Enhanced Vascular Endothelial Growth Factor Expression Research Articles

Adipose-mesenchymal stem cell-derived extracellular vesicles enhance angiogenesis and skin wound healing via bFGF-mediated VEGF expression.

This study aimed to investigate whether extracellular vesicles (EVs) derived from adipose-derived mesenchymal stem cells (ASCs) promote skin wound healing by delivering basic fibroblast growth factor (bFGF) to enhance vascular endothelial growth factor (VEGF) expression. ASCs were isolated and transfected with either a bFGF knockdown lentivirus (Lv-sh-bFGF) or a control lentivirus (Lv-sh-NC). EVs were extracted from ASCs cultures and characterized by transmission electron microscopy, nanoparticle tracking analysis, and Western blotting for surface markers. EVs were extracted from the conditioned mediums of ASCs and subjected to different treatments. These EVs or control treatments were injected at the wound edges. Wound healing was assessed using histological techniques, including H&E and Masson's trichrome staining to evaluate tissue regeneration, collagen organization, and immunohistochemistry for CD31 to quantify microvessel density. Protein expression of bFGF and VEGF was measured by Western blotting. ASC-derived EVs significantly promoted angiogenesis and improved skin wound healing. EVs encapsulating bFGF enhanced VEGF expression in the wound tissue, while knockdown of bFGF reduced both bFGF and VEGF expression, leading to delayed wound healing. Further knockdown of VEGF partially reversed the pro-angiogenic and wound-healing effects of bFGF-encapsulated EVs. This study demonstrates that ASC-derived EVs promoted skin wound repair by enhancing angiogenesis and accelerating tissue regeneration through the bFGF/VEGF axis. These findings highlight the therapeutic potential of ASCs-derived EVs in improving skin wound healing.

The Potency of Centella asiatica Leaf Extract on VEGF Expression and Angiogenesis in Second-Degree Burn Wound in Mice

Burn injuries present a significant global health challenge, with the highest incidence rates reported in Southeast Asia, including Indonesia. Healing burn wounds is a complex and dynamic process involving various cellular and molecular mechanisms, prominently featuring the role of Vascular Endothelial Growth Factor (VEGF) in tissue regeneration and wound repair. VEGF is crucial for inducing and regulating angiogenesis and supplying oxygen and nutrients to the healing tissue. This study aims to evaluate the potential of pegagan (Centella asiatica) leaf extract cream 1%, 3%, and 5% daily for 14 days in enhancing VEGF expression and angiogenesis in second-degree burn wounds in mice (Mus musculus). This study investigates the application of C. asiatica extract cream on second-degree burn wounds in mice, comparing its effects on VEGF protein expression and angiogenesis to those of base cream and silver sulfadiazine cream, with outcomes evaluated using immunohistochemistry (IHC) and hematoxylin and eosin (HE) staining methods. Our findings suggest that C. asiatica extract cream promotes reduced burn wound size, significant upregulated VEGF expression, and enhanced angiogenesis in treating burn wounds compared to positive control, with a 5% dose having the best result. The study concludes that C. asiatica extract cream may effectively treat burn wound healing through enhancing VEGF expression and angiogenesis.

YTHDF1-regulated ALOX5 in retinal pigment epithelial cells under hypoxia enhances VEGF expression and promotes viability, migration, and angiogenesis of vascular endothelial cells

Upregulation of vascular endothelial growth factor (VEGF) and enhanced angiogenesis have been implicated in the severe progression of age-related macular degeneration (AMD). Abnormal arachidonate 5-lipoxygenase (ALOX5) is associated with AMD pathogenesis. However, no reports have shown the causal role of ALOX5 in angiogenesis during AMD. In the present study, ARPE-19 cells were exposed to hypoxia, an inducer of VEGF expression. Potential proteins implicated in AMD progression were predicted using bioinformatics. RNA affinity antisense purification-mass spectrometry (RAP-MS) was applied to identify the binding proteins of ALOX5 3′UTR. Expression of ALOX5 and YTH N6-methyladenosine RNA-binding protein 1 (YTHDF1) was detected by qRT-PCR and western blotting. VEGF expression and secretion were assessed by immunofluorescence and ELISA, respectively. The chicken embryo chorioallantoic membrane (CAM) was used to analyze the effect of ALOX5 on angiogenesis. RNA stability was assayed using the Actinomycin D assay. The results show that hypoxia promoted cell growth and increased VEGF expression in ARPE-19 cells. ALOX5 was associated with AMD progression, and hypoxia upregulated ALOX5 expression in ARPE-19 cells. ALOX5 silencing reduced VEGF expression induced by hypoxia in ARPE-19 cells. Moreover, the conditioned medium of ALOX5-silenced ARPE-19 cells could suppress the viability and migration of HUVECs and diminish angiogenesis in the CAM. Furthermore, YTHDF1 was validated to bind to ALOX5 3′UTR, and YTHDF1 promoted ALOX5 expression by elevating the stability of ALOX5 mRNA. In conclusion, our findings demonstrate that YTHDF1-regulated ALOX5 increases VEGF expression in hypoxia-exposed ARPE-19 cells and enhances the viability, migration, and angiogenesis of vascular endothelial cells.

MiR-34a ameliorates arterial blood flow in rats with lower limb arteriosclerosis obliterans via Sirt1 signaling pathway.

In this study, we investigated the impact of microRNA-34a (miR-34a) on lower limb arteriosclerosis obliterans in rats through the Sirtuin 1 (Sirt1) signaling pathway. Thirty-six Sprague-Dawley rats were divided into normal, model, and miR-34a mimics groups. Rats in the normal group were raised normally, while the model group underwent lower limb arteriosclerosis obliterans induction and received saline injections. The miR-34a mimics group also underwent arteriosclerosis obliterans modeling but received miR-34a mimics injections. Immunohistochemistry revealed significantly increased vascular endothelial growth factor (VEGF) expression in both model and miR-34a mimics groups compared to the normal group, with the miR-34a mimics group showing higher levels. Western blotting indicated elevated Sirt1 protein expression in both non-normal groups, with the miR-34a mimics group exhibiting significantly higher levels. Quantitative polymerase chain reaction (qPCR) demonstrated higher levels of miR-34a, VEGF mRNA, and Sirt1 mRNA in the model group compared to the normal group, but significantly lower levels than the miR-34a mimics group. Enzyme-linked immunosorbent assay (ELISA) showed increased VEGF content in the model group compared to the normal group but decreased compared to the miR-34a mimics group. Hemorrheological detection revealed a reduced PU index in both non-normal groups compared to the normal group, with a significant increase in the miR-34a mimics group compared to the model group. Overall, miR-34a upregulation enhanced VEGF expression in rat blood vessels, ameliorating arterial blood flow in lower limb arteriosclerosis obliterans through the Sirt1 signaling pathway.

LncRNA-loc391533 is involved in the progression of preeclampsia through VEGF.

Preeclampsia (PE) is a leading cause of maternal death worldwide, which is one of the most major pregnancy complications. The effects of vascular endothelial growth factor (VEGF) and lncRNA-loc391533 on PE were evaluated in the present study. Expression of VEGF in pregnant women with PE was determined using immunohistochemical and enzyme linked immunosorbent assay (ELISA). The effects of lncRNA-loc391533 knockdown and overexpression on VEGF expression was detected using quantitative polymerase chain reaction (qPCR) and western blotting. Loss/gain-of-function assays were performed to evaluate the role of lncRNA-loc391533 on proliferation, cell cycle and migration of trophoblasts HTR-8/SVneo cells. We found that VEGF and its receptor VEGFR1/2 were low expressed in PE. Knockdown of lncRNA-loc391533 enhanced VEGF expression, while overexpression of lncRNA-loc391533 downregulated VEGF. Moreover, lncRNA-loc391533 was required for proliferation and migration of HTR-8/SVneo cells. In conclusion, our findings emphasized that lncRNA-loc391533 exhibited a critical role in progression of PE through VEGF, which might as a novel therapeutic target for PE treatment.

Combination of Carbonate Hydroxyapatite and Stem Cells from Human Deciduous Teeth Promotes Bone Regeneration by Enhancing BMP-2, VEGF and CD31 Expression in Immunodeficient Mice.

The objective of this study was to clarify the efficiency of a combination of stem cells from human deciduous teeth and carbonate apatite in bone regeneration of calvarial defects. Immunodeficient mice (n = 5 for each group/4 groups) with artificial calvarial bone defects (5 mm in diameter) were developed, and stem cells from human deciduous teeth (SHEDs) and carbonate hydroxyapatite (CAP) granules were transplanted with an atelocollagen sponge as a scaffold. A 3D analysis using microcomputed tomography, and 12 weeks after transplantation, histological and immunohistochemical evaluations of markers of bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), and cluster of differentiation (CD) 31 were performed. In the 3D analysis, regenerated bone formation was observed in SHEDs and CAP, with the combination of SHEDs and CAP showing significantly greater bone regeneration than that in the other groups. Histological and immunohistochemical evaluations showed that combining SHEDs and CAP enhanced the expression of BMP-2, VEGF, and CD31, and promoted bone regeneration. This study demonstrates that the combination of SHEDs and CAP transplantation may be a promising tool for bone regeneration in alveolar defects.

Long non-coding RNA MCM3AP antisense RNA 1 silencing upregulates microRNA-24-3p to accelerate proliferation and migration of vascular endothelial cells in myocardial infarction rats by reducing EIF4G2

ABSTRACT Long non-coding RNAs (lncRNAs) are crucial drivers in the progression of human diseases such as myocardial infarction (MI). However, the impact of lncRNA MCM3AP antisense RNA 1 (MCM3AP-AS1) on MI remains unknown. This research was determined to explore the effect of MCM3AP-AS1 modulating microRNA-24-3p (miR-24-3p) and eukaryotic translation initiation factor 4 gamma 2 (EIF4G2) on MI. The rat MI models were constructed and, respectively, treated with altered MCM3AP-AS1, miR-24-3p or/and EIF4G2. Subsequently, the cardiac function, myocardial pathological injury, malondialdehyde content and superoxide dismutase activity were determined. The vascular endothelial cells (VECs) were isolated and treated severally, and then proliferation and migration of VECs were measured. MCM3AP-AS1, miR-24-3p, EIF4G2 and vascular endothelial growth factor (VEGF) expressions in myocardial tissues and VECs were assessed. MCM3AP-AS1 and EIF4G2 were upregulated while miR-24-3p and VEGF were downregulated in MI rat myocardial tissues. MCM3AP-AS1 silencing or miR-24-3p elevation improved cardiac function and myocardial pathological injury, suppressed malondialdehyde content, and also enhanced VEGF expression and superoxide dismutase activity in MI rats. In VECs, downregulated MCM3AP-AS1 or upregulated miR-24-3p accelerated cell proliferation and migration. These effects of miR-24-3p upregulation were reversed by overexpressed EIF4G2. Our study summarizes that reduced MCM3AP-AS1 elevates miR-24-3p to promote proliferation and migration of MI rat VECs by inhibiting EIF4G2.

Voluntary Wheel Running Exercise Improves Aging-Induced Sarcopenia via Activation of Peroxisome Proliferator-Activated Receptor Gamma Coactivator-1α/Fibronectin Type III Domain-Containing Protein 5/Adenosine Monophosphate-Activated Protein Kinase Signaling Pathway.

PurposeIn this study, the protective effect of voluntary wheel running exercise on muscle loss and muscle weakness in gastrocnemius of old rats was investigated. The association of voluntary wheel exercise with the peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α)/fibronectin type III domain-containing protein 5 (FNDC5)/adenosine monophosphate- activated protein kinase (AMPK) signaling pathway and vascular endothelial growth factor (VEGF) expression was also evaluated.MethodsSix-month-old and 22-month-old male rats were used for this experiment. The rats in voluntary wheel running exercise groups were performed wheel running for 2 months. Weight bearing test for walking strength, rotarod test for motor coordination and balance, hematoxylin and eosin (H&E) staining for histological changes in the muscle tissues, Western blot analysis for PGC-1α, FNDC5, AMPK, immunofluorescence for VEGF were conducted.ResultsDecreased muscle mass, strength, and coordination due to aging were associated with a decrease in the PGC-1α/FNDC5/AMPK signaling pathway in the gastrocnemius. Voluntary wheel running exercise enhanced VEGF expression by activating the PGC-1α/FNDC5/AMPK signaling pathway, then increased muscle mass, strength, and coordination.ConclusionsIt has been suggested that voluntary wheel running exercise alleviates symptoms of urological diseases that are difficult to treat. Wheel running exercise is a good therapeutic strategy to prevent or treat aging-related sarcopenia.

Visfatin Increases VEGF-dependent Angiogenesis of Endothelial Progenitor Cells during Osteoarthritis Progression.

Osteoarthritis (OA) pannus contains a network of neovascularization that is formed and maintained by angiogenesis, which is promoted by vascular endothelial growth factor (VEGF). Bone marrow-derived endothelial progenitor cells (EPCs) are involved in VEGF-induced vessel formation in OA. The adipokine visfatin stimulates the release of inflammatory cytokines during OA progression. In this study, we found significantly higher visfatin and VEGF serum concentrations in patients with OA compared with healthy controls. We describe how visfatin enhanced VEGF expression in human OA synovial fibroblasts (OASFs) and facilitated EPC migration and tube formation. Treatment of OASFs with PI3K and Akt inhibitors or siRNAs attenuated the effects of visfatin on VEGF synthesis and EPC angiogenesis. We also describe how miR-485-5p negatively regulated visfatin-induced promotion of VEGF expression and EPC angiogenesis. In our OA rat model, visfatin shRNA was capable of inhibiting visfatin and rescuing EPC angiogenesis and pathologic changes. We detail how visfatin affected VEGF expression and EPC angiogenesis in OASFs by inhibiting miR-485-5p synthesis through the PI3K and Akt signaling pathways.

Extensive Unfolded Protein Response Stimulation in Colon Cancer Cells Enhances VEGF Expression and Secretion

Endoplasmic Reticulum (ER) is an important site for protein folding and maturation. Accumulation of unfolded or misfolded proteins in the ER leads to “ER stress”. The unfolded protein response (UPR) is a finely regulated cell-signaling program to re-establish ER folding capacity for building up cellular homeostasis. Although ER stress modulators have emerged as promising treatment options with their potential to trigger ER stress-mediated apoptosis in cancer cells, here we show that ER stress upregulates VEGF (Vascular Endothelial Growth Factor) expression and secretion in colon cancer cells, which may result in resistance against the treatment through enhanced autocrine/intracrine VEGF signaling and through supporting further angiogenesis.

Apelin Affects the Progression of Osteoarthritis by Regulating VEGF-Dependent Angiogenesis and miR-150-5p Expression in Human Synovial Fibroblasts.

Synovium-induced angiogenesis is central to osteoarthritis (OA) pathogenesis and thus a promising therapeutic target. The adipokine apelin (APLN) is involved in both OA pathogenesis and angiogenesis. We examined the role of APLN in synovium-induced angiogenesis by investigating the crosstalk between APLN and vascular endothelial growth factor (VEGF) expression in human OA synovial fibroblasts (OASFs). We found higher levels of APLN and VEGF expression in OA samples compared with normal samples. APLN-induced stimulation of VEGF expression and VEGF-dependent angiogenesis in OASFs was mitigated by FAK/Src/Akt signaling. APLN also inhibited levels of microRNA-150-5p (miR-150-5p), which represses VEGF production and angiogenesis. Analyses of an OA animal model showed that shAPLN transfection of OASFs rescued pathologic changes in OA cartilage and histology. Here, we found APLN enhances VEGF expression and angiogenesis via FAK/Src/Akt cascade and via downstream suppression of miR-150-5p expression. These findings help to clarify the pathogenesis of adipokine-induced angiogenesis in OA synovium.

Plasminogen/thrombomodulin signaling enhances VEGF expression to promote cutaneous wound healing.

Plasminogen (Plg) and thrombomodulin (TM) are glycoproteins well known for fibrinolytic and anticoagulant functions, respectively. Both Plg and TM are essential for wound healing. However, their significance during the reparative process was separately demonstrated in previous studies. Here, we investigate the interaction between Plg and epithelial TM and its effect on wound healing. Characterization of the wound margin revealed that Plg and TM were simultaneously upregulated at the early stage of wound healing and the two molecules were bound together. In vitro, TM silencing or knockout in keratinocytes inhibited Plg activation. Plg treatment enhanced keratinocyte proliferation and migration, and these actions were abolished by TM antibody. Keratinocyte-expressed vascular endothelial growth factor (VEGF), which presented a dose-response relationship with Plg treatment, can be suppressed by TM silencing. Moreover, treatment with VEGF antibody inhibited Plg-enhanced keratinocyte proliferation and wound recovery. In vivo, TM antibody treatment and keratinocyte-specific TM knockout can impede Plg-enhanced wound healing in mice. In high-glucose environments, Plg-enhanced VEGF expression and wound healing were suppressed due at least in part to downregulation of keratinocyte-expressed TM. Taken together, our findings suggest that activation of Plg/TM signaling may hold therapeutic potential for chronic wounds in diabetic or non-diabetic individuals. KEY MESSAGES: Plg binds to TM in cutaneous wound healing. TM facilitates the activation of Plg to Plm in keratinocytes. Epithelial TM regulates Plg-enhanced wound healing through VEGF expression.

Nilotinib Enhances Tumor Angiogenesis and Counteracts VEGFR2 Blockade in an Orthotopic Breast Cancer Xenograft Model with Desmoplastic Response

Vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR)-targeted therapies predominantly affect nascent, immature tumor vessels. Since platelet-derived growth factor receptor (PDGFR) blockade inhibits vessel maturation and thus increases the amount of immature tumor vessels, we evaluated whether the combined PDGFR inhibition by nilotinib and VEGFR2 blockade by DC101 has synergistic therapy effects in a desmoplastic breast cancer xenograft model. In this context, besides immunohistological evaluation, molecular ultrasound imaging with BR55, the clinically used VEGFR2-targeted microbubbles, was applied to monitor VEGFR2-positive vessels noninvasively and to assess the therapy effects on tumor angiogenesis. DC101 treatment alone inhibited tumor angiogenesis, resulting in lower tumor growth and in significantly lower vessel density than in the control group after 14 days of therapy. In contrast, nilotinib inhibited vessel maturation but enhanced VEGFR2 expression, leading to markedly increased tumor volumes and a significantly higher vessel density. The combination of both drugs led to an almost similar tumor growth as in the DC101 treatment group, but VEGFR2 expression and microvessel density were higher and comparable to the controls. Further analyses revealed significantly higher levels of tumor cell–derived VEGF in nilotinib-treated tumors. In line with this, nilotinib, especially in low doses, induced an upregulation of VEGF and IL-6 mRNA in the tumor cells in vitro, thus providing an explanation for the enhanced angiogenesis observed in nilotinib-treated tumors in vivo. These findings suggest that nilotinib inhibits vessel maturation but counteracts the effects of antiangiogenic co-therapy by enhancing VEGF expression by the tumor cells and stimulating tumor angiogenesis.

Roles of HIF-1α, VEGF, and NF-κB in Ischemic Preconditioning-Mediated Neuroprotection of Hippocampal CA1 Pyramidal Neurons Against a Subsequent Transient Cerebral Ischemia.

Ischemic preconditioning (IPC) provides neuroprotection against subsequent severe ischemic insults by specific mechanisms. We tested the hypothesis that IPC attenuates post-ischemic neuronal death in the gerbil hippocampal CA1 region (CA1) throughout hypoxia inducible factor-1α (HIF-1α) and its associated factors such as vascular endothelial growth factor (VEGF) and nuclear factor-kappa B (NF-κB). Lethal ischemia (LI) without IPC increased expressions of HIF-1α, VEGF, and p-IκB-α (/and translocation of NF-κB p65 into nucleus) in CA1 pyramidal neurons at 12h and/or 1-day post-LI; thereafter, their expressions were decreased in the CA1 pyramidal neurons with time and newly expressed in non-pyramidal cells (pericytes), and the CA1 pyramidal neurons were dead at 5-day post-LI, and, at this point in time, their immunoreactivities were newly expressed in pericytes. In animals with IPC subjected to LI (IPC/LI)-group), CA1 pyramidal neurons were well protected, and expressions of HIF-1α, VEGF, and p-IκB-α (/and translocation of NF-κB p65 into nucleus) were significantly increased compared to the sham-group and maintained after LI. Whereas, treatment with 2ME2 (a HIF-1α inhibitor) into the IPC/LI-group did not preserve the IPC-mediated increases of HIF-1α, VEGF, and p-IκB-α (/and translocation of NF-κB p65 into nucleus) expressions and did not show IPC-mediated neuroprotection. In brief, IPC protected CA1 pyramidal neurons from LI by upregulation of HIF-1α, VEGF, and p-IκB-α expressions. This study suggests that IPC increases HIF-1α expression in CA1 pyramidal neurons, which enhances VEGF expression and NF-κB activation and that IPC may be a strategy for a therapeutic intervention of cerebral ischemic injury.

SFRP2 Is Associated with Increased Adiposity and VEGF Expression.

AimsThe aim of this study was to assess depot-specific expression and secretion of secreted frizzled-related protein 2 (sFRP2) by adipose tissue and its effect on adipocyte biology. We measured serum sFRP2 concentrations in 106 patients in vivo to explore its relationship to fat mass, glycaemia and insulin resistance.MethodsExpression of sFRP2 in mouse and human tissues was assessed using polymerase chain reaction and Western blot. Western blot confirmed secretion of sFRP2 by adipose tissue into cell culture medium. Effects of recombinant sFRP2 on lipogenesis and preadipocyte proliferation were measured. Preadipocyte expression of the angiogenic genes vascular endothelial growth factor (VEGF) and nuclear factor of activated T-cells 3 (NFATC3) was measured after recombinant sFRP2 exposure. Complementary clinical studies correlating human serum sFRP2 with age, gender, adiposity and insulin secretion were also performed.ResultssFRP2 messenger RNA (mRNA) was expressed in mouse and human adipose tissue. In humans, sFRP2 mRNA expression was 4.2-fold higher in omental than subcutaneous adipose. Omental adipose tissue secreted 63% more sFRP2 protein than subcutaneous. Treatment with recombinant sFRP2 did not impact on lipogenesis or preadipocyte proliferation but was associated with increased VEGF mRNA expression. In human subjects, circulating insulin levels positively correlated with serum sFRP2, and levels were higher in patients with abnormal glucose tolerance (34.2ng/ml) compared to controls (29.5ng/ml). A positive correlation between sFRP2 and BMI was also observed.ConclusionsCirculating sFRP2 is associated with adipose tissue mass and has a potential role to drive adipose angiogenesis through enhanced VEGF expression.

Cancer-associated fibroblasts promote angiogenesis in gastric cancer through galectin-1 expression

Galectin-1, an evolutionarily conserved glycan-binding protein with angiogenic potential, was recently identified as being overexpressed in cancer-associated fibroblasts (CAFs) of gastric cancer. The role of endogenous CAF-derived galectin-1 on angiogenesis in gastric cancer and the mechanism involved remain unknown. Immunohistochemical staining was used to investigate the correlation between galectin-1 and vascular endothelial growth factor (VEGF) and CD31 expression in gastric cancer tissues and normal gastric tissues. Galectin-1 was knocked down in CAFs isolated from gastric cancer using small interfering ribonucleic acid (RNA), or overexpressed using recombinant lentiviruses, and the CAFs were co-cultured with human umbilical vein endothelial cells (HUVECs) or cancer cells. Subsequently, proliferation, migration, tube formation, and VEGF/VEGF receptor (VEGFR) 2 expression were detected. The role of CAF-derived galectin-1 in tumor angiogenesis in vivo was studied using the chick chorioallantoic membrane (CAM) assay. Galectin-1 was highly expressed in the CAFs and was positively associated with VEGF and CD31 expression. In the co-culture, high expression of galectin-1 in the CAFs increased HUVEC proliferation, migration, tube formation, and VEGFR2 phosphorylation and enhanced VEGF expression in gastric cancer cells. The CAM assay indicated that high expression of galectin-1 in the CAFs accelerated tumor growth and promoted angiogenesis. In contrast, galectin-1 knockdown in the CAFs significantly inhibited this effect. CAF-derived galectin-1 significantly promotes angiogenesis in gastric cancer and may be a target for angiostatic therapy.

VEGF in the nervous system: an important target for research in neurodevelopmental and regenerative medicine.

Vascular endothelial growth factor (VEGF) in neurodevelopment and regeneration: VEGF is a well-known factor that promotes vascularization and angiogenesis. Besides this it participates in the pathogenesis of several diseases, such as colorectal carcinoma, lung cancer or diabetic retinopathy. Within the last decade, VEGF has been successfully integrated into the treatment of such diseases, for example as a therapy for colorectal cancer with the VEGF-receptor (VEGFR)-inhibitor axitinib. VEGF effects in those diseases are primarily exerted via neovascularization and angiogenesis, which are mainly initiated by hypoxia to support tumor growth. VEGF is expressed by a high number of different cells, amongst others its expression was confirmed in different tumor cell-lines as well as in common, physiological cells. A well known initiator of VEGF (over-) expression is the hypoxia inducible factor (HIF), which is a transcriptional factor, leading to an enhanced VEGF expression.

The Effect of Smoking on Eosinophilic Rhinosinusitis with Nasal Polyp in Mouse Model

Objectives: Chronic rhinosinusitis with nasal polyp is a clinically common disease, and cigarette smoking is a major risk factor for airway inflammation. However, little is known about the effects of smoking on eosinophilic rhinosinusitis (ERS) with nasal polyp. A histopathologic and molecular study was performed to investigate the effects of smoking using mouse model of ERS with nasal polyp. Methods: Mice were divided into 4 groups: control group, smoking group, ERS group, and ERS + smoking group (N = 8, each). Histopathologic changes were investigated using several special stainings: Hemotoxylin and eosin for overall inflammation and polyp-like lesions, Sirius red for eosinophils, Toluidine blue for mast cells, Alcian blue for secretory goblet cells, and Masson’s trichrome for collagen fiber. Serum IgE levels and systemic cytokines were determined using enzyme-linked immunosorbent assay. The expression of vascular endothelial growth factor (VEGF) and HIF-1a were evaluated by immunohistochemical staining. Results: Smoking enhanced the degree of polyp-like lesions, eosinophil infiltrations, goblet cell deposition, and subepithelial fibrosis in ERS model. Total and OVA-specific IgE levels were increased in both ERS groups, however, smoking had no additive effect on them. The systemic cytokines (IL-4, IL-6, IL-17, and IFN-γ) were also elevated significantly by smoking. Smoking enhanced VEGF expression in nasal epithelial cells, suggesting underlying mechanism of increased polyp formation. Conclusions: Smoking exposure had additive effects on experimental ERS. It resulted from increased local and systemic inflammation, and airway remodeling. Increased VEGF might be its underlying mechanism.

MicroRNA regulates vascular endothelial growth factor expression in chondrosarcoma cells.

Systemic treatments to prevent or treat chondrosarcoma metastasis are lacking and targeted therapy has yet to be developed. Hypoxia develops in tumors as they grow and hypoxia-related alterations in gene expression underlie some of the traits of cancer. One critical trait is the ability to induce sustained angiogenesis, which is usually related to expression of vascular endothelial growth factor (VEGF). A potential hypoxia-related mechanism resulting in altered gene expression involves microRNA. Little is known about microRNA expression in chondrosarcoma and its potential role in regulation of VEGF expression. Our purposes were (1) to determine if there is hypoxia-regulated microRNA overexpressed in chondrosarcoma; (2) if that contributes to increased VEGF expression; and (3) can VEGF expression be inhibited with a specific antagomir? MicroRNA expression was analyzed in two primary human chondrosarcomas and articular cartilage using array analysis and a cutoff of a fourfold difference in expression between tumor and normal tissue. The effects of hypoxia and hypoxia-inducible factor-1α (HIF-1α) transfection and silencing with siRNA on expression of candidate microRNAs were analyzed in chondrosarcoma cell line JJ. VEGF expression was measured with quantitative polymerase chain reaction and enzyme-linked immunosorbent assay after specific microRNA transfection and knockdown. miR-181a was identified by array analysis and confirmed with quantitative reverse transcription-polymerase chain reaction, which showed that miR-181a was overexpressed in both human chondrosarcomas (33- and 55-fold) and the JJ cell line (sixfold) compared with cartilage and chondrocytes, respectively. In vitro, hypoxia and HIF-1α transfection each further increased miR-181a expression twofold in JJ cells. miR-181a transfection of JJ cells doubled expression of VEGF mRNA and increased secreted VEGF protein by 46% in normoxia, an effect that could be either direct or indirect. Similar enhancement of VEGF expression by miR-181a was found during hypoxia. Transfection with the antagomir anti-miR-181a decreased VEGF protein by 27% in normoxia and 23% in hypoxia. miR-181a is a hypoxia-regulated microRNA that is overexpressed in chondrosarcoma and enhances VEGF expression, an effect that could be inhibited by anti-miR-181a. Systemic treatment options for chondrosarcoma are limited. Antiangiogenic strategies could potentially be effective in limiting tumor progression. One method of inhibiting VEGF expression and associated angiogenesis could be an antagomir-based therapy targeted at miR-181a or other oncogenic microRNAs, although methods of systemic delivery are still under development. The effectiveness of antagomirs also needs to be compared with other antiangiogenic modalities in preclinical models.

TGF-β/Smad3 inhibit vascular smooth muscle cell apoptosis through an autocrine signaling mechanism involving VEGF-A.

We have previously shown that in the presence of elevated Smad3, transforming growth factor-β (TGF-β) transforms from an inhibitor to a stimulant of vascular smooth muscle cell (SMC) proliferation and intimal hyperplasia (IH). Here we identify a novel mechanism through which TGF-β/Smad3 also exacerbates IH by inhibiting SMC apoptosis. We found that TGF-β treatment led to inhibition of apoptosis in rat SMCs following viral expression of Smad3. Conditioned media from these cells when applied to naive SMCs recapitulated this effect, suggesting an autocrine pathway through a secreted factor. Gene array of TGF-β/Smad3-treated cells revealed enhanced expression of vascular endothelial growth factor (VEGF), a known inhibitor of endothelial cell apoptosis. We then evaluated whether VEGF is the secreted mediator responsible for TGF-β/Smad3 inhibition of SMC apoptosis. In TGF-β/Smad3-treated cells, VEGF mRNA and protein as well as VEGF secretion were increased. Moreover, recombinant VEGF-A inhibited SMC apoptosis and a VEGF-A-neutralizing antibody reversed the inhibitory effect of conditioned media on SMC apoptosis. Stimulation of SMCs with TGF-β led to the formation of a complex of Smad3 and hypoxia-inducible factor-1α (HIF-1α) that in turn activated the VEGF-A promoter and transcription. In rat carotid arteries following arterial injury, Smad3 and VEGF-A expression were upregulated. Moreover, Smad3 gene transfer further enhanced VEGF expression as well as inhibited SMC apoptosis. Finally, blocking either the VEGF receptor or Smad3 signaling in injured carotid arteries abrogated the inhibitory effect of Smad3 on vascular SMC apoptosis. Taken together, our study reveals that following angioplasty, elevation of both TGF-β and Smad3 leads to SMC secretion of VEGF-A that functions as an autocrine inhibitor of SMC apoptosis. This novel pathway provides further insights into the role of TGF-β in the development of IH.