Migration Of Human Endothelial Cells Research Articles

Bispecific VEGF-A and Angiopoietin-2 Antagonist RO-101 Preclinical Efficacy in Model of Neovascular Eye Disease

ObjectiveTo investigate preclinical data regarding efficacy and biocompatibility of a bispecific protein, RO-101, with effects on Vascular Endothelial Growth Factor-A (VEGF-A) and Angiopoietin-2 (Ang-2) for use in retinal diseases. Study DesignExperimental study. SubjectsBrown Norway rats, New Zealand White Cross rabbits. MethodsPreclinical study data of RO-101 in terms of target-specific ELISA binding affinity to VEGF-A and Ang-2, vitreous half-life, inhibition of target-receptor interaction, laser Choroidal Neovascular Membrane (CNVM) animal model, Human Umbilical Vein Endothelial Cell (HUVEC) migration, and biocompatibility was obtained. Where applicable, study data was compared to other anti-VEGF agents. Main Outcome MeasuresBinding affinity, half-life, biocompatibility, and efficacy of RO-101. Neovascularization prevention by RO-101. ResultsRO-101 demonstrated a strong binding affinity for VEGF-A and Ang-2 and in vitro was able to inhibit binding to the receptor with higher affinity than faricimab. The half-life of RO-101 is comparable or longer than current VEGF inhibitors used in retinal disease. RO-101 was found to be biocompatible with retinal tissue in Brown Norway rats. RO-101 was as effective or more effective than current anti-VEGF therapeutics in causing regression of neovascular growth in vivo. ConclusionRO-101 is a promising candidate for use in retinal diseases. In preclinical models, RO-101 demonstrated similar or higher regression of neovascular growth to current anti-VEGF therapeutics with comparable or longer half-life. It also demonstrates strong binding affinity for VEGF-A and Ang-2. It also was shown to be biocompatible with retinal tissue in animal studies, indicating potential compatibility for use in humans.

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.

GelMA - PVAMA hydrogel loaded with MYDGF accelerates wound healing by promoting angiogenesis

Angiogenesis within the wound site is a necessary prerequisite for skin tissue regeneration during the process of wound healing. Skin vessels provide adequate nutrients and soluble bioactive factors to the wounded area and remove metabolites. In this study, we prepared a double-cross-linked hydrogel based on GelMA and PVAMA to load Myeloid-derived growth factor (MYDGF). Additionally, we evaluated the healing-promoting effect of the hydrogel using a full-thickness skin injury mouse model. Compared with the control and MYDGF-free hydrogels, the MYDGF-loaded hydrogels significantly increased the percentage of wound healing and promoted granulation tissue proliferation and epidermization. In vitro experiments have shown that MYDGF may augment the migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) by upregulating the expression of phosphorylated ERK and phosphorylated P38. These findings indicate that MYDGF-loaded hydrogels promote wound healing by accelerating angiogenesis.

Uncovering the Anti-Angiogenic Mechanisms of Centella asiatica via Network Pharmacology and Experimental Validation.

Centella asiatica (CA) has been used to address cancer for centuries in traditional Chinese medicine (TCM). Previous studies demonstrated its anti-angiogenesis efficacy, but the underlying mechanism of its action remains to be further clarified. This study aims to investigate the underlying mechanisms of CA and its triterpenes in anti-angiogenesis for cancer therapeutics through network pharmacology and experimental validation. Cytoscape was used to construct a network of compound-disease targets and protein-protein interactions (PPIs) from which core targets were identified. GO and KEGG analyses were performed using Metascape, and the AutoDock-Vina program was used to realize molecular docking for further verification. Then, VEGF165 was employed to establish an induced angiogenesis model. The anti-angiogenic effects of CA were evaluated through assays measuring cell proliferation, migration, and tubular structure formation. Twenty-five active ingredients in CA had potential targets for anti-angiogenesis including madecassoside, asiaticoside, madecassic acid, asiatic acid, and asiaticoside B. In total, 138 potential targets for CA were identified, with 19 core targets, including STAT3, SRC, MAPK1, and AKT1. A KEGG analysis showed that CA is implicated in cancer-related pathways, specifically PD-1 and AGE-RAGE. Molecular docking verified that the active components of CA have good binding energy with the first four important targets of angiogenesis. In experimental validation, the extracts and triterpenes of CA improved VEGF165-induced angiogenesis by reducing the proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). Our results initially demonstrate the effective components and great anti-angiogenic activity of CA. Evidence of the satisfactory anti-angiogenic action of the extracts and triterpenes from CA was verified, suggesting CA's significant potential as a prospective agent for the therapy of cancer.

TET2-mediated ECM1 hypomethylation promotes the neovascularization in active proliferative diabetic retinopathy

BackgroundStudies have shown that tet methylcytosine dioxygenase 2 (TET2) is highly expressed in diabetic retinopathy (DR), which reduces the DNA methylation of downstream gene promoters and activates the transcription. Abnormally expressed TET2 and downstream genes in a high-glucose environment are associated with retinal capillary leakage and neovascularization. Here, we investigated the downstream genes of TET2 and its potential association with neovascularization in proliferative diabetic retinopathy (PDR).MethodsGSE60436, GSE57362, and GSE158333 datasets were analyzed to identify TET2-related hypomethylated and upregulated genes in PDR. Gene expression and promoter methylation of these genes under high glucose treatment were verified. Moreover, TET2 knockdown was used to assess its impact on tube formation and migration in human retinal microvascular endothelial cells (HRMECs), as well as its influence on downstream genes.ResultsOur analysis identified three key genes (PARVB, PTPRE, ECM1) that were closely associated with TET2 regulation. High glucose-treated HRMECs exhibited increased expression of TET2 and ECM1 while decreasing the promoter methylation level of ECM1. Subsequently, TET2 knockdown led to decreased migration ability and tube formation function of HRMECs. We further found a decreased expression of PARVB, PTPRE, and ECM1, accompanied by an increase in the promoter methylation of ECM1.ConclusionsOur findings indicate the involvement of dysregulated TET2 expression in neovascularization by regulating the promoter methylation and transcription of downstream genes (notably ECM1), eventually leading to PDR. The TET2-induced hypomethylation of downstream gene promoters represents a potential therapeutic target and offers a novel perspective on the mechanism underlying neovascularization in PDR.Graphical

Diagnostic and prognostic potential of long non-coding RNA NORAD in patients with acute deep vein thrombosis and its role in endothelial cell function

BackgroundDeep venous thrombosis (DVT) is the common clinical cardiovascular disease, and easily develops into post-thrombotic syndrome (PTS). The study aimed to examine the clinical value of long non-coding RNA NORAD gene in the development of DVT and PTS. In vitro, the underlying mechanism was explored.MethodsSerum levels of lncRNA NORAD gene in 85 DVT cases and 85 healthy individuals were tested. The role of lncRNA NORAD gene in human umbilical vein endothelial cells (HUVECs) proliferation, migration and inflammation was examined. The candidate downstream target gene was predicted via bioinformatic analysis. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were done for the function annotation and pathway enrichment.ResultsLncRNA NORAD gene was at high expression in the serum of DVT patients, it can distinguish DVT patients from healthy controls with the area under the curve of 0.919. Elevated expression of lncRNA NORAD gene in PTS patients was detected, DVT cases with high expression of lncRNA NORAD gene were more susceptible to PTS. LncRNA NORAD gene knockdown promoted HUVECs’ proliferation, migration while suppressing cell apoptosis and inflammation. MiR-93-5p served as a target of lncRNA NORAD gene, and its overexpression reversed the role of lncRNA NORAD gene in the biological function of HUVECs. The target genes of miR-93-5p were enriched in HIF-1 signaling, TGF-beta signaling and PI3K-Akt signaling, protein-protein interaction (PPI) network indicated STAT3, MAPK1 to be the key targets.ConclusionsUpregulation of expression of lncRNA NORAD gene was a potential diagnostic biomarker for DVT and related to the development of PTS. LncRNA NORAD/miR-93-5p axis was involved in the progress of DVT through regulating endothelial cell function.

Self-assembled peptide hydrogel loaded with functional peptide Dentonin accelerates vascularized bone tissue regeneration in critical-size bone defects.

Regeneration of oral craniofacial bone defects is a complex process, and reconstruction of large bone defects without the use of exogenous cells or bioactive substances remains a major challenge. Hydrogels are highly hydrophilic polymer networks with the potential to promote bone tissue regeneration. In this study, functional peptide Dentonin was loaded onto self-assembled peptide hydrogels (RAD) to constitute functionally self-assembling peptide RAD/Dentonin hydrogel scaffolds with a view that RAD/Dentonin hydrogel could facilitate vascularized bone regeneration in critical-size calvarial defects. The functionalized peptide RAD/Dentonin forms highly ordered β-sheet supramolecular structures via non-covalent interactions like hydrogen bonding, ultimately assembling into nano-fiber network. RAD/Dentonin hydrogels exhibited desirable porosity and swelling properties, and appropriate biodegradability. RAD/Dentonin hydrogel supported the adhesion, proliferation and three-dimensional migration of bone marrow mesenchymal stem cells (BMSCs) and has the potential to induce differentiation of BMSCs towards osteogenesis through activation of the Wnt/β-catenin pathway. Moreover, RAD/Dentonin hydrogel modulated paracrine secretion of BMSCs and increased the migration, tube formation and angiogenic gene expression of human umbilical vein endothelial cells (HUVECs), which boosted the angiogenic capacity of HUVECs. In vivo, RAD/Dentonin hydrogel significantly strengthened vascularized bone formation in rat calvarial defect. Taken together, these results indicated that the functionalized self-assembling peptide RAD/Dentonin hydrogel effectively enhance osteogenic differentiation of BMSCs, indirectly induce angiogenic effects in HUVECs, and facilitate vascularized bone regeneration in vivo. Thus, it is a promising bioactive material for oral and maxillofacial regeneration.

Topical Application of Cell-Penetrating Peptide Modified Anti-VEGF Drug Alleviated Choroidal Neovascularization in Mice.

Age-related macular degeneration (AMD) stands as the foremost cause of irreversible central vision impairment, marked by choroidal neovascularization (CNV). The prevailing clinical approach to AMD treatment relies on intravitreal injections of anti-vascular endothelial growth factor (VEGF) drugs. However, this method is encumbered by diverse complications, prompting exploration of non-invasive alternatives such as ocular administration via eye drops for anti-VEGF therapy. Two complexes, 5-FITC-CPP-Ranibizumab (5-FCR) and 5-FITC-CPP-Conbercept (5-FCC), were synthesized by incorporating the anti-VEGF drugs Ranibizumab (RBZ) or Conbercept (CBC) with cell-penetrating peptide (CPP). Circular dichroism spectrum (CD) facilitated complexes characterization. Eye drops was utilized to address laser-induced CNV in mice. Fluorescein fundus angiography (FFA) observe the CNV lesion, while FITC-dextran and IB4 dual fluorescent staining, along with hematoxylin-eosin (HE) staining, assessed in lesion size. Tissue immunofluorescence examined CD31 and VEGF expression in choroidal/retinal pigment epithelial (RPE) tissues. Biocompatibility and biosafety of 5-FCR and 5-FCC was evaluated through histological examination of various organs or cell experiments. Both 5-FCR and 5-FCC exhibited favorable biocompatibility and safety profiles. VEGF-induced migration of Human umbilical vein endothelial cells (HUVECs) significantly decreased post-5-FCR/5-FCC treatment. Additionally, both complexes suppressed VEGF-induced tube formation in HUVECs. FFA results revealed a significant improvement in retinal exudation in mice. Histological examination unveiled the lesion areas in the 5-FCR and 5-FCC groups showed a significant reduction compared to the control group. Similar outcomes were observed in histological sections of the RPE-choroid-sclera flat mounts. In this study, utilizing the properties of CPP and two anti-VEGF drugs, we successfully synthesized two complexes, 5-FCR and 5-FCC, through a straightforward approach. Effectively delivering the anti-VEGF drugs to the target area in a non-invasive manner, suppressing the progression of laser-induced CNV. This offers a novel approach for the treatment of wet AMD.

A Model to Predict Prognosis of Renal Cell Clear Cell Carcinoma Based on 3 Angiogenesis-related Long Non-coding RNAs.

Background: Tumor angiogenesis is closely related to the progression of clear cell renal cell carcinoma (ccRCC). Long non-coding RNAs (lncRNAs) regulating angiogenesis could be potential biomarkers for predicting ccRCC prognosis. With this study, we aimed to construct a prognostic model based on lncRNAs and explore its underlying mechanisms. Methods: RNA data and clinical information were obtained from The Cancer Genome Atlas (TCGA) database. Angiogenesis-related genes (ARGs) were extracted from the Molecular Signatures database. Pearson correlation and LASSO and COX regression analyses were performed to identify survival-related AR-lncRNAs (sAR-lncRNAs) and construct a prognostic model. The predictive power of the prognostic model was verified according to Kaplan‒Meier curve, receiver operating characteristic (ROC) curve and nomogram analyses. The correlation between the prognostic model and clinicopathological characteristics was assessed via univariate and multivariate analyses. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was subsequently performed to elucidate the mechanisms of the sAR-lncRNAs. In vitro qPCR, immunohistochemistry, migration and invasion assays were conducted to confirm the angiogenic function of sAR-lncRNAs. Results: Three sAR-lncRNAs were used to construct a prognostic model. The model was moderately accurate in predicting 1- , 3- and 5-year ccRCC prognosis, and the risk score according to this model was closely related to clinicopathological characteristics such as T grade and T stage. A nomogram was constructed to precisely estimate the overall survival of ccRCC patients. KEGG enrichment analysis indicated that the MAPK and Notch pathways were highly enriched in high-risk patients. Additionally, we found that the expression of the lncRNAs AC005324.4 and AC104964.4 in the prognostic model was lower in ccRCC cell lines and cancer tissues than in the HK-2 cell line and paracancerous tissues, while the expression of the lncRNA AC087482.1 showed the opposite trend. In a coculture model, knockdown of lncRNA AC005324.4 and lncRNA AC104964.4 significantly promoted the migration and invasion of human umbilical vein endothelial cells (HUVECs), but siR-AC087482.1 transfection alleviated these effects. Conclusions: We constructed a prognostic model based on 3 sAR-lncRNAs and validated its value in clinicopathological characteristics and prognostic prediction of ccRCC patients, providing a new perspective for ccRCC treatment decision making.

(R,S)-Equol 7-β-D-glucuronide, but not other circulating isoflavone metabolites, modulates migration and tubulogenesis in human aortic endothelial cells targeting the VEGF pathway.

Current knowledge indicates that the consumption of isoflavone-rich foodstuffs can have a beneficial impact on cardiovascular health. To what extent these isoflavones act as the main actors of that benefit is less clear. Genistein (GEN), daidzein (DAZ), and the DAZ-derived microbial metabolite equol (Eq) exhibit antiangiogenic effects in vitro, but their low bloodstream concentrations make it difficult to rationalize the in vivo effects. Their derived phase-II metabolites (glucuronides and sulfates) are major metabolites found in plasma, but their role as antiangiogenic molecules remains unexplored. We aimed here to first assess the anti-angiogenic activities of the main circulating isoflavone metabolites (glucuronides and sulfates) and compare them with their corresponding free forms at physiological concentrations (0.1-10 μM). The effects of the conjugated vs. free forms on tubulogenesis, cell migration, and VEGF-induced signalling were investigated in primary human aortic endothelial cells (HAECs). While (R,S)-equol 7-β-D-glucuronide (Eq 7-glur) exerted dose-dependent inhibition of tubulogenesis and endothelial migration comparable to that exerted by the free forms (GEN, DAZ, and Eq), the rest of the phase-II conjugates exhibited no significant effects. The underlying molecular mechanisms were independent of the bFGF but related to the modulation of the VEGF pathway. Besides, the observed dissimilar cellular metabolism (conjugation/deconjugation) places the phase-II metabolites as precursors of the free forms; however, the question of whether this metabolism impacts their biological activity requires additional studies. These new insights suggest that isoflavones and their circulating metabolites, including Eq 7-glur, may be involved in cardiovascular health (e.g., targeting angiogenesis).

Nano-enzyme functionalized hydrogels promote diabetic wound healing through immune microenvironment modulation.

Non-healing diabetic wounds often culminate in amputation and mortality. The main pathophysiological features in diabetic wounds involve the accumulation of M1-type macrophages and excessive oxidative stress. In this study, we engineered a nano-enzyme functionalized hydrogel by incorporating a magnesium ion-doped molybdenum-based polymetallic oxide (Mg-POM), a novel bioactive nano-enzyme, into a GelMA hydrogel, to obtain the GelMA@Mg-POM system to enhance diabetic wound healing. GelMA@Mg-POM was crosslinked using UV light, yielding a hydrogel with a uniformly porous three-dimensional mesh structure. In vitro experiments showed that GelMA@Mg-POM extraction significantly enhanced human umbilical vein endothelial cell (HUVEC) migration, scavenged ROS, improved the inflammatory microenvironment, induced macrophage reprogramming towards M2, and promoted HUVEC regeneration of CD31 and fibroblast regeneration of type I collagen. In in vivo experiments, diabetic rat wounds treated with GelMA@Mg-POM displayed enhanced granulation tissue genesis and collagen production, as evidenced by HE and Masson staining. Immunohistochemistry demonstrated the ability of GelMA@Mg-POM to mitigate macrophage-associated inflammatory responses and promote angiogenesis. Overall, these findings suggest that GelMA@Mg-POM holds significant promise as a biomaterial for treating diabetic wounds.

Extended siponimod release via low-porosity PLGA fibres: a comprehensive three-month in vitro evaluation for neovascular ocular diseases.

Neovascular ocular diseases are among the most common causes of preventable or treatable vision loss. Their management involves lifelong, intravitreal injections of anti-vascular endothelial growth factor (VEGF) therapeutics to inhibit neovascularization, the key pathological step in these diseases. Anti-VEGF products approved for ocular administration are expensive biological agents with limited stability and short half-life. Additionally, their therapeutic advantages are hindered by high treatment resistance, poor patient compliance and the need for frequent, invasive administration. Herein, we used electrospinning to develop a unique, non-porous, PLGA implant for the ocular delivery of siponimod to improve ocular neovascular disease management. Siponimod is an FDA-approved drug for multiple sclerosis with a novel indication as a potential ocular angiogenesis inhibitor. The electrospinning conditions were optimised to produce a microfibrous, PLGA matte that was cut and rolled into the desired implant size. Physical characterisation techniques (Raman, PXRD, DSC and FTIR) indicated siponimod was distributed uniformly within the electrospun fibres as a stabilised, amorphous, solid dispersion with a character modifying drug-polymer interaction. Siponimod dispersion and drug-polymer interactions contributed to the formation of smooth fibres, with reduced porous structures. The apparent reduced porosity, coupled with the drug's hydrophobic dispersion, afforded resistance to water penetration. This led to a slow, controlled, Higuchi-type drug diffusion, with ∼30% of the siponimod load released over 90 days. The released drug inhibited human retinal microvascular endothelial cell migration and did not affect the cells' metabolic activity at different time points. The electrospun implant was physically stable after incubation under stress conditions for three months. This novel siponimod intravitreal implant broadens the therapeutic possibilities for neovascular ocular diseases, representing a potential alternative to biological, anti-VEGF treatments due to lower financial and stability burdens. Additionally, siponimod interaction with PLGA provides a unique opportunity to sustain the drug release from the electrospun fibres, thereby reducing the frequency of intravitreal injection and improving patient adherence.

In Vitro Experimental Approach for Studying Human Pulmonary Artery Smooth Muscle Cells and Endothelial Cells Proliferation and Migration.

Pulmonary arterial hypertension (PAH) is a severe vascular disease characterized by persistent precapillary pulmonary hypertension, leading to right heart failure and death. Despite intense research in the last decades, PAH remains an incurable disease with high morbidity and mortality. New directions and therapies to improve understanding and treatment of PAH are desperately needed. The pathological mechanisms leading to this fatal disorder remain mostly undetermined, although structural remodeling of the pulmonary vessels is known to be an early feature of PAH. Pulmonary vascular remodeling includes proliferation and migration of pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial cells (PAECs). The use of in vitro approaches is useful to delineate the mechanisms involved in the pathogenesis of PAH and to identify new therapeutic strategies for PAH. In this chapter, we describe protocols for culturing and assessing proliferation and migration of human PASMCs and PAECs.

Endothelium-Mimicking Bilayer Vascular Grafts with Dual-Releasing of NO/H2S for Anti-Inflammation and Anticalcification.

Vascular complications caused by diabetes impair the activities of endothelial nitric oxide synthase (eNOS) and cystathionine γ-lyase (CSE), resulting in decreased physiological levels of nitric oxide (NO) and hydrogen sulfide (H2S). The low bioavailability of NO and H2S hinders the endothelialization of vascular grafts. In this study, endothelium-mimicking bilayer vascular grafts were designed with spatiotemporally controlled dual releases of NO and H2S for in situ endothelialization and angiogenesis. Keratin-based H2S donor was synthesized and electrospun with poly(l-lactide-co-ε-caprolactone) (PLCL) as the outer layer of the graft to release H2S. Hyaluronic acid, one of the major glycosaminoglycans in endothelial glycocalyx, was complexed with Cu ions as the inner layer to mimic glutathione peroxidase (GPx) and maintain long-term physiological NO flux. The synergistic effects of NO and H2S of bilayer grafts selectively promoted the regeneration and migration of human umbilical vascular endothelial cells (HUVECs), while inhibiting the overproliferation of human umbilical artery smooth muscle cells (HUASMCs). Bilayer grafts could effectively prevent vascular calcification, reduce inflammation, and alleviate endothelial dysfunction. The in vivo study in a rat abdominal aorta replacement model for 1 month showed that the graft had a good patency rate and had potential for vascular remodeling in situ.

Ultrahigh-Efficacy VEGF Neutralization Using Carbonized Nanodonuts: Implications for Intraocular Anti-Angiogenic Therapy.

Ocular angiogenesis, associated with diseases such as retinopathy of prematurity and diabetic retinopathy, is a leading cause of irreversible vision loss. Herein, carbon nanodonuts (CNDs) with a donut-shaped structure are synthesized using sodium alginate (SA) and 1,8-diaminooctane (DAO) through a one-step thermal process. The formation of SA/DAO-CNDs occurs through a crosslinking reaction between SA and DAO, creating amide bonds followed by partial carbonization. In human retinal pigment epithelialcells exposed to H2 O2 or lipopolysaccharide, the SA/DAO-CNDs display a more than fivefold reduction in reactive oxygen species and proinflammatory cytokines, such as IL-6 and IL-1β, when compared to carbonized nanomaterials produced exclusively from SA. Furthermore, the CNDs effectively inhibit vascular endothelial growth factor A-165 (VEGF-A165 )-induced cell migration and tube formation in human umbilical vein endothelial cells due to their strong affinity for VEGF-A165 , with a dissociation constant of 2.2 × 10-14 M, over 1600 times stronger than the commercial drug bevacizumab (Avastin). Trypsin digestion coupled with LC-MS/MS analysis reveals that VEGF-A165 interacts with SA/DAO-CNDs through its heparin-binding domain, leading to activity loss. The SA/DAO-CNDs demonstrate excellent biocompatibility and potent anti-angiogenic effects in chicken embryos and rabbit eyes. These findings suggest that SA/DAO-CNDs hold promise as a therapeutic agent for treating various angiogenesis-related ocular diseases.

Effects of preconditioning with TNFα and IFNγ in angiogenic potential of mesenchymal stromal cell-derived extracellular vesicles.

Introduction: Mesenchymal stromal cells (MSCs) have demonstrated therapeutic properties both in vitro and in vivo to treat various diseases, including anti-inflammatory, immunomodulatory and pro-angiogenic effects. These therapeutic effects are mediated by their secretome composed of soluble factors and extracellular vesicles (EVs). The composition of EVs reflects the molecular and functional characteristics of parental cells. MSC preconditioning can alter the composition of EVs, thereby influencing their therapeutic potential. Methods: MSCs were subjected to preconditioning with two cytokines, TNFα and IFNγ. Following 24h of preconditioning, MSC-EVs secreted into the culture supernatant were isolated through tangential filtration. Particle concentration and size distribution were measured by nanoparticle tracking analysis, and the surface antigen expression of the EV-specific CD63 was quantified via Enzyme Linked ImmunoSorbent Assay. The angiogenic potential of MSCEVs obtained after preconditioning MSCs was assessed by the analysis of their protein composition and their influence on human umbilical vein endothelial cell (HUVECs) proliferation, migration, and tube-forming ability. Results: Preconditioning with TNFα and IFNγ did not influence the MSC-EV profile but did induce changes in their protein content. Indeed, the expression of pro-angiogenic proteins increased in EVs from preconditioned MSCs compared to EVs from no-preconditioned MSCs. EVs from preconditioned MSCs tend to stimulate HUVEC migration, proliferation and tubeforming ability. These observations imply the presence of a pro-angiogenic potential in EVs obtained after preconditioning of MSCs with TNFα and IFNγ. Discussion: In conclusion, it appears that the pro-angiogenic potential of EVs is enhanced through preconditioning of MSCs with TNFα and IFNγ. The use of these MSCs-EVs in therapy would circumvent the limitations of current cell-based therapies. Indeed, the therapeutic potential of MSC-EVs presents an attractive strategy for exploiting the clinical benefits of MSC therapy. For example, in the field of regenerative medicine, the exploitation of cell-free therapy using highly pro-angiogenic MSC-EVs is of great interest.

LncRNA SNHG8 regulates the migration and angiogenesis of pHUVECs induced by high glucose via the TRPM7/ERK1/2 signaling axis

This study aimed to evaluate the regulatory effect and molecular mechanism of long noncoding RNA small nucleolus RNA host gene 8 (LncRNA SNHG8) in the migration and angiogenesis of primary human umbilical vein endothelial cells (pHUVECs) under high-glucose (HG) conditions. The HG-induced endothelial injury model was established in vitro.The cell model of silencing SNHG8, overexpressing SNHG8, and silencing TRPM7 was established by transfecting SNHG8-siRNA, SNHG8 plasmid and TRPM7-siRNA into cells with liposomes.The SNHG8 level was determined through reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The expression levels of transient receptor potential melastatin 7 (TRPM7), endothelial nitric oxide synthase (eNOS), p-eNOS, extracellular signal-regulated kinase 1/2(ERK1/2), and p-ERK1/2 were assessed through western blot. Nitric oxide (NO) levels were measured with DAF-FM. pHUVEC migration was examined through wound healing and Transwell assay, and pHUVEC angiogenesis was observed through a tube formation assay. Results showed that HG promoted the expression of lncRNA SNHG8 and TRPM7 and decreased the ratio of p-eNOS/eNOS and p-ERK1/2/ERK1/2 in pHUVECs . NO production, migration , and angiogenesis were inhibited in pHUVECs under HG conditions. Silencing lncRNA SNHG8 and TRPM7 could significantly reverse the HG-induced decrease in eNOS activation, NO production , migration, and angiogenesis . SNHG8 and U0126 (ERK pathway inhibitor) overexpression enhanced the HG effects, whereas using U0126 did not affect the TRPM7 expression. In conclusion, lncRNA SNHG8 participates in HG-induced endothelial cell injury and likely regulates NO production, migration, and angiogenesis of pHUVECs via the TRPM7/ERK1/2 signaling axis.

CD73-positive pediatric urethral mesenchymal stem-like cell-derived small extracellular vesicles stimulate angiogenesis

IntroductionAngiogenesis plays an important role in the repair of urethral injury, and stem cells and their secretomes can promote angiogenesis. We obtained pediatric urethral mesenchymal stem-like cells (PU-MSLCs) in an earlier study. This project studied the pro-angiogenic effect of PU-MSLC-derived small extracellular vesicles (PUMSLC-sEVs) and the underlying mechanisms. Materials and methodsPUMSLCs and PUMSLC-sEVs were cultivated and identified. Then, biological methods such as the ethynyl deoxyuridine (EdU) incorporation assay, Cell Counting Kit-8 (CCK-8) assay, scratch wound assay, Transwell assay, and tube formation assay were used to study the effect of PUMSLC-sEVs on the proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). We explored whether the proangiogenic effect of PUMSLC-sEVs is related to CD73 and whether adenosine (ADO, a CD73 metabolite) promoted angiogenesis. GraphPad Prism 8 software was used for data analysis. ResultsWe observed that PUMSLC-sEVs significantly promoted the proliferation, migration, and tube-forming abilities of HUVECs. PUMSLC-sEVs delivered CD73 molecules to HUVECs to promote angiogenesis. The angiogenic ability of HUVECs was enhanced after treatment with extracellular ADO produced by CD73, and PUMSLC-sEVs further promoted angiogenesis by activating Adenosine Receptor A2A (A2AR). ConclusionsThese observations suggest that PUMSLC-sEVs promote angiogenesis, possibly through activation of the CD73/ADO/A2AR signaling axis.

Verteporfin-Loaded Bioadhesive Nanoparticles for the Prevention of Hypertrophic Scar.

Hypertrophic scarring (HS) is a common skin injury complication with unmet needs. Verteporfin (VP) should be an ideal HS-targeted therapeutic drug due to its efficient fibrosis and angiogenesis inhibitory abilities. However, its application is restricted by its side effects such as dose-dependent cytotoxicity on normal cells. Herein, the bioadhesive nanoparticles encapsulated VP (VP/BNPs) are successfully developed to attenuate the side effects of VP and enhance its HS inhibition effects by limiting VP releasing slowly and stably in the lesion site but not diffusing easily to normal tissues. VP/BNPs displayed significant inhibition on the proliferation, migration, collagen deposition, and vessel formation of human hypertrophic scar fibroblasts (HSFBs) and dermal vascular endothelial cells (HDVECs). In a rat tail HS model, VP/BNPs treated HS exhibits dramatic scar repression with almost no side effects compared with free VP or VP-loaded non-bioadhesive nanoparticles (VP/NNPs) administration. Further immunofluorescence analysis on scar tissue serial sections validated VP/BNPs effectively inhibited the collagen deposition and angiogenesis by firmly confined in the scar tissue and persistently releasing VP targeted to nucleus Yes-associated protein (nYAP) of HSFBs and HDVECs. These findings collectively suggest that VP/BNPs can be a promising and technically advantageous agent for HS therapies.

Multi-crosslinked hydrogel built with hyaluronic acid-tyramine, thiolated glycol chitosan and copper-doped bioglass nanoparticles for expediting wound healing

The migration of fibroblasts and endothelial cells is a critical determinant of wound-healing outcomes for skin injuries. Here, hyaluronic acid-tyramine (HAT) and thiolated glycol chitosan (TGC) conjugates were combined with copper-doped bioglass (ACuBG) nanoparticles to build a novel type of multi-crosslinked hydrogel for stimulating the migration of cells, and thus, expediting wound healing. The optimally devised HAT/TGC/ACuBG gels had markedly improved strength and stiffness compared to the gels built from either HAT or TGC while showing sufficient elasticity, which contributes to stimulating the migration of fibroblasts. The sustainable release of silicon and copper ions from the gels was found to jointly induce the migration of human umbilical vein endothelial cells. The results based on mouse full-thickness skin defects demonstrated that they were able to fully restore the skin defects with formation of complete appendages within two weeks, suggesting their promising potency for use in expediting wound healing.