A single dose of VEGF-A circular RNA sustains in situ long-term expression of protein to accelerate diabetic wound healing.

Diabetic foot ulcer (DFU), which is characterised by damage to minute blood vessels or capillaries around wounds, is one of the most serious and dreaded complications of diabetes. It is challenging to repair chronic non-healing DFU wounds. Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis and promotes wound healing in DFU. However, it is difficult to sustainably deliver VEGF to the wound site owing to its poor stability and easy degradation. To overcome this challenge, lipid nanoparticles (LNP) encapsulating circular RNA (circRNA) encoding VEGF-A have been developed to continuously generate and release VEGF-A and accelerate diabetic wound healing. First, VEGF-A circRNA was synthesized using group I intron autocatalysis strategy and confirmed by enzyme digestion, polymerase chain reaction, and sequencing assay. VEGF-A circRNA was encapsulated in ionizable lipid U-105-derived LNP (U-LNP) using microfluidic technology to fabricate U-LNP/VEGF-A circRNA. For comparison, a commercially ionizable lipid ALC-0315-derived LNP (A-LNP) encapsulating circRNA (A-LNP/circRNA) was used. Dynamic light scattering and transmission electron microscopy characterization indicated that U-LNP/circRNA had spherical structure with an average diameter of 108.5 nm, a polydispersity index of 0.22, and a zeta potential of -3.31 mV. The messenger RNA (mRNA) encapsulation efficiency (EE%) of U-LNP was 87.12%. In vitro transfection data confirmed better stability and long-term VEGF-A expression of circRNA compared with linear mRNA. Assessment of cytotoxicity and innate immunity further revealed that U-LNP/circRNA was biocompatible and induced a weak congenital immune response. Cell scratch and angiogenesis tests demonstrated the bioactivity of U-LNP/VEGF-A circRNA owing to its VEGF-A expression. In situ bioluminescence imaging of firefly luciferase (F-Luc) probe and ELISA demonstrated that circRNA had long-term and strong expression of VEGF-A in the first week, and a gradual decrease in the next week at the wound site and surrounding areas. Finally, a diabetic mouse model was used to validate the healing effect of U-LNP/VEGF-A circRNA formulation. The results showed that a single dose of U-LNP/VEGF-A circRNA administered by dripping resulted in almost complete wound recovery on day 12, which was significantly superior to that of U-LNP/VEGF-A linear mRNA, and it also outperformed recombinant human vascular endothelial growth factor (rhVEGF) injection and A-LNP/circRNA dripping. Histological analysis confirmed the healing efficiency and low toxicity of U-LNP/VEGF-A circRNA formulation. Together, VEGF-A circRNA delivered by U-105-derived LNP showed good performance in wound healing, which was ascribed to the long-term expression and continuous release of VEGF-A, and has potential applications for the treatment of diabetic foot ulcer wounds.

Elevated VEGF-A Levels in the Aqueous Humor of Patients With Primary Open Angle Glaucoma.

BACKGROUND/AIM: The purpose of the current study was to compare the vascular endothelial growth factor-A (VEGF-A) levels in the aqueous humor of patients with primary open angle glaucoma (POAG) and non-glaucomatous eyes and reveal any potential statistically significant correlations. PATIENTS AND METHODS: This was an observational cross-sectional study. Aqueous humor samples (50-100 µl) were collected under aseptic conditions, from the anterior chamber at the start of glaucoma or cataract surgery. The levels of VEGF-A were measured using a multiplex bead-based immunoassay. RESULTS: Aqueous humor samples were obtained from 76 participants: 39 with POAG and 36 with age-related cataracts as controls. VEGF-A levels were significantly elevated in the POAG group (166.37±110.04 pg/ml, p=0.011) compared to the control group (119.02±49.09 pg/ml). The receiver operating characteristic (ROC) analysis showed that VEGF-A had significant prognostic ability for POAG (AUC=0.67; p=0.006). An optimal cut-off for VEGF-A was found to be 148.5 pg/ml with a sensitivity of 54%, specificity of 81.1%, positive prognostic value (PPV) of 75% and negative prognostic value (NPV) of 62.5%. Logistic regression analysis showed that after adjusting for sex and age, patients with VEGF-A higher than 148.5 pg/ml had almost 10 times greater likelihood for POAG. CONCLUSION: VEGF-A is elevated in patients with POAG and can potentially have a prognostic ability for these patients.

Local complement activation and modulation in mucosal immunity.

The complement system is an evolutionarily conserved arm of innate immunity, which forms one of the first lines of host response to pathogens and assists in the clearance of debris. A deficiency in key activators/amplifiers of the cascade results in recurrent infection, whereas a deficiency in regulating the cascade predisposes to accelerated organ failure, as observed in colitis and transplant rejection. Given that there are over 60 proteins in this system, it has become an attractive target for immunotherapeutics, many of which are United States Food and Drug Administration-approved or in multiple phase 2/3 clinical trials. Moreover, there have been key advances in the last few years in the understanding of how the complement system operates locally in tissues, independent of its activities in circulation. In this review, we will put into perspective the abovementioned discoveries to optimally modulate the spatiotemporal nature of complement activation and regulation at mucosal surfaces.

MiR-9-3 hypermethylation is associated with stages of diabetic retinopathy.

Purpose: To investigate the potential relation between methylation of miR-9-3 and stages of diabetic retinopathy (DR). Additionally, we explored whether miR-9-3 methylation impacts the serum levels of Vascular Endothelial Growth Factor (VEGF). Methods: A cross-sectional study was conducted with 170 participants with type 2 diabetes, including a control group (n = 64) and a diabetes retinopathy group (n = 106), which was further divided into NPDR (n = 58) and PDR (n = 48) subgroups. Epidemiological, clinical, anthropometric, biochemical ELISA assay were analysed. DNA extracted from leukocytes was used to profile miR-9-3 methylation using PCR-MSP. Results: MiR-9-3 hypermethylated profile was higher in the DR group (p < 0.001) and PDR subgroup compared to DM2 control group (p < 0.001). The hypermethylated profile in the PDR subgroup was also higher compared to NPDR subgroup (p < 0.001). There was no difference between DM2 control and NPDR group (p = 0.234). Logistic regression showed that miR-9-3 hypermethylation increases the odds of presenting DR (OR: 2.826; p = 0.002) and PDR (OR: 5.472; p < 0.001). In addition, hypermethylation of miR-9-3 in the DR and NPDR subgroup was associated with higher serum VEGF-A levels (p = 0.012 and p = 0.025, respectively). Conclusion: The methylation profile of the miR-9-3 promoter increases the risk of developing PDR. Higher levels of VEGF-A are associated with miR-9-3 hypermethylated profile in patients in the DR and NPDR stages. Supplementary Information: The online version contains supplementary material available at 10.1007/s40200-024-01411-9.

Metastatic adult Wilms' tumor managed by chemotherapy, immunotherapy and target therapy: a case report.

Wilms' tumor is a rare type of tumor in adult. Herein, we reported a case of 37-year-old female with adult Wilms' tumor (AWT) admitted in our institution. After a multidisciplinary team discussion, she underwent receiving immunotherapy plus chemotherapy and VEGF-targeted therapy. The tumor got smaller obviously after eight cycles of treatment. Our present case suggested that immunotherapy and anti-angiogenesis combined with chemotherapy is promising new approach for treating AWT. Moreover, we review the literatures reporting AWT with the purpose to improve the understanding of AWT treatment.

A 37-year-old woman discovered a huge renal mass with multiple lymph node metastases. After ultrasound-guided needle biopsy of tumor tissue in the right kidney, she was found to be a rare adult Wilms' tumor. After a multidisciplinary team discussion, she underwent systemic therapy. Then, we gave her two cycles of treatment, as the tumor got smaller. Then, we continued to give her six cycles of treatment. Now, she is in good condition.

Effect of Stanozolol and/or Cannabis Abuse on Hypertrophic Mechanism and Oxidative Stress of Male Albino Rat Cardiac Tissue in Relation to Exercise: A Sport Abuse Practice.

Adolescents commonly co-abuse many drugs including anabolic androgenic steroids either they are athletes or non-athletes. Stanozolol is the major anabolic used in recent years and was reported grouped with cannabis. The current study aimed at evaluating the biochemical and histopathological changes related to the hypertrophic effects of stanozolol and/or cannabis whether in condition of exercise practice or sedentary conditions. Adult male Wistar albino rats received either stanozolol (5 mg/kg, s.c), cannabis (10 mg/kg, i.p.), and a combination of both once daily for two months. Swimming exercise protocol was applied as a training model. Relative heart weight, oxidative stress biomarkers, cardiac tissue fibrotic markers were evaluated. Left ventricular morphometric analysis and collagen quantification was done. The combined treatment exhibited serious detrimental effects on the heart tissues. It increased heart tissue fibrotic markers (Masson's trichrome stain (p < 0.001), cardiac COL3 (p < 0.0001), and VEGF-A (p < 0.05)), lowered heart glutathione levels (p < 0.05) and dramatically elevated oxidative stress (increased malondialdehyde (p < 0.0001) and 8-OHDG (p < 0.0001)). Training was not ameliorating for the observed effects. Misuse of cannabis and stanozolol resulted in more hypertrophic consequences of the heart than either drug alone, which were at least largely assigned to oxidative stress, heart tissue fibrotic indicators, histological alterations, and morphometric changes.

Pharmacological inhibition of PDGF-C/neuropilin-1 interaction: A novel strategy to reduce melanoma metastatic potential.

Activation of neuropilin-1 (NRP-1) by platelet derived growth factor (PDGF)-C sustains melanoma invasiveness. Therefore, in the search of novel agents capable of reducing melanoma spreading, PDGF-C/NRP-1 interaction was investigated as a potential druggable target. Since the PDGF-C region involved in NRP-1 binding is not yet known, based on the sequence and structural homology between PDGF-C and vascular endothelial growth factor-A (VEGF-A), we hypothesized that the NRP-1 b1 domain region involved in the interaction with VEGF-A might also be required for PDGF-C binding. Hence, this region was selected from the protein crystal structure and used as target in the molecular docking procedure. In the following virtual screening, compounds from a DrugBank database were used as query ligands to identify agents potentially capable of disrupting NRP-1/PDGF-C interaction. Among the top 45 candidates with the highest affinity, five drugs were selected based on the safety profile, lack of hormonal effects, and current availability in the market: the antipsychotic pimozide, antidiabetic gliclazide, antiallergic cromolyn sodium, anticancer tyrosine kinase inhibitor entrectinib, and antihistamine azelastine. Analysis of drug influence on PDGF-C in vitro binding to NRP-1 and PDGF-C induced migration of human melanoma cells expressing NRP-1, indicated gliclazide and entrectinib as the most specific agents that were active at clinically achievable and non-toxic concentrations. Both drugs also reverted PDGF-C ability to stimulate extracellular matrix invasion by melanoma cells resistant to BRAF inhibitors. The inhibitory effect on tumor cell motility involved a decrease of p130Cas phosphorylation, a signal transduction pathway activated by PDGF-C-mediated stimulation of NRP-1.

Thymic Stromal Lymphopoietin (TSLP) Is Cleaved by Human Mast Cell Tryptase and Chymase.

Thymic stromal lymphopoietin (TSLP), mainly expressed by epithelial cells, plays a central role in asthma. In humans, TSLP exists in two variants: the long form TSLP (lfTSLP) and a shorter TSLP isoform (sfTSLP). Macrophages (HLMs) and mast cells (HLMCs) are in close proximity in the human lung and play key roles in asthma. We evaluated the early proteolytic effects of tryptase and chymase released by HLMCs on TSLP by mass spectrometry. We also investigated whether TSLP and its fragments generated by these enzymes induce angiogenic factor release from HLMs. Mass spectrometry (MS) allowed the identification of TSLP cleavage sites caused by tryptase and chymase. Recombinant human TSLP treated with recombinant tryptase showed the production of 1-97 and 98-132 fragments. Recombinant chymase treatment of TSLP generated two peptides, 1-36 and 37-132. lfTSLP induced the release of VEGF-A, the most potent angiogenic factor, from HLMs. By contrast, the four TSLP fragments generated by tryptase and chymase failed to activate HLMs. Long-term TSLP incubation with furin generated two peptides devoid of activating property on HLMs. These results unveil an intricate interplay between mast cell-derived proteases and TSLP. These findings have potential relevance in understanding novel aspects of asthma pathobiology.

Dysregulated VEGF/VEGFR-2 Signaling and Plexogenic Lesions in the Embryonic Lungs of Chickens Predisposed to Pulmonary Arterial Hypertension.

Plexiform lesions are a hallmark of pulmonary arterial hypertension (PAH) in humans and are proposed to stem from dysfunctional angioblasts. Broiler chickens (Gallus gallus) are highly susceptible to PAH, with plexiform-like lesions observed in newly hatched individuals. Here, we reported the emergence of plexiform-like lesions in the embryonic lungs of broiler chickens. Lung samples were collected from broiler chickens at embryonic day 20 (E20), hatch, and one-day-old, with PAH-resistant layer chickens as controls. Plexiform lesions consisting of CD133+/vascular endothelial growth factor receptor type-2 (VEGFR-2)+ angioblasts were exclusively observed in broiler embryos and sporadically in layer embryos. Distinct gene profiles of angiogenic factors were observed between the two strains, with impaired VEGF-A/VEGFR-2 signaling correlating with lesion development and reduced arteriogenesis. Pharmaceutical inhibition of VEGFR-2 resulted in enhanced lesion development in layer embryos. Moreover, broiler embryonic lungs displayed increased activation of HIF-1α and nuclear factor erythroid 2-related factor 2 (Nrf2), indicating a hypoxic state. Remarkably, we found a negative correlation between lung Nrf2 activation and VEGF-A and VEGFR-2 expression. In vitro studies indicated that Nrf2 overactivation restricted VEGF signaling in endothelial progenitor cells. The findings from broiler embryos suggest an association between plexiform lesion development and impaired VEGF system due to aberrant activation of Nrf2.

On Connective Tissue Mast Cells as Protectors of Life, Reproduction, and Progeny.

The connective tissue mast cell (MC), a sentinel tissue-residing secretory immune cell, has been preserved in all vertebrate classes since approximately 500 million years. No physiological role of the MC has yet been established. Considering the power of natural selection of cells during evolution, it is likely that the MCs exert essential yet unidentified life-promoting actions. All vertebrates feature a circulatory system, and the MCs interact readily with the vasculature. It is notable that embryonic MC progenitors are generated from endothelial cells. The MC hosts many surface receptors, enabling its activation via a vast variety of potentially harmful exogenous and endogenous molecules and via reproductive hormones in the female sex organs. Activated MCs release a unique composition of preformed and newly synthesized bioactive molecules, like heparin, histamine, serotonin, proteolytic enzymes, cytokines, chemokines, and growth factors. MCs play important roles in immune responses, tissue remodeling, cell proliferation, angiogenesis, inflammation, wound healing, tissue homeostasis, health, and reproduction. As recently suggested, MCs enable perpetuation of the vertebrates because of key effects-spanning generations-in ovulation and pregnancy, as in life-preserving activities in inflammation and wound healing from birth till reproductive age, thus creating a permanent life-sustaining loop. Here, we present recent advances that further indicate that the MC is a specific life-supporting and progeny-safeguarding cell.

The vascular footprint in cardiac homeostasis and hypertensive heart disease-A link between apelin receptor, vascular endothelial growth factor, and neuronal nitric oxide synthase.

Recent studies have suggested a connection between disturbances of the apelin system and various cardiac pathologies, including hypertension, heart failure, and atherosclerosis. Vascular endothelial growth factor is crucial for cardiac homeostasis as a critical molecule in cardiac angiogenesis. Neuronal nitric oxide synthase is an essential enzyme producing nitric oxide, a key regulator of vascular tone. The present study aims to shed light upon the complex interactions between these three vital signaling molecules and examine their changes with the progression of hypertensive heart disease. We used two groups of spontaneously hypertensive rats and age-matched Wistar rats as controls. The expression of the apelin receptor, vascular endothelial growth factor, and neuronal nitric oxide synthase were assessed immunohistochemically. We used capillary density and cross-sectional area of the cardiomyocytes as quantitative parameters of cardiac hypertrophy. Immunoreactivity of the molecules was more potent in both ventricles of spontaneously hypertensive rats compared with age-matched controls. However, capillary density was lower in both ventricles of the two age groups of spontaneously hypertensive rats compared with controls, and the difference was statistically significant. In addition, the cross-sectional area of the cardiomyocytes was higher in both ventricles of the two age groups of spontaneously hypertensive rats compared with controls, and the difference was statistically significant. Our study suggests a potential link between the apelin receptor, vascular endothelial growth factor, and neuronal nitric oxide synthase in cardiac homeostasis and the hypertensive myocardium. Nevertheless, further research is required to better comprehend these interactions and their potential therapeutic implications.

Regulation of VEGF-A expression and VEGF-A-targeted therapy in malignant tumors.

Vascular endothelial growth factor A (VEGF-A), a highly conserved dimeric glycoprotein, is a key regulatory gene and a marker molecule of angiogenesis. The upregulation of VEGF-A facilitates the process of tumor vascularization, thereby fostering the initiation and progression of malignant neoplasms. Many genes can adjust the angiogenesis of tumors by changing the expression of VEGF-A. In addition, VEGF-A also exhibits immune regulatory properties, which directly or indirectly suppresses the antitumor activity of immune cells. The emergence of VEGF-A-targeted therapy alone or in rational combinations has revolutionized the treatment of various cancers. This review discusses how diverse mechanisms in various tumors regulate VEGF-A expression to promote tumor angiogenesis and the role of VEGF-A in tumor immune microenvironment. The application of drugs targeting VEGF-A in tumor therapy is also summarized including antibody molecule drugs and traditional Chinese medicine.

Peritumor Mucosa in Advanced Laryngeal Carcinoma Exhibits an Aberrant Proangiogenic Signature Distinctive from the Expression Pattern in Adjacent Tumor Tissue.

The field cancerization theory is an important paradigm in head and neck carcinoma as its oncological repercussions affect treatment outcomes in diverse ways. The aim of this study is to assess the possible interconnection between peritumor mucosa and the process of tumor neoangiogenesis. Sixty patients with advanced laryngeal carcinoma were enrolled in this study. The majority of patients express a canonical HIF-upregulated proangiogenic signature with almost complete predominancy of HIF-1α overexpression and normal expression levels of the HIF-2α isoform. Remarkably, more than 60% of the whole cohort also exhibited an HIF-upregulated proangiogenic signature in the peritumoral benign mucosa. Additionally, the latter subgroup had a distinctly shifted phenotype towards HIF-2α upregulation compared to the one in tumor tissue, i.e., a tendency towards an HIF switch is observed in contrast to the dominated by HIF-1α tumor phenotype. ETS-1 displays stable and identical significant overexpression in both the proangiogenic phenotypes present in tumor and peritumoral mucosa. In the current study, we report for the first time the existence of an abnormal proangiogenic expression profile present in the peritumoral mucosa in advanced laryngeal carcinoma when compared to paired distant laryngeal mucosa. Moreover, we describe a specific phenotype of this proangiogenic signature that is significantly different from the one present in tumor tissue as we delineate both phenotypes, quantitively and qualitatively. This finding is cancer heterogeneity, per se, which extends beyond the "classical" borders of the malignancy, and it is proof of a strong interconnection between field cancerization and one of the classical hallmarks of cancer-the process of tumor neoangiogenesis.

The Combination of Vascular Endothelial Growth Factor A (VEGF-A) and Fibroblast Growth Factor 1 (FGF1) Modified mRNA Improves Wound Healing in Diabetic Mice: An Ex Vivo and In Vivo Investigation.

BACKGROUND: Diabetic foot ulcers (DFU) pose a significant health risk in diabetic patients, with insufficient revascularization during wound healing being the primary cause. This study aimed to assess microvessel sprouting and wound healing capabilities using vascular endothelial growth factor (VEGF-A) and a modified fibroblast growth factor (FGF1). METHODS: An ex vivo aortic ring rodent model and an in vivo wound healing model in diabetic mice were employed to evaluate the microvessel sprouting and wound healing capabilities of VEGF-A and a modified FGF1 both as monotherapies and in combination. RESULTS: The combination of VEGF-A and FGF1 demonstrated increased vascular sprouting in the ex vivo mouse aortic ring model, and topical administration of a combination of VEGF-A and FGF1 mRNAs formulated in lipid nanoparticles (LNPs) in mouse skin wounds promoted faster wound closure and increased neovascularization seven days post-surgical wound creation. RNA-sequencing analysis of skin samples at day three post-wound creation revealed a strong transcriptional response of the wound healing process, with the combined treatment showing significant enrichment of genes linked to skin growth. CONCLUSION: f-LNPs encapsulating VEGF-A and FGF1 mRNAs present a promising approach to improving the scarring process in DFU.

Changes in the Concentrations of Proangiogenic Cytokines in Human Brain Glioma and Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) and glioma are some of the most common malignancies, with ALL most often affecting children and glioma affecting adult men. Proangiogenic cytokines and growth factors play an important role in the development of both of these tumors. Glioma is characterized by an extremely extensive network of blood vessels, which continues to expand mainly in the process of neoangiogenesis, the direct inducers of which are cytokines from the family of vascular endothelial growth factors, i.e., vascular endothelial growth factor (VEGF-A) and its receptor vascular endothelial growth factor receptor 2 (VEGF-R2), as well as a cytokine from the fibroblast growth factor family, fibroblast growth factor 2 (FGF-2 or bFGF). Growth factors are known primarily for their involvement in the progression and development of solid tumors, but there is evidence that local bone marrow angiogenesis and increased blood vessel density are also present in hematological malignancies, including leukemias. The aim of this study was to examine changes in the concentrations of VEGF-A, VEGF-R2, and FGF-2 (with a molecular weight of 17 kDa) in a group of patients divided into specific grades of malignancy (glioma) and a control group; changes of VEGF-A and FGF-2 concentrations in childhood acute lymphoblastic leukemia and a control group; and to determine correlations between the individual proteins as well as the influence of the patient's age, diet, and other conditions that may place the patient in the risk group. During the statistical analysis, significant differences in concentrations were found between the patient and control groups in samples from people with diagnosed glioma and from children with acute lymphoblastic leukemia, but in general, there are no significant differences in the concentrations of VEGF-A, VEGF-R2, and FGF-2 between different grades of glioma malignancy. Among individuals treated for glioma, there was no significant impact from the patient's gender and age, consumption of food from plastic packaging, frequency of eating vegetables and fruit, smoking of tobacco products, the intensity of physical exercise, or the general condition of the body (Karnofsky score) on the concentrations of the determined cytokines and receptor. The listed factors do not bring about an actual increase in the risk of developing brain glioma.

ILT4 facilitates angiogenesis in non-small cell lung cancer.

Antiangiogenic therapy targeting VEGF-A has become the standard of first-line therapy for non-small cell lung cancer (NSCLC). However, its clinical response rate is still less than 50%, and most patients eventually develop resistance, even when using combination therapy with chemotherapy. The major cause of resistance is the activation of complex bypass signals that induce angiogenesis and tumor progression. Therefore, exploring novel proangiogenic mechanisms and developing promising targets for combination therapy are crucial for improving the efficacy of antiangiogenic therapy. Immunoglobulin-like transcript (ILT) 4 is a classic immunosuppressive molecule that inhibits myeloid cell activation. Recent studies have shown that tumor cell-derived ILT4 drives tumor progression via the induction of malignant biologies and creation of an immunosuppressive microenvironment. However, whether and how ILT4 participates in NSCLC angiogenesis remain elusive. Herein, we found that enriched ILT4 in NSCLC is positively correlated with high microvessel density, advanced disease, and poor overall survival. Tumor cell-derived ILT4 induced angiogenesis both in vitro and in vivo and tumor progression and metastasis in vivo. Mechanistically, ILT4 was upregulated by its ligand angiopoietin-like protein 2 (ANGPTL2). Their interaction subsequently activated the ERK1/2 signaling pathway to increase the secretion of the proangiogenic factors VEGF-A and MMP-9, which are responsible for NSCLC angiogenesis. Our study explored a novel mechanism for ILT4-induced tumor progression and provided a potential target for antiangiogenic therapy in NSCLC.

Melatonin and its bioisosteres as potential therapeutic agents for the treatment of retinopathy of prematurity.

We conducted a study on the impact of intraperitoneal injections of melatonin and its three bioisosteres (compounds 1-3) on the development of oxygen-induced retinopathy in newborn rats during a 21-day experiment. It was demonstrated that melatonin and its analogues 1-3 effectively reduce the total protein concentration in the vitreous body of rat pups, decrease concentration of VEGF-A, and lower the level of oxidative stress (as indicated by normalization of antioxidant activity in the vitreous body). Melatonin and its analogues 1-3 equally normalize the level of VEGF-A. Analogues 1 and 2 even exceed melatonin in their ability to reduce protein influx into the vitreous body. However, analogue 2 had no effect on antioxidant activity, while analogues 1 and 3 caused a significant increase in this parameter, with analogue 3 even slightly exceeding melatonin. Thus, it can be concluded that analogues 1-3 are comparable to melatonin and can be utilized as potential therapeutic agents for the treatment of retinopathy of prematurity.

Serum VEGF-A levels on admission in COVID-19 patients correlate with SP-D and neutrophils, reflecting disease severity: A prospective study.

BACKGROUND AND OBJECTIVE: The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in significant global morbidity and mortality. This study aimed to investigate the clinical significance of serum vascular endothelial growth factor A (VEGF-A) in COVID-19 patients and its association with disease severity and pulmonary injury. METHODS: We prospectively collected data from 71 hospitalized COVID-19 patients between June 2020 and January 2021. Patients were classified as either mild or severe based on their oxygen requirements during hospitalization. Serum VEGF-A levels were measured using an ELISA kit. RESULTS: In comparison to mild cases, significantly elevated serum VEGF-A levels were observed in severe COVID-19 patients. Furthermore, VEGF-A levels exhibited a positive correlation with white blood cell count, neutrophil count, and lymphocyte count. Notably, serum surfactant protein-D (SP-D), an indicator of alveolar epithelial cell damage, was significantly higher in patients with elevated VEGF-A levels. CONCLUSION: These results suggest that elevated serum VEGF-A levels could serve as a prognostic biomarker for COVID-19 as it is indicative of alveolar epithelial cell injury caused by SARS-CoV-2 infection. Additionally, we observed a correlation between VEGF-A and neutrophil activation, which plays a role in the immune response during endothelial cell injury, indicating a potential involvement of angiogenesis in disease progression. Further research is needed to elucidate the underlying mechanisms of VEGF-A elevation in COVID-19.

Expression of VEGFR2 Ligand Binding Domain in Pichia pink™ 4 Cells and Evaluation of Its Interactions with VEGF-A165 Receptor Binding Domain.

Vascular endothelial growth factor A165 (VEGF-A165) and VEGF receptor 2 (KDR) are important mediators of angiogenesis. We aimed to express the soluble KDR ligand-binding domain (sKDR1-3) and evaluate its interaction with the VEGF-A165 receptor-binding domain (VEGFA165-RBD). sKDR1-3 DNA was designed and subcloned into pPinkα-HC plasmid. The cassette was transfected into the Pichia pink™ 4 genome by homologous recombination. We optimized the expression of sKDR1-3 under the induction of different methanol concentrations. VEGFA165-RBD was expressed in E. coli BL21 harboring pET28a( +)─VEGFA165-RBD vector under induction with IPTG with/without lactose. Interaction and biological activity of sKDR1-3 and VEGFA165-RBD were investigated by ELISA and anti-proliferation tests. sKDR1-3 migrated on SDS-PAGE gel as a 35-180 kDa protein due to glycosylation. The relative expression level of sKDR1-3 under 1% methanol was higher than 0.5% and 4% methanol induction. IPTG and cysteine were suitable for induction and refolding of VEGFA165-RBD. 25 ng sKDR1-3 and 20 ng VEGFA165-RBD showed strong binding. sKDR1-3 bound to VEGFA165-RBD and VEGF-A165 with dissociation constants of 0.148 and 0.2 nM, respectively. 4-10 nM concentrations of sKDR1-3 inhibited the proliferation of HUVE cells induced by 5 nM VEGFA165-RBD. In consideration, sKDR1-3 in the nanomolar concentration range, is a promising anticancer drug to inhibit angiogenesis.

Plasma Angiogenic Factors as Predictors of the Efficacy of Second-line Chemotherapy Combined with Angiogenesis Inhibitors in Metastatic Colorectal Cancer: Results From the GI-SCREEN CRC-Ukit Study.

BACKGROUND: The significance of angiogenic factors as predictors of second-line (2L) chemotherapy efficacy when combined with angiogenesis inhibitors for metastatic colorectal cancer (mCRC) remains unestablished. PATIENTS AND METHODS: In this multicenter prospective observational study, 17 angiogenic factors were analyzed in plasma samples collected at pretreatment and progression stages using a Luminex multiplex assay. Patients who received chemotherapy plus bevacizumab (BEV group), FOLFIRI plus ramucirumab (RAM group), or FOLFIRI plus aflibercept (AFL group) as the 2L treatment were included. Interactions between pretreatment and treatment groups for progression-free survival (PFS), overall survival (OS), and response rate (RR) were assessed using the propensity-score weighted Cox proportional hazards model. RESULTS: From February 2018 to September 2020, 283 patients were analyzed in the 2L cohort. A strong interaction was observed for PFS between BEV and RAM with HGF, sNeuropilin-1, sVEGFR-1, and sVEGFR-3. Interactions for RR between the BEV and RAM groups were observed for sNeuropilin-1 and sVEGFR-1. Contrarily, OS, PlGF, sVEGFR-1, and sVEGFR-3 differentiated the treatment effect between BEV and AFL. Plasma samples were evaluable for dynamic analysis in 203 patients. At progression, VEGF-A levels significantly decreased in the BEV group and increased in the RAM and AFL groups. CONCLUSION: The pretreatment plasma sVEGFR-1 and sVEGFR-3 levels could be predictive biomarkers for distinguishing BEV and RAM when combined with chemotherapy in 2L mCRC treatment. Based on the VEGF-A dynamics at progression, selecting RAM or AFL for patients with significantly elevated VEGF-A levels may be a 2L treatment strategy, with BEV considered for the third-line treatment. CLINICAL TRIAL NUMBER: UMIN000028616.