Reduced VEGF Expression Research Articles

Abstract Tu023: The VEGF-A splice variants as a mechanism of impaired Inflammatory Angiogenesis in the context of advanced aging.

Background and Objectives: Aging and age-related diseases like peripheral artery disease (PAD) is associated with impairment of angiogenesis responses to injury. Critical ischemic limb and wound injury animal models have shown impact of monocytes/macrophages as a major source of angiogenic mediators leading to new arterial growth. Aging has also been associated with impairment of blood flow recovery and reduced VEGF-A expression in animals. However, recombinant VEGF-A administration or clinical trials involving VEGF-A have not fully overcome the foot perfusion and prevent limb/leg amputation. We sought to understand molecular mechanisms of reduced VEGF-A expression which remain incomplete in the context of advanced aging. Results: Firstly, in hindlimb ischemia mouse model, our data from liposomes containing-clodronate treated mice (12-weeks C57Bl6 with macrophage depletion model ) ( Fig. 1a ) demonstrate impairment of blood flow recovery compared to Control group. On day 3 post-surgery, VEGF-A and VEGF-A isoforms expression (VEGF-A 165 a and VEGF-A 165 b, respectively) were reduced in ischemic muscle. Fluorescence staining showed a reduction of macrophages (CD68 + ) was also associated with reduced endothelial cells (CD31 + ). Using a myeloid-specific and inducible deleted VEGF fl/fl mice, we then have shown a key role of early inflammatory macrophages stage as a major source of VEGF-A required for angiogenesis in young mice (Fig. 1b). Secondly, bone marrow-derived-macrophages (BMDMs) from C57Bl6 104-weeks mice also demonstrate reduced VEGF-A expression. Aged BMDMs demonstrate reduced VEGF-A 165 a while VEGF-A 165 b was highly increased. Polarized BMDMs from 12-weeks demonstrate increased VEGFR1 in “M2" macrophages while VEGFR2 was increased in “M1” macrophages. On the other hand, BMDMs from 104-weeks demonstrate reduced VEGFR2 expression while VEGFR1 was unaffected compared to young. Lastly, 104-weeks mice also demonstrate severe impairment of blood flow recovery ( Fig. 2 ) after surgery and reduced VEGF-A and VEGF-A 165 a in ischemic muscle tissue. VEGF-A 165 b was increased and both receptors, VEGF-R1/R2 levels ( Fig. 3 ) were also reduced. Fluorescence staining on day 3 post ligation demonstrates reduced endothelial cells recruitment in muscle tissue while macrophage (CD68+) number was similar. Conclusion: Our investigations will contribute to define mechanisms whereby vascular injury is associated with VEGF-A isoforms specific switch in the context of advanced aging.

Exosomal circ_0084043 derived from colorectal cancer-associated fibroblasts promotes in vitro endothelial cell angiogenesis by regulating the miR-140–3p/HIF-1α/VEGF signaling axis

BackgroundCircular RNAs (circRNAs) hold potential as diagnostic markers for colorectal cancer (CRC); however, their functional mechanisms remain incompletely elucidated. This work investigates the clinical implications of a unique set comprising six circRNAs derived from serum in CRC. Furthermore, we delve into the role of exosomal circ_0084043, originating from colorectal cancer-associated fibroblasts (CAFs), with a specific focus on its contribution to endothelial cell angiogenesis. MethodsThe study analyzed circRNA levels in serum samples obtained from both CRC and control groups using qRT-PCR. Additionally, exosomes originating from colorectal CAFs and normal fibroblasts (NFs) were purified and confirmed by electron microscopy and Western blotting techniques. The proangiogenic effects of CAF-derived exosomal circ_0084043 were assessed in endothelial cells through proliferation, migration, and in vitro capillary tube formation assays. Gain- and loss-of-function experiments were employed to clarify the role of the circ_0084043/miR-140–3p/HIF-1α axis in endothelial cell angiogenesis, utilizing luciferase reporter assay, Western blotting, and ELISA for mechanism elucidation. ResultsThe candidate circRNAs (circ_0060745, circ_001569, circ_007142, circ_0084043, Circ_BANP, and CiRS-7) exhibited notably elevated expression in CRC patient sera compared to the levels observed in healthy individuals. Except for CiRS-7, all circRNAs showed elevated expression in CRC patients with positive lymph node metastasis and advanced tumor stages. Exosomes released by colorectal CAFs augmented endothelial cell proliferation, migration, and angiogenesis by upregulating VEGF expression and secretion. Circ_0084043 was highly detected in endothelial cells treated with CAF-derived exosomes. Silencing circ_0084043 reduced VEGFA expression and diminished CAF exosome-induced endothelial cell processes, indicating its pivotal role in angiogenesis. Circ_0084043 sponges miR-140–3p, regulating HIF-1α, and a reverse relationship was also identified between miR-140–3p and VEGFA in endothelial cells. Inhibiting miR-140–3p mitigated circ_0084043 knockdown effects in CAF exosome-treated endothelial cells. Co-transfection of si-circ_0084043 and a miR-140–3p inhibitor reversed the inhibited migration and angiogenesis caused by circ_0084043 knockdown in CAF exosome-treated endothelial cells. Inhibiting miR-140–3p rescued reduced VEGFA expression due to circ_0084043 knockdown in endothelial cells exposed to CAF-derived exosomes, indicating modulation of the circ_0084043/miR-140–3p/VEGF signaling in CAF-derived exosome-induced angiogenesis. ConclusionsThis study unveiled a distinctive signature of six serum-derived circular RNAs, indicating their potential as promising diagnostic biomarkers for CRC. Importantly, exosomal circ_0084043 originating from colorectal CAFs was identified as playing a crucial role in endothelial cell angiogenesis, exerting its influence through the modulation of the miR-140–3p/HIF-1α/VEGF signaling axis.

β-hydroxybutyrate restrains colitis-associated tumorigenesis by inhibiting HIF-1α-mediated angiogenesis

Decreased levels of β-hydroxybutyrate (BHB), a lipid metabolic intermediate known to slow the progression of colorectal cancer (CRC), have been observed in the colon mucosa of patients with inflammatory bowel diseases (IBD). In particular, patients with recurrent IBD present an increased risk of developing colitis-associated colorectal cancer (CAC). The role and molecular mechanism of BHB in the inflammatory and carcinogenic process of CAC remains unclear. Here, the anti-tumor effect of BHB was investigated in the Azoxymethane (AOM)/Dextran Sulfate Sodium (DSS)-induced CAC model and tumor organoids derivatives. The underlying mechanisms were studied using transcriptome and non-target metabolomic assay and further validated in colon tumor cell lineage CT26 in vitro. The tumor tissues and the nearby non-malignant tissues from colon cancer patients were collected to measure the expression levels of ketogenic enzymes. The exogenous BHB supplement lightened tumor burden and angiogenesis in the CAC model. Notably, transcriptome analysis revealed that BHB effectively decreased the expression of VEGFA in the CAC tumor mucosa. In vitro, BHB directly reduced VEGFA expression in hypoxic-treated CT26 cells by targeting transcriptional factor HIF-1α. Conversely, the deletion of HIF-1α largely reversed the inhibitory effect of BHB on CAC tumorigenesis. Additionally, decreased expression of ketogenesis-related enzymes in tumor tissues were associated with poor survival outcomes in patients with colon cancer. In summary, BHB carries out anti-angiogenic activity in CAC by regulating HIF-1α/VEGFA signaling. These findings emphasize the role of BHB in CAC and may provide novel perspectives for the prevention and treatment of colonic tumors.

Investigation of the anticancer, antimigration and antiangiogenesis effects of an oxadiazole derivative in two- and three-dimensional cultured Ishikawa and Huvec cells; in vitro and in silico studies

Introduction: There are various methods used in cases of metastatic endometrial cancer. However, the prognosis is generally poor. Therefore, new anticancer agents are expected to exhibit the potential to prevent metastasis, as well as their effects on the viability of cancer cells. In in vitro conditions, the Ishikawa cell line represents endometrial adenocarcinoma and the Huvec cell line represents human umbilical vein endothelial cells. Oxadiazole derivatives may be promising new agents for endometrial cancer treatments by exhibiting anticancer and antiangiogenic properties. Objective: The aim of this study was to examine the anticancer, antimigration and antiangiogenic effects of 5-[(4-Phenylpiperazine-1-yl)methyl]-1,3,4-oxadiazol-2-thiol (FP-Oxa) on co-cultured Ishikawa and Huvec cells. Materials and Methods: In silico molecular docking, ADME and toxicity analyzes were performed for FP-Oxa. The effect on the viability of two- and three-dimensional mono- and co-culture models created with HUVEC and Ishikawa cells was evaluated by MTT analysis and IC50 values were calculated. The effect on the migration of two-dimensional cultured cells was determined by wound healing assay. Changes in VEGF expression in three-dimensional co-culture models were evaluated by immunofluorescence staining. Results: As a result of in silico analyses, it was determined that FP-Oxa was within the oral bioavailability limits, exhibited class 4 toxicity, and had inhibition potential by binding to VEGR2. While FP-Oxa clearly inhibited migration in two-dimensionally cultured Ishikawa cells, it did not show the same level of success in Huvec and co-cultured cells. It was effective in reducing VEGF expression in three-dimensional co-cultures. Conclusion: FP-Oxa may have various therapeutic effects on endometrial adenocarcinoma cells. It will be important to conduct biological activity studies in three-dimensional models that mimic the tumor structure created with different types of cancer cells.

Exosomes Derived from Adipose Stem Cells Enhance Angiogenesis in Diabetic Wound Via miR-146a-5p/JAZF1 Axis

Chronic trauma in diabetes is a leading cause of disability and mortality. Exosomes show promise in tissue regeneration. This study investigates the role of exosomes derived from adipose stem cells (ADSC-Exos) in angiogenesis. MiRNA-seq analysis revealed significant changes in 47 genes in human umbilical vein endothelial cells (HUVECs) treated with ADSC-Exos, with miR-146a-5p highly expressed. MiR-146a-5p mimics enhanced the pro-angiogenic effects of ADSC-Exos, while inhibitors had the opposite effect. JAZF1 was identified as a direct downstream target of miR-146a-5p through bioinformatics, qRT-PCR, and dual luciferase assay. Overexpress of JAZF1 resulted in decreased proliferation, migration, and angiogenic capacity of HUVECs, and reduced VEGFA expression. This study proposes that ADSC-Exos regulate angiogenesis partly via the miR-146a-5p/JAZF1 axis.Graphical

Fluorinated Cell-Penetrating Peptide for Co-Delivering siHIF-1α and Sorafenib to Enhance In Vitro Anti-Tumor Efficacy.

Antiangiogenic therapy with sorafenib (SF) alone is ineffective in eradicating tumors, and its long-term application can exacerbate tumor hypoxia, which in turn restricts SF's therapeutic efficacy. Here, a redox-responsive fluorinated peptide (DEN-TAT-PFC) consisting of dendritic poly-lysine, cell-penetrating peptide TAT, and perfluorocarbon was designed and synthesized to co-load siRNA-targeting hypoxia-inducible factors (siHIF-1α) and SF. The unique architecture of the peptide and fluorinated modifications enhanced the siRNA delivery efficiency, including increased siRNA binding, GSH-responsive release, cellular uptake, endosomal escape, and serum resistance. Simultaneously, the DEN-TAT-PFC/SF/siHIF-1α co-delivery system achieved efficient knockdown of HIF-1α at mRNA and protein levels, thus alleviating hypoxia and further substantially reducing VEGF expression. Additionally, the excellent oxygen-carrying ability of DEN-TAT-PFC may facilitate relief of the hypoxic microenvironment. As a result of these synergistic effects, DEN-TAT-PFC/SF/siHIF-1α exhibited considerable anti-tumor cell proliferation and anti-angiogenesis effects. Therefore, DEN-TAT-PFC can be a versatile platform for fabricating fluorine-containing drugs/siRNA complex nano-systems.

Exosome-transmitted ANGPTL1 suppresses angiogenesis in glioblastoma by inhibiting the VEGFA/VEGFR2/Akt/eNOS pathway

ObjectiveGlioblastoma (GBM) is a highly vascularized malignancy that relies on new vessel generation, and thus targeting angiogenesis has been a promising anti-GBM approach. ANGPTL1 is well-known for its anti-angiogenic property; nevertheless, its role in GBM is yet to be explored. Recently, the crucial role of exosomes (Exos) as intercellular communication mediators has gained prominence in GBM therapy. This work aimed to explore the role of exosomal ANGPTL1 in GBM angiogenesis and its mechanisms. MethodsBioinformatic analysis was performed to evaluate ANGPTL expression in GBM. Human GBM cell lines (U87 and U251) and a xenograft mouse model were employed. Exos were isolated from oe-NC- and oe-ANGPTL-transfected bone mesenchymal stem cells and identified. Cell proliferation, migration, and apoptosis were detected. Immunofluorescence, qRT-PCR, western blotting, co-immunoprecipitation, and immunohistochemistry were used to determine the molecular mechanisms underlying exosomal ANGPTL1 against GBM angiogenesis. Besides, tube generation and transmission electron microscope assays were conducted to assess GBM angiogenesis. ResultsLow ANGPTL1 expression was observed in GBM tumor tissues and cells. Functionally, e-ANGPTL-Exos inhibited GBM malignant progression and angiogenesis in vitro and in vivo. Mechanically, e-ANGPTL-Exos reduced VEGFA expression and blocked the VEGFR2/Akt/eNOS pathway in GBM cells and tumor tissues. Co-immunoprecipitation revealed a link between ANGPTL1 and VEGFA in GBM cells. Notably, oe-VEGFA abolished the suppressive functions of e-ANGPTL-Exos in GBM progression and angiogenesis and the VEGFR2/Akt/eNOS axis. The VEGFR2 inhibitor, vandetanib, eliminated the promotive effects of oe-VEGFA on GBM angiogenesis with suppressed VEGFR2/Akt/eNOS pathway. ConclusionsExosomal ANGPTL1 suppressed GBM angiogenesis by inhibiting the VEGFA/VEGFR2/Akt/eNOS axis.

Improved antitumor activity through a tyramidyl maslinic acid derivative. Design and validation as drug-loaded electrospun polymeric nanofibers

Among the most harmful tumors detected in the human body, such as breast, colon, brain or pancreas, breast (BC) and colorectal cancer (CRC) are the first and third most frequent cancer worldwide, respectively. The current existing chemotherapeutic treatments present serious side effects due to their intravenous administration can induce cytotoxicity in healthy cells. Thus, new treatment methods based on drug-loaded polymeric nanofibers (NFs) have gained significant potential for their use in localized cancer chemotherapy. Here, a deep in vitro comparative analysis between maslinic acid (MA) and a tyramine-maslinic acid (TMA) derivative is initially performed. This analysis includes a proliferation, and a cell cycle assay, and a genotoxicity, antiangiogenic and apoptosis study. Then, the TMA derivative has been incorporated into electrospun polymeric NFs obtaining an implantable dressing material with antitumor activity. Two types of patches containing TMA-loaded polymeric NFs of poly(caprolactone) (PCL), and a mixture of polylactic acid/poly(4-vinylpyridine) (PLA/PVP) were fabricated by the electrospinning technique. The characterization of the drug-loaded NFs showed an encapsulation capacity of 0.027 mg TMA/mg PCL and 0.024 mg TMA/mg PLA/PVP. Then, the cytotoxic activity of both polymeric systems was tested in CRC (T84), BC (MCF-7) and a no tumor (L929) cell lines exposed to TMA-loaded NFs and blank NFs for 48 h. Moreover, cell cycle assay, genotoxicity, angiogenesis and apoptosis tests were carried out to study the mechanism of action of TMA. Blank NFs showed no-toxicity in all cell lines tested and both drug-loaded NFs significantly reduced cell proliferation (relative proliferation of ≈44 % and ≈25 % respectively). Therefore, TMA was less genotoxic than maslinic acid (MA), and reduced VEGFA expression in MCF-7 cells (1.32 and 2.12-fold for MA and TMA respectively). These results showed that TMA-loaded NFs could constitute a promising biocompatible and biodegradable nanoplatform for the local treatment of solid tumors such as CRC or BC.

The role of natural compounds in rat mammary cancer: the beneficial effects of Santolina chamaecyparissus L. aqueous extract

Breast cancer is the most diagnosed cancer among women, and a leading cause of death worldwide. Santolina chamaecyparissus L. is a plant with multiple health benefits, including anticancer and anti-diabetic properties. This study aimed to assess the chemopreventive effects of S. chamaecyparissus aqueous extract (SCE) in an animal model of mammary cancer. A total of 28 four-week-old female Wistar rats were divided into four groups: control, MNU-induced (IND), SCE-supplemented (SCE), and SCE+IND. SCE was added to drinking water (12.72 mg/kg body weight) ad libitum. MNU was administered via the intraperitoneal route at 50 days of age. Weekly monitoring of body weight, food/drink intake, humane endpoints, and number of mammary tumours were recorded. Twenty weeks after MNU administration, animals were sacrificed by anaesthetic overdose and a necropsy was performed. Blood samples were used to determine blood count and serum biochemistry analysis, while kidney and liver samples were analysed for oxidative stress. Tumour samples were collected for gene expression and histology studies. SCE chemical composition was analysed by LC-MS and contained 19 phenolic compounds, with the most abundant being myricetin-O-glucuronide and 1,3-O-dicaffeoylquinic acid. Two animals in the IND group were sacrificed due to exceeding the humane endpoint limits. SCE supplementation delayed mammary tumour development, reducing its volume and weight. SCE had a positive impact on haematological parameters, particularly the neutrophil-lymphocyte ratio (P=0.026). No significant differences were observed in serum biochemistry, except for creatinine kinase MB, or in oxidative stress markers. Gene expression analysis showed significantly reduced VEGF expression levels (P=0.0158) in tumours from SCE+IND. These findings suggest that SCE is deserving of further study to identify the individual compounds and to understand its influence on animal models during cancer development.

Tumor-suppressive role of miR-139-5p in angiogenesis and tumorigenesis of ovarian cancer: Based on GEO microarray analysis and experimental validation

This study clarified the possible molecular mechanisms by which the miR-139-5p/SOX4/TMEM2 axis affected angiogenesis and tumorigenesis of ovarian cancer (OC) based on GEO microarray datasets and experimental support. The expression of miR-139-5p and SOX4 was examined in clinical OC samples. Human umbilical vein endothelial cells (HUVECs) and human OC cell lines were included in vitro experiments. Tube formation assay was conducted in HUVECs. The expression of SOX4, SOX4, and VEGF in OC cells was identified using Western blot and immunohistochemistry. Luciferase assays were conducted to validate the targeting relationship between miR-139-5p and SOX4 and between SOX4 and TMEM2. A RIP assay assessed the binding of SOX4 and miR-139-5p. The impact of miR-139-5p and SOX4 on OC tumorigenesis in vivo was evaluated in nude mice. SOX4 was up-regulated, while miR-139-5p was down-regulated in OC tissues and cells. Ectopic miR-139-5p expression or SOX4 knockdown inhibited angiogenesis and tumorigenicity of OC. By targeting SOX4 in OC, miR-139-5p lowered VEGF expression, angiogenesis, and TMEM2 expression. The miR-139-5p/SOX4/TMEM2 axis also reduced VEGF expression and angiogenesis, which might curtail OC growth in vivo. Collectively, miR-139-5p represses VEGF expression and angiogenesis by targeting the transcription factor SOX4 and down-regulating TMEM2 expression, thereby impeding OC tumorigenesis.

Thrombospondin-1 induction and VEGF reduction by proteasome inhibition

The present study focuses on investigating the expression of thrombospondin-1 (TSP-1), a natural inhibitor of neovascularization. Immunofluorescent staining was used to detect the expression of TSP-1 in rabbit corneal tissue with vascularization induced by limbectomy. TSP-1 was detected in healthy and Cultured Autologous Oral Mucosal Epithelial Cell Sheet (CAOMECS) grafted rabbit corneas. TSP-1 was not detected in diseased corneas. Rabbit and human primary oral mucosal and corneal epithelial cells were cultured and treated with proteasome inhibitor (PI) in vitro. Changes in the expression of TSP-1, HIF-1 alpha and 2 alpha, VEGF-A, and VEGF receptor were analyzed by Western blotting. Neovascularization developed in rabbits’ corneas as early as 1 month after limbectomy and was stable for at least 3 months. HIF-1 alpha and VEGF-A expression was reduced in CAOMECS grafted corneas, as compared to sham corneas. While TSP-1 expression was decreased in injured corneas, it was expressed in CAOMECS grafted corneas, but still less expressed compared to healthy corneas. PI treatment, of human oral mucosal and corneal epithelial cells increased TSP-1 expression and reduced VEGF-A expression. The results showed that TSP-1 expression was lost in injured corneal surface and that CAOMECS grafting restored TSP-1 expression to certain extent. Proteasome inhibition treatment increased TSP-1 and decreased VEGF-A expression in human oral mucosal and corneal epithelial cells. The result suggests that corneal neovascularization could be managed with the inhibition of the proteasome after CAOMECS grafting and increase corneal transparency.

Inhibition of activin-signalling reduces the growth of LβT2 gonadotroph pituitary tumours in mouse.

Pituitary tumours are benign neoplasms that derive from hormone-producing cells of the pituitary gland. While medical treatments have emerged for most subtypes, Gonadotroph tumours that express FSH, and/or LH still lack therapeutic options apart from surgery and radiotherapy. Activin-ligands are physiological regulators of production and secretion of FSH by gonadotroph cells, but their role in gonadotroph tumorigenesis remains little explored. Using the LT2 mouse gonadotroph-cell line which produces FSH under Activin-stimulation, we first tested whether sub-cutaneous xenografts of LT2 cells resulted in tumour formation in Rag2KO mice. Histological analysis confirmed the presence of LT2-tumours with endothelial cells and macrophages in their microenvironment. FSH expression was found in a subset of clusters of LT2 cells in the tumours. We subsequently addressed the consequences of targeting activin-signalling via injection of a soluble activin decoy receptor (sActRIIB-Fc). sActRIIB-Fc treatment resulted in significantly decreased LT2 tumour volume. Reduced Smad2-phosphorylation as well as inhibition of tumour-induced FSH production confirmed the efficient targeting of activin-downstream signalling in treated tumours. More interestingly, treated tumours showed significantly fewer endothelial cells, associated with reduced Vegfa expression. In-vitro treatment of LT2 cells with sActRIIB-Fc had no effect on cell proliferation or apoptosis, but Vegfa expression was inhibited, pointing to a likely paracrine effect of LT2 cells on endothelial cells through activin-mediated Vegfa regulation. Further in-vitro and in-vivo studies are now needed to pinpoint the exact roles of activin-signalling in these processes prior to translating these observations to the clinic.

Tumor-Derived Exosomal miR-29b Reduces Angiogenesis in Pancreatic Cancer by Silencing ROBO1 and SRGAP2.

Background Exosomal miR-29b reportedly plays a role during cancer metastasis. However, its exact function and underlying mechanism during pancreatic cancer (PC) have not been investigated. Methods Exosomes from PC cells were prepared and identified. Transmission electron microscopy (TEM) and confocal microscopy were used to examine structural characteristics of the exosomes and verify their internalization by human umbilical vein endothelial cells (HUVECs). The tube formation and migration abilities of HUVECs were detected. VEGF content was assessed by ELISA. GW4869 was used to suppress exosome release. Luciferase reporter assays were performed to verify the predicted interaction of miR-29b with ROBO1 and SRGAP2 mRNA. Results Exosomal miRNA-29b was differentially expressed in the conditioned medium of PC cells. Exosomes from PC cells were verified by TEM and western blotting. Treatment with the exosomal inhibitor (GW4869) prevented an increase in miR-29b expression and recused the reduced VEGF expression and tube formation and migration abilities of HUVECs cocultured with BxPC3 and AsPC-1 cells that overexpressed miR-29b. Furthermore, the downregulation of ROBO1 and SRGAP2 in cocultured HUVECs was also reduced after additional treatment with GW4869. After incubation with miR-29b exosomes, HUVECs had lower VEGF concentrations and reduced migration and tube formation rates; however, those effects were eliminated by subsequent transfection with the miR-29b inhibitor. Luciferase reporter assays verified the interaction of miR-29b with ROBO1 and SRGAP2. That interaction was also supported by rescue assays showing that overexpression of ROBO1 and SRGAP2 also reduced the antiangiogenic effect of exosomal miR-29b in HUVECs. Conclusion Exosomal miR-29b originating from PC cells protected HUVECs from PC cell-induced angiogenesis by attenuating ROBO1 and SRGAP2 expression. Our findings suggest a strategy for treating PC.

484-P: Uncoupling of IL-1beta and VEGF-A Signaling Axis Contributes to Impaired Arteriogenesis in the Context of Aging and Diabetes Mellitus

Peripheral artery disease (PAD) caused by atherosclerosis leads to considerable morbidity and mortality throughout the world, in large part, due to tissue damage from both acute and chronic occlusive ischemia. Preclinical studies have identified mechanisms involving bone marrow derived macrophage (BMDM) -dependent angiogenesis in an inflammation suppressed state and we recently defined a novel IL-1beta-dependent transcriptional regulation of the pro-angiogenic isoform of VEGF-A. We sought to understand the impact of diabetes on inflammatory angiogenesis in the context of aging. We hypothesized that the uncoupling of IL-1beta and VEGF-A expression with consequent impairments in angio/arteriogenesis. Control mice at 26-week-old of age or mice with experimental diabetes have reductions of angio/arteriogenesis, using a PAD model of femoral artery ligation that involves macrophage-directed blood flow recovery. Combined aging with chronic diabetes led to further reductions in blood flow recovery consequent to impaired angiogenesis. We also found elevated VEGF-R2 (relative to VEGF-R1) expression in the inflammatory “M1” state which was associated with elevations of IL-1beta and VEGF-A. Interestingly selective inhibition of VEGF-R2 led to reduced VEGF-A expression despite stable IL-1beta levels, suggesting an uncoupling of the relationship between IL-1beta and VEGF-A. Lastly, BMDMs from aged, diabetic mice demonstrated an uncoupling of IL-1beta and VEGF-A expression and VEGF-R2 was decreased in aged, diabetic BMDMs. Defining inflammatory macrophages as key, early drivers of angio/arteriogenesis via IL-1beta and VEGF-A/VEGF-R2 signaling supports a paradigm shift away from inflammation-suppression for adequate healing, allowing for macrophage reprograming strategies that promote appropriate inflammation-dependent healing responses. Disclosure C.S.Mantsounga: None. S.Sharma: None. C.Lee: None. R.Carley: None. G.Choudhary: None. A.R.Morrison: None. Funding National Institute of Health NIGMS (P20GM103652)

Recombinant human erythropoietin accelerated the proliferation of non-small cell lung cancer cell lines and reduced the expression of VEGF, HIF-1α, and PD-L1 under a simulated hypoxic environment in vitro.

BackgroundAs erythropoietin (EPO) has been used to treat anemia in cancer patients, negative controversy has continued. Unfortunately, its effects on non‐small‐cell lung carcinoma (NSCLC) cell lines are uncertain and the phenomenon of inducing immune escape of tumor cells remains to be explored. This study aimed to provide an important basis for the application of exogenous EPO in the treatment of tumor‐associated anemia.MethodsCells were cultured in 1% O2, 5% CO2, and 94% N2 to simulate a hypoxic environment of the tumor. A549 cell line (lower expression EPOR) and NCI‐H838 cell line (higher expression EPOR) were treated with 2 and 8 U/ml recombinant human EPO (rhEPO). CCK‐8 method was used to determine the logarithmic growth phase of the cells and to detect cell proliferation. The expression levels of VEGF, HIF‐1α, and PD‐L1 were determined by western blot. One‐way ANOVA was used for statistical analysis between groups, with p < 0.05 indicating a significant difference.ResultsHypoxia itself could decrease the survival rate of NSCLC cells. Under the hypoxic condition, rhEPO induced tumor cells proliferation, especially in the NCI‐H838 cell line, where 2 U/ml rhEPO increased the total number of surviving cells (Hypoxia + rhEPO 2 U/ml vs. Hypoxia, p < 0.05). Western blot analysis showed that hypoxia upregulated the expression of VEGF, HIF‐1α, and PD‐L1 in NSCLC cell lines (Normoxia vs. Hypoxia, p < 0.05), but may not be dependent on the expression levels of EPOR. RhEPO decreased the expression levels of VEGF and HIF‐1α. In the A549 cell line, it depended on the concentration of rhEPO and was particularly obvious in HIF‐1α (Hypoxia vs. Hypoxia + rhEPO 2 U/ml vs. Hypoxia + rhEPO 8 U/ml, p < 0.05). A low concentration of rhEPO may not reduce VEGF expression. In the NCI‐H838 cell line, the effect of rhEPO on VEGF was more obvious, but it may be independent of rhEPO concentrations. The downregulation of PD‐L1 expression by rhEPO was only presented in the A549 cell line and required higher rhEPO concentrations (Hypoxia + rhEPO 8 U/ml vs. Hypoxia&Hypoxia + rhEPO 2 U/ml, p < 0.05).ConclusionThe effect of prolonged high concentrations of rhEPO under hypoxic conditions resulted in accelerated cells proliferation of non‐small‐cell lung cancer and was independent of EPOR expression levels on the cell lines surface. Hypoxia resulted in increased expression of VEGF, HIF‐1α, and PD‐L1 on the NSCLC cell lines. Under normoxic conditions, rhEPO did not affect the expression of VEGF, HIF‐1α, and PD‐L1; but under hypoxic conditions, the application of rhEPO reduced the expression of VEGF, HIF‐1α, and PD‐L1, producing an impact on the biological behavior of tumor cells.

Effect of Artemisia vulgaris Extract on VEGF Expression, CD34, Microvascular Density, and Diameter of Mammary Adenocarcinoma Tumors: In vivo Study

Background. Breast cancer in Indonesia still has a high incidence. Surgery remains the main choice with other modalities such as chemotherapy, radiation, and immunotherapy such as Artemisia vulgaris (AV). This study aimed to prove that administration of AV extract reduced VEGF expression, CD34 and microvascular density (MVD), and tumor diameter in mammary adenocarcinoma. &#x0D; Methods. This study used a "Posttest only control group design" design for 24 female C3H mice which were selected randomly and divided into four groups, namely: group K (control), P1 (chemotherapy), P2 (extract), and P3. (combination). Mammary adenocarcinoma was derived from the inoculation of donor mice. AC chemotherapy (Adriamycin 0.18 mg and Cyclophosphamide 1.8 mg) was given in 2 cycles. AV is given at a dose of 13 mg (0.2 ml) once a day orally. VEGF and CD34 expression were assessed by immunohistochemical staining whereas MVD was assessed by the hematoxylin-eosin stain. Tumor diameter was measured using tumor calipers.&#x0D; Results. There was a significant relationship between CD34 expression and tumor diameter (p&lt;0.001; r=0.927). There was a significant relationship between CD34 expression and MVD features (p&lt;0.001; r=0.906). There was a significant relationship between CD34 and VEGF expression (p&lt;0.001; r=0.986). There was a significant relationship between tumor diameter and VEGF expression (p&lt;0.001; r=0.903). There was a significant relationship between tumor diameter and the appearance of MVD (p&lt;0.001; r=0.882). There was a significant relationship between VEGF expression and MVD features (p&lt;0.001; r=0.893).&#x0D; Conclusion. AV extract gave a higher response to chemotherapy in mammary adenocarcinoma of C3H mice given AC. a chemotherapy regimen (p=&lt;0.05).

Anti-Cancer Effects of α-Cubebenoate Derived from Schisandra chinensis in CT26 Colon Cancer Cells.

α-Cubebenoate derived from Schisandra chinensis has been reported to possess anti-allergic, anti-obesity, and anti-inflammatory effects and to exhibit anti-septic activity, but its anti-cancer effects have not been investigated. To examine the anti-cancer activity of α-cubebenoate, we investigated its effects on the proliferation, apoptosis, and metastasis of CT26 cells. The viabilities of CT26 cells (a murine colorectal carcinoma cell line) and HCT116 cells (a human colon cancer cell line) were remarkably and dose-dependently diminished by α-cubebenoate, whereas the viability of CCD-18Co cells (a normal human fibroblast cell line) were unaffected. Furthermore, α-cubebenoate treatment increased the number of apoptotic CT26 cells as compared with Vehicle-treated cells and increased Bax, Bcl-2, Cas-3, and Cleaved Cas-3 protein levels by activating the MAP kinase signaling pathway. α-Cubebenoate also suppressed CT26 migration by regulating the PI3K/AKT signaling pathway. Furthermore, similar reductions were observed in the expression levels of some migration-related proteins including VEGFA, MMP2, and MMP9. Furthermore, reduced VEGFA expression was found to be accompanied by the phosphorylations of FAK and MLC in the downstream signaling pathway of adhesion protein. The results of the present study provide novel evidence that α-cubebenoate can stimulate apoptosis and inhibit metastasis by regulating the MAPK, PI3K/AKT, and FAK/MLC signaling pathways.

Therapeutic Potential of Exosomal circRNA Derived from Synovial Mesenchymal Cells via Targeting circEDIL3/miR-485-3p/PIAS3/STAT3/VEGF Functional Module in Rheumatoid Arthritis.

BackgroundSynovial inflammation and its associated activation of angiogenesis play critical roles in rheumatoid arthritis (RA). Exosomes, as carriers of genetic information including circular RNAs (circRNAs), have been explored as delivery vehicles for therapeutic molecules. However, the effects of synovial mesenchymal stem cells (SMSCs)-derived exosomal circRNAs and their mechanisms of action in RA progression remain unclear.MethodsSMSCs-derived exosomes (SMSCs-Exos) were administered to a co-culture of RA fibroblast-like synoviocytes (RA-FLS) and human dermal microvascular endothelial cells (HDMECs) in vitro as well as to a collagen-induced arthritis (CIA) mouse model in vivo. Their effects on VEGF expression and angiogenic activity in vitro and the therapeutic efficacy in vivo were evaluated. Exosomes from circEDIL3-overexpressing SMSCs (Ad-circEDIL3-SMSCs-Exos) were used to further determine the role of circEDIL3 in SMSCs-Exo-based therapy.ResultsBoth SMSCs-Exos and Ad-circEDIL3-SMSCs-Exos significantly downregulated the expression of VEGF induced by the IL-6/sIL-6R complex in the supernatants of RA-FLS and HDMECs co-culture as well as in the cell lysate of co-cultured RA-FLS, and the extent of reduction was more pronounced in the latter. Subsequent experiments showed that angiogenic activity was significantly downregulated by SMSCs-Exos and Ad-circEDIL3-SMSCs-Exos due to reduced VEGF expression. CircEDIL3 functioned as a sponge for miR-485-3p, which targeted PIAS3. PIAS3 is known to suppress STAT3 activity and reduce downstream VEGF. Injection of SMSCs-Exos or Ad-circEDIL3-SMSCs-Exos reduced synovial VEGF and consequently ameliorated arthritis severity in the CIA mouse model.ConclusionThe intracellular transfer of circEDIL3 by SMSCs-Exos may be a potential novel therapeutic strategy for RA.

Curcumin Alters VEGF Expression in Human Pterygium Fibroblast

Background: Recurrence remains a great challenge for pterygium management. Recurrence pterygium is also known to be more difficult to manage. Thus, this study focuses on recurrence prevention by exploring the potential of curcumin through its anti-angiogenesis properties. Methods: An experimental study was performed using cultured Human Pterygium Fibroblast (HPF). HPF was cultured to 90% confluency rate on cell culture and divided into three groups. Control group as a negative control, curcumin 100 µmol/L and curcumin 200 µmol/L were applied to each group of cell cultures. After 48 hours, VEGF expression was analyzed using immunofluorescence staining and fluorescein microscope, then the intensity was measured using ImageJ software. Results: Curcumin successfully decreases VEGF expression with a mean in control group 88.61 ?20.05 pixels; curcumin 100 µmol/L 52.64 ?2.74 pixels; curcumin 200 µmol/L 36.30 ?2.74 pixels on HPF. The Tukey HSD posthoc test found significant decrease between control group and each treatment group (p=0.000, p=0.000, and p=0.004; respectively) Conclusions: In conclusion, Curcumin has a potential effect on reducing VEGF expression in human pterygium fibroblast.

Associating Graphene Oxide Derivatives to Treat Non-Muscle Invasive Bladder Cancer (NMIBC)

According to estimates, there were 17,670 bladder cancer-associated deaths in the USA by 2019. Intravesical Bacillus Calmette-Guerin (BCG) instillation is used to treat non-muscle invasive bladder cancer (NMIBC). However, more than 40% of BCG administration cases fail to avoid NMIBC relapse and progression. Our aim is to use synthesized GO derivatives to enable the transport of doxorubicin (DOX), a conventional cancer drug, and siRNA to reduce VEGF. Several platforms were tested, and their effects on NMIBC progression were assessed in vivo based on histo- and immune analyses. siRNA and GO were covalently bonds to polyethyleneimine (PEI) and polyethylene glycol (PEG) (GO-PEG-PEI/siRNA). DOX bond to oxidized GO was also synthesized. Hybrids were administered in vivo (rats) against NMIBC. Histopathology results have shown that hybrids have reduced bladder cancer. The GO-COOH-DOX/GO-PEG-PEI/siRNA potentiated tumor aggressiveness (60%) reduction in animals that did not show signs of lesions. Immunohistochemistry results have shown that the GO hybrid reduced VEGF expression, increased endostatin levels, and low p53 levels were observed. Data analyzed in the current study have shown that hybrid graphene oxides were capable of reducing VEGF expression and have great potential to treat NMIBC.