VEGFR2 Gene Research Articles

Knockout of histone deacetylase 8 gene in breast cancer cells may alter the expression pattern of the signaling molecules

PurposeBreast cancer (BC) is the most common cancer diagnosed in the world and it is also the main leading cause of cancer deaths in women. Change in epigenetic mechanisms promotes BC initiation and progression. Histone deacetylase 8 (HDAC8) was found to act as a potential oncogene in different malignancies. For better understanding of the HDAC8 function in BC development, we investigated the effect of HDAC8 deletion on the expression of genes involved in signaling pathways. Materials and methodsIn this study, CRISPR technology was used to knockout the HDAC8 gene in MDA-MB-468, MDA-MB-231 and MCF-7 ​cell lines. For this purpose, two gRNAs were designed and cloned into the PX459 vector. The gRNA-containing vectors were transfected into the BC cell lines and then the effect of this deletion on the expression of genes involved in signaling pathway was determined using quantitative real-time PCR (qRT-PCR). ResultsAnalysis of qRT-PCR results showed a reduction in the expression of studied genes in BC cell lines after deletion of the HDAC8 gene compared to untreated controls. Although this decline was not significant for FGF2 and FGFR1 genes, however the mTOR, IGF1R, INSR, VEGFA and VEGFR2 genes showed statistically significant reduction in the studied BC cell lines. In addition, the down-regulation of PDGFC and PDGFRA genes were only significant in the TNBC cell lines. ConclusionOverall, our study showed that HDAC8 can exert its oncogenic effects by altering the expression level of molecules involved in some signaling pathways, and inhibiting HDAC8 can revert these effects.

A Recombinant Lentiviral Vegfr2-Silencing Vector Attenuates Roxarsone-Promoted Growth of Rat Vascular Endothelial Cells and Angiogenesis in Matrigel Plug and B16F10 Xenograft Models.

Roxarsone (ROX) is widely used as a feed addictive for livestock and poultry. ROX promotes angiogenesis, which can lead to health problems, and it is necessary to identify methods to counter this angiogenic effect of ROX. The VEGF/VEGFR2 signaling pathway is involved in the growth and reconstruction of new blood vessels during angiogenesis. In this study, a recombinant lentiviral vector encoding Vegfr2 shRNA was transfected into rat vascular endothelial cells and used in mouse matrigel plug and melanoma xenograft models to investigate its potential to regulate ROX-induced angiogenesis and tumor growth. Treating endothelial cells with ROX increased cell proliferation, migration, and a tube-like structure of growth relative to the control group. The addition of the lentiviral Vegfr2-silencing vector significantly attenuated the effects of ROX on endothelial cells. The hemoglobin content of mouse matrigel plugs treated with ROX was increased significantly. This effect was dramatically attenuated by the co-administration of shRNA targeting Vegfr2. The volume, weight and CD34 staining of the melanoma xenograft tumors increased by ROX were also attenuated by Vegfr2 silence. These results indicate that the down-regulation of VEGFR2 protein plays an inhibitory role in the ROX-promoted angiogenesis in vivo and in vitro. These data support the targeting of Vegfr2 gene as an effective means to treat ROX-induced angiogenesis and tumor growth.

HNRNPA2B1 stabilizes NFATC3 levels to potentiate its combined actions with FOSL1 to mediate vasculogenic mimicry in GBM cells

BackgroundVasculogenic mimicry (VM) is an enigmatic physiological feature that influences blood supply within glioblastoma (GBM) tumors for their sustained growth. Previous studies identify NFATC3, FOSL1 and HNRNPA2B1 as significant mediators of VEGFR2, a key player in vasculogenesis, and their molecular relationships may be crucial for VM in GBM.AimsThe aim of this study was to understand how NFATC3, FOSL1 and HNRNPA2B1 collectively influence VM in GBM.MethodsWe have investigated the underlying gene regulatory mechanisms for VM in GBM cell lines U251 and U373 in vitro and in vivo. In vitro cell-based assays were performed to explore the role of NFATC3, FOSL1 and HNRNPA2B1 in GBM cell proliferation, VM and migration, in the context of RNA interference (RNAi)-mediated knockdown alongside corresponding controls. Western blotting and qRT-PCR assays were used to examine VEGFR2 expression levels. CO-IP was employed to detect protein–protein interactions, ChIP was used to detect DNA–protein complexes, and RIP was used to detect RNA–protein complexes. Histochemical staining was used to detect VM tube formation in vivo.ResultsFocusing on NFATC3, FOSL1 and HNRNPA2B1, we found each was significantly upregulated in GBM and positively correlated with VM-like cellular behaviors in U251 and U373 cell lines. Knockdown of NFATC3, FOSL1 or HNRNPA2B1 each resulted in decreased levels of VEGFR2, a key growth factor gene that drives VM, as well as the inhibition of proliferation, cell migration and extracorporeal VM activity. Chromatin immunoprecipitation (ChIP) studies and luciferase reporter gene assays revealed that NFATC3 binds to the promoter region of VEGFR2 to enhance VEGFR2 gene expression. Notably, FOSL1 interacts with NFATC3 as a co-factor to potentiate the DNA-binding capacity of NFATC3, resulting in enhanced VM-like cellular behaviors. Also, level of NFATC3 protein in cells was enhanced through HNRNPA2B1 binding of NFATC3 mRNA. Furthermore, RNAi-mediated silencing of NFATC3, FOSL1 and HNRNPA2B1 in GBM cells reduced their capacity for tumor formation and VM-like behaviors in vivo.ConclusionTaken together, our findings identify NFATC3 as an important mediator of GBM tumor growth through its molecular and epistatic interactions with HNRNPA2B1 and FOSL1 to influence VEGFR2 expression and VM-like cellular behaviors.Graphical 1. NFATC3 binds to the promoter region of VEGFR2 to enhance VEGFR2 gene expression which leads to an increase in VM of GBM.2. FOSL1 interacts with NFATC3 to further facilitate VEGFR2 gene expression and VM.3. HNRNPA2B1 enhances NFATC3 mRNA stability to increase VEGFR2 expression and VM.

Effects of Hyperglycemia on Angiogenesis in Human Placental Endothelial Cells.

The placenta is a temporary organ that provides communication between the mother and fetus. Maternal diabetes and abnormal placental angiogenesis may be linked. We investigated the angiogenesis mechanism resulting from VEGF and glucose stimulation in PECs obtained from human term placenta. Immunohistochemistry was performed to characterize PECs obtained from human term placenta. D-glucose was added to the medium containing PECs to establish normoglycemic and hyperglycemic conditions. The expression levels of VEGF, VEGFR-1 and VEGFR-2 genes and proteins in PECs from the control and experimental groups were analyzed by RT-PCR and Western blotting, respectively. With 48-hours incubation, gene expressions increased due to hyperglycemia, while protein levels increased due to the combined effect of VEGF and hyperglycemia. While VEGFR-2 gene expression and protein amounts increased in 24-hours due to the combined effect of VEGF and hyperglycemia, the effect of VEGF stimulation and glucose level on VEGFR-2 decreased in 48-hour incubation with time. VEGF, VEGFR-1 and VEGFR-2 genes and proteins were affected by hyperglycemic conditions in PECs. Hyperglycemia occurring in various conditions such as gestational diabetes mellitus and diabetes mellitus may affect VEGF, VEGFR-1 and VEGFR-2 genes and proteins of PECs derived from human term placenta.

Effect of Orthosiphon stamineus Extract on HIF-1Α, Endothelin-1, and VEGFR-2 Gene Expression in NRK-52E Renal Tubular Cells Subjected to Glucotixicity

This study aimed to investigate the impact of Orthosiphon stamineus extract on gene expression in NRK-52E cells under conditions of glucotoxicity. Gene expression analysis using RT-PCR was conducted following exposure of cultured NRK-52E cells to glucotoxic conditions and various concentrations of Orthosiphon stamineus extract. The results revealed a dose-dependent decrease in HIF-1α, ET-1 and VEGFR-2 gene expression levels upon treatment with Orthosiphon stamineus extract. The diverse array of phytochemicals found within Orthosiphon demonstrates a significant impact on the biochemical pathways implicated in the pathogenesis of the disease. Additionally, molecular docking studies suggested the potential inhibition of Rho-kinase as a mechanistic explanation for this observed effect. These findings suggest that Orthosiphon stamineus extract may possess renoprotective properties against glucotoxicity-induced cellular damage. This study contributes to understanding potential therapeutic interventions for managing renal complications associated with conditions such as diabetes.

Effect of Borreria hispida Extract on SIRT1, HIF-1α, ET-1 and VEGFR-2 Gene Expression in NRK-52E Cells Subjected to Glucotoxicity

This study aimed to investigate the impact of Borreria hispida extract on gene expression in NRK-52E cells under conditions of glucotoxicity. Gene expression analysis was conducted using RT-PCR following the exposure of cultured NRK-52E cells to glucotoxic conditions and varying concentrations of Borreria hispida extract. The results demonstrated a dose-dependent increase in SIRT1 gene expression and a concomitant decrease in HIF-1λ, ET-1, and VEGFR-2 gene expressions upon treatment with Borreria hispida extract. Additionally, molecular docking studies suggested the potential inhibition of Rho-kinase as a mechanistic explanation for these effects. Borreria hispida extract may confer renoprotective benefits against glucotoxicity-induced cellular damage. The potential therapeutic utility of Borreria hispida extracts in managing renal complications associated with conditions such as diabetes. Furthermore, our docking studies shed light on the potential molecular mechanisms underlying the observed effects, suggesting interactions between phytochemicals present in Borreria hispida extract and the JNK-1 protein. These interactions may contribute to the augmentation of SIRT1 activity, further bolstering the extract's therapeutic utility in DKD.

A Randomized, Double-Blind, Placebo-Controlled Trial Evaluating the Effect of Topical Urea for Secondary Prophylaxis of Hand Foot Skin Reaction in Renal Cell Cancer Patients on Sunitinib Therapy

IntroductionHand foot skin reaction (HFSR) is a common dose-limiting adverse effect of multi kinase inhibitors (MKI) whose mechanism is not fully understood, and the prophylaxis is inadequate. ObjectiveIn this pilot study, a double-blind, randomized placebo-controlled trial was conducted to evaluate the effect of topical urea in secondary prevention of sunitinib-induced HFSR in renal cell cancer patients. MethodsOut of 55 screened patients, 14 were randomized to receive topical urea or placebo for four weeks. The association of HFSR with drug levels of sunitinib and its metabolite (n-desethyl sunitinib), genetic polymorphism of VEGFR2 gene, quality of life (QOL) and biochemical markers was also assessed. ResultsThe results showed that urea-based cream was not superior to placebo (P = .075). There was no change in the QOL in both the groups. Single nucleotide polymorphism was checked for two nucleotides rs1870377 and rs2305948 located in VEGFR2 gene on chromosome 4. SNP (variant T > A) at rs1870377 was associated with appearance of new HFSR as compared to the wild type, although the association was not statistically significant (OR 0.714). There was no statistically significant difference between mean plasma levels of sunitinib and N-desethyl sunitinib in urea arm as compared to placebo arm as compared to placebo. The best fit population pharmacokinetic model for sunitinib was one compartment model with first order absorption and linear elimination. The median (IQR) of population parameters calculated from the population pharmacokinetics model for Ka, V and Cl was 0.22 (0.21-0.24) h-1, 4.4 (4.09-4.47) L, 0.049 (0.042-0.12) L/hr, respectively. ConclusionThe study suggested that the urea-based cream was not superior to placebo in decreasing the appearance of new HFSR in renal cancer patients receiving 4:2 regimen of sunitinib.

Genetic polymorphisms in VEGFA and VEGFR2 genes associated with coronary heart disease susceptibility and severity.

​Vascular endothelial growth factor A (VEGFA) is well acknowledged as a powerful angiogenesis-promoting agent mainly through its receptor VEGFR2. Ischemia stimulates VEGFA/VEGFR2 signaling pathway and elevated serum levels of VEGFA were detected in coronary heart disease (CHD) patients. The goal of the current study is to determine how four SNPs in the genes for VEGFA (rs3025039 and rs699947) and VEGFR2 (rs2305948 and rs1870377) contribute to the development of CHD. We also wanted to use the Gensini score to confirm if these four SNPs have an effect on the severity of coronary lesions. In this case-control research, we used the restriction fragment length polymorphism of the polymerase chain reaction to genotype 239 CHD patients and 200 controls. Age, sex, smoking behavior, and obesity were taken into account in the statistical analysis. Two VEGFA/VEFGR2 signaling pathway SNPs (rs699947 and rs1870377) were found to be associated with CHD (C vs. A, P = 0.002; OR = 1.47 (1.12-1.93); A vs. T, P = 0.001; OR = 1.58 (1.17-2.13) respectively). The rs2305948 showed no allelic associations with CHD susceptibility, although we noticed a slight association under the recessive model of rs3025039 TT genotype (p = 0.023; OR = 6.41 (1.14-36.12)) only under adjusted analyses. In addition, both VEGFA SNPs (rs699947and rs3025039) were found to be associated with high Gensini score (p < 0.001). Our research helps to shed further light on the pathophysiology of CHD. The VEGFA/VEGFR2 signaling pathway may have been downregulated, increasing CHD susceptibility and risk.

Synthesis, molecular modeling Insights, and anticancer assessment of novel polyfunctionalized Pyridine congeners

The present study describes synthesizing a novel series of polyfunctionalized pyridine congeners 1–18 and assessed for cytotoxic efficacies versus HCT-116, MCF-7, and HepG-2 among one non-cancerous BJ-1 human normal cell. Most compounds were precisely potent anticancer candidate drugs. The molecular impact of the most active compounds 9, 10, 11, 13, 15, and 17 was evaluated after MCF-7 treatment. The gene expression of pro- and ant-apoptosis markers P53, Bax, Caspase-3 and Bcl-2 as well as VEGFR-2 and HER2 were determined. Compounds 13 and 15 induced upregulation of pro-apoptosis of P53, Bax, Caspase-3 and downregulation of anti-apoptosis Bcl-2 gene. However, compound 15 showed higher effect compared to 13 and respective control. Moreover, a slight reduction in HER2 gene expression was detected due to compound 15 treatment, while VEGFR-2 gene was upregulated. In agreement, the immunoblotting analysis showed higher accumulation of P53, Bax, Caspase-3 proteins and of decrease the Bcl-2 protein levels. Furthermore, docking studies united with molecular dynamic simulation exposed compounds 13 and 15 fitting in the middle of the active site at the interface linking the ATP binding site and the allosteric hydrophobic binding pocket. Finally, we performed Petra/Osiris/ Molinspiration (POM) analysis for the newly synthesized compounds. The evaluation of primary in silico parameters revealed significant differences among individual polyfunctionalized pyridine compounds, highlighting the most promising candidates. These preliminary results may help in coordinating and initiating other research projects focused on polyfunctionalized pyridine compounds, especially those with predicted bioactivity, low toxicity, optimal ADME parameters, and promising perspectives.

New proapoptotic chemotherapeutic agents based on the quinolone-3-carboxamide scaffold acting by VEGFR-2 inhibition

In the current study, we designed and synthesized a series of new quinoline derivatives 10a-p as antiproliferative agents targeting cancer through inhibition of VEGFR-2. Preliminary molecular docking to assess the interactions of the designed derivatives with the binding site of VEGFR-2 (PDB code: 4ASD) displayed binding poses and interactions comparable to sorafenib. The synthesized compounds exhibited VEGFR-2 inhibitory activity with IC50 ranging from 36 nM to 2.23 μM compared to sorafenib (IC50 = 45 nM), where derivative 10i was the most potent. Additionally, the synthesized derivatives were evaluated in vitro for their cytotoxic activity against HepG2 cancer cell line. Seven compounds 10a, 10c, 10d, 10e, 10i, 10n and 10o (IC50 = 4.60, 4.14, 1.07, 0.88, 1.60, 2.88 and 2.76 μM respectively) displayed better antiproliferative activity than sorafenib (IC50 = 8.38 μM). Compound 10i was tested against Transformed Human Liver Epithelial-2 normal cell line (THLE-2) to evaluate its selective cytotoxicity. Furthermore, 10i, as a potent representative of the series, was assayed for its apoptotic activity and cell cycle kinetics’ influence on HepG2, its effects on the gene expression of VEGFR-2, and protein expression of the apoptotic markers Caspase-7 and Bax. Compound 10i proved to have a potential role in apoptosis by causing significant increase in the early and late apoptotic quartiles, a remarkable activity in elevating the relative protein expression of Bax and Caspase-7 and a significant reduction of VEGFR-2 gene expression. Collectively, the obtained results indicate that compound 10i has a promising potential as a lead compound for the development of new anticancer agents.

Expression of angiogenic and lymphangiogenic genes in primary cutaneous melanoma: relationship with angiolymphatic invasion and disease-free survival.

Melanoma is one of the most common cancers in the world. The main routes of tumor progression are related to angiogenesis and lymphangiogenesis. These routes can occur by local invasion, which is called angiolymphatic invasion (ALI). In this study, we assess gene expression of relevant biomarkers of angiogenesis and lymphangiogenesis in 80 FFPE melanoma samples to determine a molecular profile that correlates with ALI, tumor progression, and disease-free survival. The results were enhanced by a posttranscriptional analysis by an immunofluorescence assay. Three SNPs in the VEGFR-2 gene were genotyped in 237 malignant melanoma (MM) blood DNA samples by qPCR. A significant correlation was found for LYVE -1 and ALI, qualitative ( P = 0.017) and quantitative ( P = 0.005). An increased expression of protein LIVE-1 in ALI samples supported these results ( P = 0.032). VEGFR2 was lower in patients who showed disease progression ( P = 0.005) and protein VEGFR2 posttranscriptional expression decreased ( P = 0.016). DFS curves showed differences ( P = 0.023) for VEGFR2 expression detected versus the absence of VEGFR2 expression. No significant influence on DFS was detected for the remaining analyzed genes. Cox regression analysis suggested that VEGFR2 expression has a protective role (HR = 0.728; 95% CI = 0.552-0.962; P = 0.025) on disease progression. No significant association was found between any of the studied SNPs of VEGFR2 and DFS or progression rate. Our main results suggest that LYVE-1 gene expression is closely related to ALI; the relationship with the development of metastases in MM deserves further studies. Low expression of VEGFR2 was associated with disease progression and the expression of VEGFR2 correlates with an increased DFS.

Lanthanum Oxide Nanoparticles Reinforced Collagen ƙ-Carrageenan Hydroxyapatite Biocomposite as Angio-Osteogenic Biomaterial for In Vivo Osseointegration and Bone Repair.

A composite biomatrix fabricated with collagen, ƙ-carrageenan, hydroxyapatite reinforced with lanthanum oxide nanoparticles is explored as proangiogenic and osteogenic bone tissue repair biomaterial. The biomatrix shows increased physical and biological stability as observed from proteolytic degradation and thermal stability studies. The addition of lanthanum oxide nanoparticles facilitates good osseointegration coupled with simultaneous activation of proangiogenic properties to act as a bone mimicking material. The minimal level of reactive oxygen species and superior cytocompatibility help the as-synthesized biomatrix in achieving capillary migration into the bone micro environment. The composite biomatrix upregulates the expression of VEGF, VEGF-R2 genes in endothelial cells and osteopontin, osteocalcin in osteoblasts cells, respectively. The in vivo hard tissue repair experiment conducted in a rat model shows complete healing of the bone defect by eight weeks with the application of collagen-ƙ-carrageenan-hydroxyapatite-lanthanum oxide nanoparticle biomaterial when compared to the biomaterial made out of individual constituents alone. The biomaterial matrix gets biointegrated into the bone tissue and exerts its therapeutic value in bringing a faster osseo repair process. The study shows the feasibility of using rare-earth metal nanoparticles in combination with protein-polysaccharide biopolymers for bone regeneration.

Abstract 4592: In vivo efficacy evaluation of anti-human VEGFR2 antibodies in humanized B-hVEGFR2 mice

Abstract Angiogenesis, vascular development, vascular permeability, and embryonic hematopoiesis is partly regulated by VEGF binding to VEGFR2, which forms receptor dimers and activates downstream signaling pathways. In addition to developmental cues, VEGFR2 is also associated with disease progression for psoriasis, rheumatoid arthritis, diabetic retinopathy, and more significantly, tumor growth and drug resistance. Given this, inhibition of VEGFR2 signaling is actively being studied, and multiple VEGFR2 inhibitors have entered various phases of clinical studies and have shown promising results. To evaluate in vivo efficacy of anti-human VEGFR2 antibodies, we generated a human VEGFR2 knock-in (B-hVEGFR2) mouse model by replacing the murine Vegfr2 extracellular domain sequence with the corresponding human sequence. We validated human VEGFR2 gene and protein expression in B-hVEGFR2 mice by RT-PCR and flow cytometry, respectively. Furthermore, we observed an anti-tumor effect with anti-human VEGFR2 antibodies in homozygous B-hVEGFR2 mice subcutaneously implanted with MC38 colon cancer cells. Taken together, we established a novel preclinical B-hVEGFR2 mouse model for evaluation of VEGFR2-targeted immunotherapy. Citation Format: Lei Zhao, Chengzhang Shang, Rebecca Soto, Chonghui Liu, Zhaoxue Yu. In vivo efficacy evaluation of anti-human VEGFR2 antibodies in humanized B-hVEGFR2 mice. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4592.

Restricted feeding regimens improve white striping associated muscular defects in broiler chickens.

The current study investigated the effects of intermittent feeding (IF) and fasting strategies at different times post-hatch on muscle growth and white striping (WS) breast development. In the first trial, 32 one-day-old Abor Acre broilers were fed ad libitum (AL) for 3d post-hatch and then randomly allotted into 4 feeding strategies including AL, 1h-IF group (1h IF, 4 times feeding/d, 1h each time), 1.5h-IF (1.5h IF, 4 times feeding/d, 1.5h each time), and fasting (1d acute fasting, 6d free access to feed) groups and fed for 7d. Although angiogenic genes including VEGFA, VEGFR1, and VEGFR2, and myogenic genes including MYOG and MYOD were upregulated (P<0.05), the breast muscle satellite cell (SC) number and PAX7, MYF5 expression were decreased by the IF strategies (P<0.05). One-day fasting at 6d of age also upregulated angiogenic genes and MYOD expression (P<0.05), downregulated MYF5 expression (P<0.05), but did not change SC number (P>0.05). In the second trial, 384 one-day-old birds were fed AL for 1wk and then randomly allotted to the above 4 feeding strategies starting at 8d of age until 42d of age. Similarly, IF and fasting strategies upregulated the expression of angiogenic and myogenic genes (P<0.05). Both 1h-IF and 1.5h-IF increased breast muscle SC number (P<0.05). At slaughter, breast muscle fiber diameter of 1.5h-IF was smaller but the SC number was larger than that of the birds fed AL (P<0.05). The IF and fasting strategies prevented WS development, and reduced breast WS scores and triglyceride content (P<0.05) without changing the body weight (P>0.05). Fasting and 1h-IF reduced the expression of adipogenic genes ZNF423 and PDGFRα (P<0.05). Moreover, IF and fasting strategies reduced fibrosis in breast muscle and reduced skeletal muscle-specific E3 ubiquitin ligases (TRIM63 and MAFBX) (P<0.05). Fasting significantly reduced CASPASE-3 in breast muscle (P<0.05). In conclusion, IF starting in the first week decreases SC number. Compared to AL, IF or fasting promotes muscular angiogenesis, increases SC number, prevents muscle degeneration, and prevents the development of WS without impairing the growth performance of broiler chickens.

Overexpression of BCL2, BCL6, VEGFR1 and TWIST1 in Circulating Tumor Cells Derived from Patients with DLBCL Decreases Event-Free Survival.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous malignant lymphoid neoplasm and is the most common subtype of non-Hodgkin lymphoma in adults. More than half of patients with DLBCL can achieve remission with standard R-CHOP regimes; however, approximately 30-40% of patients are still failing this standard therapy, which remains as an important cause of progression and mortality of this disease. It is necessary to have diagnostic and monitoring tools that allow us to improve the accuracy of prognosis in these patients. Circulating tumor cells (CTCs) identification through molecular biomarkers is one of the novel strategies that have been used in other types of cancer, and we aim to use this tool to analyze the potential role in DLBCL. We analyzed 138 blood samples of patients with DLBCL, of which CTCs were isolated by density gradient for subsequent detection and quantitation of molecular biomarkers using RT-qPCR with TaqMan probes. Survival analysis was performed using Kaplan-Meier curves. We found overexpression of ABCB1, αSMA, BCL2, BCL6 and VEGFR1 genes, as well as the presence of CK19, EpCAM, KI67, MAGE-A4, SNAIL and TWIST1 genes. CK19 and EpCAM expression were associated with a minor OS (85.7% vs 98.1%, p = 0.002). The overexpression of BCL2, BCL6, VEGFR1 and TWIST1 was related to a minor EFS (p = 0.001). This study showed that in liquid biopsies analyzed, the presence of CTCs can be confirmed through molecular biomarkers, and it has an impact on OS and EFs, making this detection useful in the follow-up and prognosis of patients with DLBCL.

Smart chlorotoxin-functionalized liposomes for sunitinib targeted delivery into glioblastoma cells

IntroductionGlioblastoma efficient drug delivery is hurdled by several pathophysiological barriers of the tumor microenvironment and the intrinsic limitations of chemotherapeutic agents. To overcome these drawbacks, we developed smart chlorotoxin (CTX)-functionalized liposomes (LS) for sunitinib (STB) targeted delivery into glioblastoma cells. MethodsThe synthesized liposomes in terms of physicochemical parameters, drug encapsulation efficiency (EE%) and release profile, cytotoxicity, targeted cellular uptake by U87 and HEK293 cells, and their internalization mechanism were evaluated using dynamic light scattering, dialysis membrane, MTT cell viability, confocal microscopy, and flow cytometry. The qRT-PCR and scratch assays were used to evaluate VEGFR2, PECAM1, KI67, BCL2, BAX, CASP3, and BCLN1 gene expression levels and migration capacity in U87 cells in response to the CTX-LS-STB treatment. ResultsThe CTX-LS-STB formula showed biocompatibility, suitable stability, small size, and high yield EE% of 97.34 ± 1.51% with a slow-controlled release pattern of STB. CTX-LS-STB liposomes were more efficiently internalized by U87 cells, while the cellular uptake of them was significantly reduced by non-tumor HEK293 cells compared to LS-STB liposomes. A mechanistic study demonstrated that CTX-LS-STB liposomes were internalized into studied cell lines via the receptor-mediated endocytosis. Treatment with CTX-LS-STB significantly inhibited cell viability and migration ability of U87 cells and suppressed KI67, VEGFR2, PECAM1 and BCL2 expressions. While, CTX-LS-STB treatment significantly upregulated BAX, CASP3 and BCLN1 expression levels. Furthermore, it led to the induction of apoptosis and autophagy-cell death simultaneously. ConclusionThe CTX-LS-STB formula can be used as an alternative nanoplatforms to enhance the targeted delivery of STB to achieve potential synergistic therapeutic efficacy on glioblastoma cells by modulating several different pathways governing glioblastoma tumorigenesis.

Platelet-derived growth factor (PDGF) cross-signaling via non-corresponding receptors indicates bypassed signaling in colorectal cancer.

Platelet-derived growth factor (PDGF) signaling, besides other growth factor-mediated signaling pathways like vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF), seems to play a crucial role in tumor development and progression. We have recently provided evidence for upregulation of PDGF expression in UICC stage I–IV primary colorectal cancer (CRC) and demonstrated PDGF-mediated induction of PI3K/Akt/mTOR signaling in CRC cell lines. The present study sought to follow up on our previous findings and explore the alternative receptor cross-binding potential of PDGF in CRC. Our analysis of primary human colon tumor samples demonstrated upregulation of the PDGFRβ, VEGFR1, and VEGFR2 genes in UICC stage I-III tumors. Immunohistological analysis revealed co-expression of PDGF and its putative cross-binding partners, VEGFR2 and EGFR. We then analyzed several CRC cell lines for PDGFRα, PDGFRβ, VEGFR1, and VEGFR2 protein expression and found these receptors to be variably expressed amongst the investigated cell lines. Interestingly, whereas Caco-2 and SW480 cells showed expression of all analyzed receptors, HT29 cells expressed only VEGFR1 and VEGFR2. However, stimulation of HT29 cells with PDGF resulted in upregulation of VEGFR1 and VEGFR2 expression despite the absence of PDGFR expression and mimicked the effect of VEGF stimulation. Moreover, PDGF recovered HT29 cell proliferation under simultaneous treatment with a VEGFR or EGFR inhibitor. Our results provide some of the first evidence for PDGF cross-signaling through alternative receptors in colorectal cancer and support anti-PDGF therapy as a combination strategy alongside VEGF and EGF targeting even in tumors lacking PDGFR expression.

The association of umbilical coiling and angiogenesis markers: Impact assessment of gestational diabetes

IntroductionThe purpose of this study was to determine the association between the postnatal umbilical coiling index (pUCI) and vascular endothelial growth factor A (VEGFA) and its receptor (VEGFR2) in parturients with and without gestational diabetes mellitus (GDM). MethodsWithin 24 h following birth, the umbilical cord and pUCI of 29 newborns with GDM and 28 neonates with non-GDM parturients were prospectively examined. Real-time PCR tests were used to determine the expression levels of the VEGFA and VEGFR2 genes, measured from the umbilical cord. The Mann-Whitney and Chi-squared tests were used to compare continuous and discrete variables with and without GDM. ResultsThe median (IQR) of maternal age was 30 (26-34) years. There were no differences in demographic features between GDM and non-GDM parturients. While there was a marginal difference in VEGFA expression levels between the GDM and non-GDM groups (P-values = 0.07), no difference was detected for VEGFR2 (P-values = 0.75). Comparing hyper- and hypocoiling cords revealed a small difference in VEGFA levels (P-values = 0.05), but no change in VEGFR2 (P-values = 0.50). Furthermore, in both GDM and non-GDM parturients, down-regulated VEGFA was the general rule among abnormal pUCIs. DiscussionThe GDM and coiling state both are associated with the amount of VEGFA expression, but neither is related to VEGFR2. Furthermore, regardless of whether the patient has GDM or not, the abnormal coiling pattern appears to be related to the VEGFA down-regulation.

Molecular Polymorphisms of Vascular Endothelial Growth Factor Gene and Bronchopulmonary Dysplasia in Very Low Birth Weight Infants.

Background Bronchopulmonary dysplasia (BPD) is a chronic lung disease affecting primarily preterm and very low birth weight (VLBW) infants. Despite the advances in perinatal care, BPD remains a major clinical and costly complication in premature infants. The pathogenesis of BPD is complex and multifactorial. Prematurity, mechanical ventilation, oxidative stress, and inflammation are recognized as major interrelated contributing factors. Recently, some candidate genes involved in angiogenesis and alveolarization regulating mechanisms have been associated to BPD risk development. The aim of this study was to evaluate the role of vascular endothelial growth factor (VEGF) polymorphisms on BPD onset in VLBW newborns. Methods Eighty-two VLBW infants, without major anomalies, were consecutively enrolled: 33 developed BPD (BPD group) and 49 infants without BPD served as controls (control group). In all infants, two polymorphisms, respectively (VEGF receptor) VEGFR1-710 C/T and VEGF +936 C/T, were determined through salivary brush. Genomic DNA was extracted and purified from saliva samples by using the MasterAmp Buccal Swab DNA Extraction Kit (Tebu-bio, Milan, Italy). Results Significant statistic differences were found between BPD newborns and controls with regard to gestational age, birth weight, mechanical ventilation, duration of oxygen therapy, maternal preeclampsia, and chorioamnionitis. No differences were detected between genotypic and allelic levels regarding VEGFR1 and VEGF molecular polymorphisms. Conclusions Two single nucleotide polymorphisms within VEGF and VEGFR1 genes are not associated with BPD. Further researches are needed to reveal gene polymorphisms involved in vascular development as contributors to the onset of BPD.

Abstract 2210: The study of age and race genetic signatures of prostate cancer with formalin fixed paraffin embedded (FFPE) samples

Abstract Objectives: Diagnosis with prostate cancer (PCa) at younger age is a serious challenge in Military Health System. PCa onset at younger age is more likely to be aggressive in African American (AA) population. Also, application of formalin fixed paraffin embedded (FFPE) tissue in RNA based study was significantly hindered due to poor quality/quantity of RNA in archived pathology slides. This study is aimed to examine age/race associated gene signatures in military cohort with high representation of AA and younger population in FFPE samples for potential genetic mechanisms of health disparities and broaden tissue sample availability. Methods: A cohort of 41 Caucasian American (CA) and 20 AA non-familial PCa patients from the biospecimen bank of CPDR, WRNMMC were used. Specimens donated by radical prostatectomy patients who provided written consent and protocols approved by Institutional Review Boards. Total RNA was extracted from tumor and the adjacent normal epithelium of whole-mount FFPE tissue of matched specimens. Differential gene expression, in tumor versus normal tissues, between younger and older AA and CA patients were analyzed by NanoString using a customized CodeSet of 151 probes. The panel designed to detect 135 target transcripts, compiled from 34 genes implicated/associated with PCa progression, 27 PCa specific gene fusions, 25 cancer-associated genes, and 5 genes encoding ETS-family of transcription factors. The 27 over-expressed genes and 17 under-expressed genes in PCa were included as comparison to matched, benign epithelium. Additionally, 5 prostate stroma or epithelium specific genes were selected as control and 11 housekeeping genes were included for biological normalization. Results: The differentially expressed genes related to tumor-normal differences between AA older (66-73 years) and younger (42-58 years) as well as CA older and younger PCa patients with moderate Gleason score (3+3 and 3+4) sum were identified. After controlling for false discovery rates, panel of nine genes (ANXA2, C-MYC, VEGFR1, ERG, NPY, PSMA/FOLH1, TWIST1, MAOA, MYCN) uniquely associated with age/race were identified. Tumor vs normal ratios of ANXA2 and C-MYC expression in prostate tumors of older and younger patients were significantly enhanced. We found a clear association of VEGFR1 gene expression with CA young and c-MYC gene with AA young PCa. Conclusions: Using age/race as endpoint, we identified ANXA2, c-MYC and VEGFR1 as age and race associated PCa gene. Our findings highlight distinct racial and age-related differences and addresses the clinical utility of available genetic tests across old and young AA and CA men, which needs to be further validated in lager cohort. Further, our study also highlighted the utilization of FFPE tissue for NanoString assay and subsequent genetic signature analyses. Citation Format: Shashwat Sharad, Darryl Nousome, Gregory T. Chesnut, Hua Li. The study of age and race genetic signatures of prostate cancer with formalin fixed paraffin embedded (FFPE) samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2210.