cells migration ability, and tube formation assay to detect tube formation ability. The protein expression of YAP, TAZ, VEGF, matrix metalloproteinase (MMP)-8, MMP-13, vessel endothelium (VE)-cadherin and alpha smooth muscle actin (α-SMA) was measured by western blot. RESULTS: The proliferation of hRECs was significantly higher in the high glucose group compared with the normal group, as well as the protein expression of YAP and TAZ (p < 0.01). YAP and VEGF promoted the proliferation, migration and tube formation of hRECs in the high glucose environment (p < 0.01), and increased the expression of TAZ, VEGF, MMP-8, MMP-13 and α-SMA while reducing the expression of VE-cadherin (p < 0.01). Knockdown of YAP effectively reversed the above promoting effects of OE-VEGF (p < 0.01) and overexpression of YAP significantly reversed the inhibition effects of sh-VEGF on above cell function (p < 0.01). CONCLUSION: In a high-glucose environment, YAP/TAZ can significantly promote the proliferation, migration and tube formation ability of hRECs, and the mechanism may be related to the regulation of VEGF expression.

Knockdown of CCM3 promotes angiogenesis through activation and nuclear translocation of YAP/TAZ.

Angiogenesis, a finely regulated process, plays a crucial role in the progression of various diseases. Cerebral cavernous malformation 3 (CCM3), alternatively referred to as programmed cell death 10 (PDCD10), stands as a pivotal functional gene with a broad distribution across the human body. However, the precise role of CCM3 in angiogenesis regulation has remained elusive. YAP/TAZ, as core components of the evolutionarily conserved Hippo pathway, have garnered increasing attention as a novel mechanism in angiogenesis regulation. Nonetheless, whether CCM3 regulates angiogenesis through YAP/TAZ mediation has not been comprehensively explored. In this study, our primary focus centers on investigating the regulation of angiogenesis through CCM3 knockdown mediated by YAP/TAZ. Silencing CCM3 significantly enhances the proliferation, migration, and tubular formation of human umbilical vein endothelial cells (HUVECs), thereby promoting angiogenesis. Furthermore, we observe an upregulation in the expression levels of VEGF and VEGFR2 within HUVECs upon silencing CCM3. Mechanistically, the evidence we provide suggests for the first time that endothelial cell CCM3 knockdown induces the activation and nuclear translocation of YAP/TAZ. Finally, we further demonstrate that the YAP/TAZ inhibitor verteporfin can reverse the pro-angiogenic effects of siCCM3, thereby confirming the role of CCM3 in angiogenesis regulation dependent on YAP/TAZ. In summary, our findings pave the way for potential therapeutic targeting of the CCM3-YAP/TAZ signaling axis as a novel approach to promote angiogenesis.

Astragaloside IV induces endothelial progenitor cell angiogenesis in deep venous thrombosis through inactivation of PI3K/AKT signaling.

BACKGROUND: Deep vein thrombosis (DVT), referred to as venous thromboembolism, is the third most frequent cardiovascular disease. Endothelial progenitor cells (EPCs) contribute to the recanalization of DVT. Astragaloside IV (AS-IV) has been suggested to have angiogenesis-enhancing effects. Here, we investigate the roles and mechanisms of AS-IV in EPCs and DVT. METHODS: The experimental DVT model was established by inferior vena cava stenosis in rats. EPCs were collected from patients with DVT. Transwell assays were performed to detect cell migration. Tube formation was determined using Matrigel basement membrane matrix and ImageJ software. The thrombus weight and length were measured. Pathological changes were examined by hematoxylin-eosin staining. The production of proinflammatory cytokines was estimated by ELISA. The level of PI3K/AKT-related proteins was measured by western blotting. RESULTS: AS-IV administration facilitated the migrative and angiogenic functions of human EPCs in vitro. Additionally, AS-IV inhibited thrombosis and repressed the infiltration of leukocytes into the thrombus and the production of proinflammatory cytokines in rats. Mechanistically, AS-IV inactivated PI3K/AKT signaling in rats. CONCLUSION: AS-IV prevents thrombus in an experimental DVT model by facilitating EPC angiogenesis and decreasing inflammation through inactivation of PI3K/AKT signaling.

MAGP1 maintains tumorigenicity and angiogenesis of laryngeal cancer by activating Wnt/ß-catenin/MMP7 pathway.

Microfibril-associated glycoprotein-1 (MAGP1), a crucial extracellular matrix protein, contributes to the initiation and progression of different cancers. However, the role of MAGP1 in laryngeal cancer is not clear. The purpose of this study was to investigate the clinical significance and biological function of MAGP1 in laryngeal cancer. MAGP1 was upregulated in public databases and laryngeal cancer tissues, and high MAGP1 expression led to a poor prognosis and was identified as an independent prognostic marker. Knocking-down MAGP1 inhibited laryngeal cancer cell growth and metastasis. According to gene set enrichment analysis, high MAGP1 expression revealed enrichment in Wnt/ß-catenin signaling and knocking-down MAGP1 in laryngeal cancer cells also caused degradation, de-activation, re-location and loss of stability of ß-catenin. Additionally, we observed MAGP1 in laryngeal cancer cells inhibits angiogenesis in an MMP7-dependent way. In conclusion, our study suggests a clinical role of MAGP1 in laryngeal cancer, signifying its potential as a therapeutic target in the future.