Ang-2 Expression Research Articles

Baicalin regulates mRNA expression of VEGF-c, Ang-1/Tie2, TGF-β and Smad2/3 to inhibit wound healing in streptozotocin-induced diabetic foot ulcer rats.

Diabetic foot ulcer (DFU) is biggest life threats globally and increases their severity increases health complications for health of patients. The present study was investigated to recover the wound healing activity of baicalin in STZ-induced DFU rats by evaluating biochemical and molecular markers. The experimental animals induced with diabetes and excision wounds were treated with different doses of baicalin (25, 50, and 100 mg/kg). The serum glucose level, body weight and food intake were measured. In addition, DFU rat groups showed decreased food intake and increased body weight. The tissue was subjected to biochemical evaluation, histopathology, quantitative polymerase chain reaction and Western blot analysis. Histopathology reports revealed that diabetic wound control (DWC) + baicalin (100 mg/kg) treated group showed more than 90% recovery with more epithelization and remarkably improved angiogenesis and infiltration of the inflammatory cells. In this study we also proved that upregulated the p-ERK, ERK, HSP27, and p-HSP27 protein expression and mRNA expression of Ang-1, VEGF-c, TGF-β, Tie-2, and SMAD2/3 implicating the potential antidiabetic and wound healing property of baicalin. Thus, baicalin is a potential therapeutic candidate for a diabetic foot ulcer and chronic wounds treatment.

Microtubule associated protein 4 (MAP4) phosphorylation reduces cardiac microvascular density through NLRP3-related pyroptosis

Phosphorylation of MAP4 (p-MAP4) causes cardiac remodeling, with the cardiac microvascular endothelium being considered a vital mediator of this process. In the current study, we investigated the mechanism underlying p-MAP4 influences on cardiac microvascular density. We firstly confirmed elevated MAP4 phosphorylation in the myocardium of MAP4 knock-in (KI) mice. When compared with the corresponding control group, we detected the decreased expression of CD31, CD34, VEGFA, VEGFR2, ANG2, and TIE2 in the myocardium of MAP4 KI mice, accompanied by a reduced plasma concentration of VEGF. Moreover, we observed apoptosis and mitochondrial disruption in the cardiac microvascular endothelium of MAP4 KI animals. Consistently, we noted a decreased cardiac microvascular density, measured by CD31 and lectin staining, in MAP4 KI mice. To explore the underlying mechanism, we targeted the NLRP3-related pyroptosis and found increased expression of the corresponding proteins, including NLRP3, ASC, mature IL-1β, IL-18, and GSDMD-N in the myocardium of MAP4 KI mice. Furthermore, we utilized a MAP4 (Glu) adenovirus to mimic cellular p-MAP4. After incubating HUVECs with MAP4 (Glu) adenovirus, the angiogenic ability was inhibited, and NLRP3-related pyroptosis were significantly activated. Moreover, both cytotoxicity and PI signal were upregulated by the MAP4 (Glu) adenovirus. Finally, NLRP3 inflammasome blockage alleviated the inhibited angiogenic ability induced by MAP4 (Glu) adenovirus. These results demonstrated that p-MAP4 reduced cardiac microvascular density by activating NLRP3-related pyroptosis in both young and aged mice. We thus managed to provide clues explaining MAP4 phosphorylation-induced cardiac remodeling and enriched current knowledge regarding the role of MAP4.

Hydroxysafflor Yellow a Promotes Angiogenesis in Rat Brain Microvascular Endothelial Cells Injured by Oxygen-glucose Deprivation/reoxygenation(OGD/R) through SIRT1-HIF-1α-VEGFA Signaling Pathway.

Angiogenesis led by brain microvascular endothelial cells (BMECs) contributes to the remission of brain injury after brain ischemia reperfusion. In this study, we investigated the effects of hydroxysafflor yellow A(HSYA) on angiogenesis of BMECs injured by OGD/R via SIRT1-HIF-1α-VEGFA signaling pathway. The OGD/R model of BMECs was established in vitro by OGD for 2h and reoxygenation for 24h. At first, the concentrations of vascular endothelial growth factor (VEGF), Angiopoietin (ang) and platelet-derived growth factor (PDGF) in supernatant were detected by ELISA, and the proteins expression of VEGFA, Ang-2 and PDGFB in BMECs were tested by western blot; the proliferation, adhesion, migration (scratch healing and transwell) and tube formation experiment of BMECs; the expression of CD31 and CD34 were tested by immunofluorescence staining. The levels of sirtuin1(SIRT1), hypoxia-inducible factor-1α (HIF-1α), VEGFA mRNA and protein were tested. HSYA up-regulated the levels of VEGF, Ang and PDGF in the supernatant of BMECs under OGD/R, and the protein expression of VEGFA, Ang-2 and PDGFB was increased; HSYA could significantly alleviate the decrease of cell proliferation, adhesion, migration and tube formation ability of BMECs during OGD/R; HSYA enhanced the fluorescence intensity of CD31 and CD34 of BMECs during OGD/R; HSYA remarkably up-regulated the expression of SIRT1, HIF-1α, VEGFA mRNA and protein after OGD/R, and these increase decreased after SIRT1 was inhibited. SIRT1-HIF-1α-VEGFA signaling pathway is involved in HSYA improves angiogenesis of BMECs injured by OGD/R.

Relationship between quantitative contrast-enhanced ultrasonography parameters and angiogenesis in primary small hepatocellular carcinoma

To analyze the correlation between quantitative contrast-enhanced ultrasonography (CEUS) parameters and angiogenesis in primary small hepatocellular carcinoma (sHCC) with varying degrees of differentiation.According to varying degrees of differentiation, a total of 90 primary sHCC patients admitted to our hospital from July 2018 to January 2020 were selected and divided into poorly differentiated group (24 cases), moderately differentiated group (31 cases), and highly differentiated group (35 cases). All patients received real-time CEUS before surgery. The tumor diameter, microvascular morphology, grading of color blood flow, contrast-enhanced performance in different phases, quantitative CEUS parameters, expression of angiogenesis-related genes, and microvessel density (MVD) were compared among the 3 groups. The correlation between quantitative parameters of CEUS and angiogenesis indexes was analyzed by Spearman rank correlation analysis.Spearman rank correlation analysis showed that vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), epidermal growth factor receptor (EGFR), and angiopoietin-2 (Ang-2) expression and MVD were negatively correlated with the time to peak (TTP), wash-out time, and peak accelerating time (PAT) (r < 0, P < .05), and were positively correlated with enhancing slope rate (ESR) and peak intensity increasing rate (PIIR) (r > 0, P < .05).CEUS is able to identify varying degrees of differentiation in primary sHCC, and the quantitative CEUS parameters are closely related to angiogenesis.

Transcriptomal Evidence for Dysregulation of Angiogenic Factors and Inflammatory Markers after Allogeneic Hematopoietic Cell Transplantation

Introduction:Angiogenic factors (AF) are the prototypic growth factors required for wound healing, and are linked to the immune response after tissue injury. Angiogenesis and inflammation together influence recovery from organ damage after hematopoietic stem cell transplantation (HCT), as well as contribute to the risk for complications such as acute graft-versus-host disease (aGVHD). We have recently described alterations in circulating angiogenic factors occurring at the onset of aGVHD post-HCT (BBMT 2015 21(6):1029), with dysregulation in AF associated with tissue repair versus those reflective of tissue damage. We hypothesized that the AF-inflammatory signature identified in the plasma in severe aGVHD could be linked to the gene expression of circulating donor cells after HCT. We performed transcriptomal analysis of changes in the peripheral blood immune cells and correlated with plasma AF levels in order to understand if the circulating donor cells might contribute to the AF profile.Methods:Peripheral blood samples from 3 patients (uncomplicated without aGVHD, steroid responsive aGVHD, and steroid refractory aGVHD) were compared to a healthy stem cell donor. Total RNA was derived from whole blood samples, and samples were amplified and labeled using MessageAMP Premier RNA Amplification kit (Ambion). Microarray analyses were performed using Human PrimeView GeneChip arrays (Affymetrix). Plasma AF were quantified using a multiplex angiogenesis array (Millipore).Results:Patients were matched as able according to age, gender, conditioning intensity, donor characteristics, and disease (Table). All patients were 100% donor chimeras and without active infections at the time of blood draw. Compared to HCT donor, all HCT recipients had increased circulating levels of Angiopoietin-2 (Ang-2), endoglin, follistatin, placental growth factor (PlGF), and vascular endothelial growth factor (VEGF) (Figure). However, in steroid refractory aGVHD, levels of epidermal growth factor (EGF) were markedly lower compared to donor, steroid-responsive, and uncomplicated HCT recipients (1.4 pg/ml vs 32.4, 137.1, and 204.8 pg/ml respectively), while levels of VEGF-D, Ang-2, leptin, and PlGF were highest in steroid refractory aGVHD. In microarray analysis (Figure), steroid refractory aGVHD was again associated with a lower gene expression of EGF, but higher expression of only VEGF-D and leptin. Gene expression of Ang-2 and PlGF was not higher in hematopoietic cells, despite higher plasma levels, in steroid-refractory aGVHD, leading to the hypothesis that these growth factors may be predominantly host-derived. Interestingly, we found higher transcript expression of IFN-gamma, comparing steroid refractory versus steroid responsive aGVHD, along with a lower transcript expression of inflammatory cytokines TNF, IL-6, and IL-1B.Conclusions:The plasma profile of steroid-refractory aGVHD (low EGF, and high VEGF-D, Ang-2, leptin, and PlGF) appears to be only partially explained by donor hematopoietic cell gene expression. Gene expression of Ang-2 and PlGF are lower in steroid-refractory aGVHD, suggesting that the plasma elevations in these factors might be predominantly host-derived. Ang-2 likely reflects host endothelial tissue damage as previously described, while the PlGF response to aGVHD requires further investigation. The elevation in several damage-associated factors in the plasma and transcriptome in a healthy HCT recipient may reflect normal adaptation of donor cells to a new host environment. These results are hypothesis generating and mechanistic studies aimed at understanding and manipulating the dysregulated system of AF and downstream signaling pathways are indicated, with the goal of ameliorating severe aGVHD and facilitating tissue repair, without excess immunosuppression. [Display omitted] DisclosuresHoltan:Incyte: Other: One-time advisory board member.

Targeting the hallmarks of cancer: the effects of silibinin on proliferation, cell death, angiogenesis, and migration in colorectal cancer

BackgroundSilibinin, as a chemopreventive agent, has shown anti-cancer efficacy against different types of cancers. In the present study, we investigated the anti-cancer activities of silibinin on CT26 mouse colon cell line.MethodsCT26 cells were treated with different concentrations of silibinin. To examine the cytotoxic effect of silibinin on proliferation, apoptosis, autophagy, angiogenesis, and migration, MTT, colony-forming assay, Annexin V/PI flow cytometry, RT-qPCR, and Scratch assay were used.ResultsSilibinin was found to significantly reduce CT26 cells survival. Furthermore, silibinin strongly induced apoptosis and autophagy by up-regulating the expression of Bax, Caspase-3, Atg5, Atg7 and BECN1 and down-regulating Bcl-2. Silibinin considerably down-regulated the expression of COX-2, HIF-1α, VEGF, Ang-2, and Ang-4 as well as the expression of MMP-2, MMP-9, CCR-2 and CXCR-4.ConclusionsThe present study revealed that silibinin shows anticancer activities by targeting proliferation, cell survival, angiogenesis, and migration of CT26 cells.

Fucoxanthin Exerts Anti-Tumor Activity on Canine Mammary Tumor Cells via Tumor Cell Apoptosis Induction and Angiogenesis Inhibition.

Simple SummaryFucoxanthin is a carotenoid that reportedly exhibits anticancer activity against different types of cancer cells. However, the activity of fucoxanthin in canine mammary gland tumors has not been extensively investigated. In this study, we evaluated fucoxanthin against canine mammary tumor cells (CMT-U27) and human umbilical vein endothelial cells (HUVECs). Our results indicated that fucoxanthin induced apoptosis via caspase activation and suppressed angiogenesis in CMT-U27 cells. Moreover, fucoxanthin inhibited tube formation and cell migration in HUVECs and CMT-U27 cells, indicating that it possessed anti-angiogenic potential. In conclusion, fucoxanthin induced tumor cell death and inhibited angiogenesis. Therefore, we propose that fucoxanthin can be considered a prospective therapeutic agent for canine mammary gland tumors.Fucoxanthin is a carotenoid derived from brown algae. It is known to exhibit anticancer activity, including the promotion of apoptosis and cell cycle arrest in several tumors. However, it remains unclear whether fucoxanthin exhibits anticancer activity against mammary gland tumors. In this study, we evaluated fucoxanthin activity against canine mammary tumor cells (CMT-U27) and human umbilical vein endothelial cells (HUVECs) to investigate its effect on cell viability, migration, tube formation, and angiopoietin 2 (Ang2) expression. Our results showed that fucoxanthin induced apoptosis via caspase activation in CMT-U27 cells. In rat aortic ring assay, fucoxanthin suppressed endothelial cell sprouting. Furthermore, fucoxanthin inhibited tube formation and migration in HUVECs. The number of migrated cells was assessed using CMT-U27 cells. The results demonstrated that fucoxanthin exerted anti-angiogenic activity on HUVECs and CMT-U27 cells by promoting Ang2 expression. In conclusion, our results demonstrated that fucoxanthin induced tumor cell death and inhibited angiogenesis, suggesting that fucoxanthin could be considered as a promising therapeutic agent for canine mammary gland tumors.

Skeletal Muscle Extracellular Vesicles Regulate Endothelial Cells in a Fiber Type Dependent Manner

Introduction Skeletal muscle regulates endothelial cells (EC) via the secretion of angiogenic signaling factors, including extracellular vesicles (EV). Muscle can be characterized as predominantly oxidative (OXI) or glycolytic (GLY) depending on the expression of specific fiber types. OXI fibers have greater capillary density and secrete greater quantities of several angiogenic factors and EV, however it is unknown if EV signaling is regulated by fiber type. We hypothesized that EV released from OXI muscle (OXI-EV) induce a greater angiogenic response in cultured EC compared to EV from GLY muscle (GLY-EV). Methods From male CL57BL/6 mice, OXI muscle was isolated from red portions of the gastrocnemius and soleus; GLY muscle was isolated from white portions of the gastrocnemius and tibialis anterior. EVs were isolated using ultrafiltration-size exclusion chromatography. EV number and RNA content were measured. Human umbilical vein endothelial cells (HUVEC) were treated with OXI-EV or GLY-EV (10 μg/ml) for 24 h; and HUVEC functional angiogenic responses and molecular pathways related to angiogenesis were analyzed. Significance was established at P ≤ 0.05. Results OXI muscle released more EV than GLY muscle (OXI: 8.1x106; GLY: 4.6x106 particles/ml/mg muscle). Selected angiogenic mRNA (vascular endothelial growth factor (VEGF) A and B, angiopoietin (ANG) 1 and 2) and miRNAs (15a, 126, 133a) were not different between OXI-EV and GLY-EV, although there was a trend towards greater VEGFA in OXI-EV. In HUVEC, OXI-EV promoted greater tube formation (# of tubes: +34%; length: +20%), lowered intracellular superoxide (-58%), and trended (P < 0.10) toward greater growth (+22%) with no difference in migration compared to GLY-EVs. Treatment with OXI-EV promoted greater HUVEC endothelial nitric oxide synthase (eNOS) phosphorylation (+40%) and gene expression (+42%) compared to GLY-EV. In HUVEC, there was no difference in gene expression of VEGFA, VEGFB, ANG1, or Ang2 between OXI-EV and GLY-EV treatments. Conclusions EV from oxidative muscle demonstrate greater angiogenic potential than EV from glycolytic muscle consistent with greater capillarization of oxidative muscle. Thus, greater oxidative vs. glycolytic muscle capillarization is likely regulated in part by the combination of greater release of EV and greater EV angiogenic potential from oxidative muscle fibers.

The functional role of decorin in corneal neovascularization in vivo

Our earlier decorin (Dcn) gene overexpression studies found that the targeted Dcn gene transfer into the cornea inhibited corneal angiogenesis in vivo using a rabbit model. In this study, we tested the hypothesis that anti-angiogenic effects of decorin in the cornea are mediated by alterations in a normal physiologic balance of pro- and anti-angiogenic factors using decorin deficient (Dcn−/−) and wild type (Dcn+/+) mice. Corneal neovascularization (CNV) in Dcn−/− and Dcn+/+ mice was produced with a standard chemical injury technique. The clinical progression of CNV in mice was monitored with stereo- and slit-lamp microscopes, and histopathological hematoxylin and eosin (H&E) staining. Protein and mRNA expression of pro- and anti-angiogenic factors in the cornea were evaluated using immunofluorescence and quantitative real-time PCR, respectively. Slit-lamp clinical eye examinations revealed significantly more CNV in Dcn−/- mice than the Dcn+/+ mice post-injury (p < 0.05) and AAV5-Dcn gene therapy significantly reduced CNV in Dcn−/- mice compered to no AAV5-Dcn gene therapy controls (p < 0.001). H&E-stained corneal sections exhibited morphology with several neovessels in injured corneas of the Dcn−/− mice than the Dcn+/+ mice. Immunofluorescence of corneal sections displayed significantly higher expression of α-smooth muscle actin (α-SMA) and endoglin proteins in Dcn−/- mice than Dcn+/+ mice (p < 0.05). Quantitative real-time PCR found significantly increased mRNA levels of pro-angiogenic factors endoglin (2.53-fold; p < 0.05), Vegf (2.47-fold; p < 0.05), and Pecam (2.14-fold; p < 0.05) and anti-angiogenic factor Vegfr2 (1.56-fold; p < 0.05) in the normal cornea of the Dcn−/− mice than the Dcn+/+ mice. Furthermore, neovascularized Dcn−/− mice corneas showed greater increase in mRNA expression of pro-angiogenic factors endoglin (4.58-fold; p < 0.0001), Vegf (4.16-fold; p < 0.0001), and Pdgf (2.15-fold; p < 0.0001) and reduced expression of anti-angiogenic factors Ang2 (0.12-fold; p < 0.05), Timp1 (0.22-fold; p < 0.05), and Vegfr2 (0.67-fold; p > 0.05) compared to neovascularized Dcn+/+ mice corneas. These gene deficience studies carried with transgenic Dcn−/− mice revealed decorin's role in influencing a physiologic balance between pro-and anti-angiogenic factors in the normal and injured cornea. We infer that the functional deletion of Dcn promotes irregular corneal repair and aggravates CNV.

Anti-Angiogenic Efficacy of PSORI-CM02 and the Associated Mechanism in Psoriasis In Vitro and In Vivo.

Psoriasis is a chronic proliferative autoimmune dermatologic disease characterised by abnormal angiogenesis. Thus, regulating angiogenesis in the skin is an important treatment strategy for psoriasis. PSORI-CM02, an empirical Chinese medicine formula optimised from Yin Xie Ling, was created by the Chinese medicine specialist, Guo-Wei Xuan. Clinical studies have shown that PSORI-CM02 is safe and effective for the treatment of psoriasis. However, its anti-psoriatic mechanisms remain to be further explored. In this study, we investigated the effects of PSORI-CM02 on angiogenesis in the skin and the underlying mechanisms in IL-17A-stimulated human umbilical vein endothelial cells (HUVECs) and a murine model of imiquimod (IMQ)-induced psoriasis. In vitro, PSORI-CM02 significantly inhibited the proliferation and migration of IL-17A-stimulated HUVECs in a dose-dependent manner. Further, it markedly regulated the antioxidative/oxidative status and inflammation; suppressed the expression of VEGF, VEGFR1, VEGFR2, ANG1, and HIF-1α; and reduced the phosphorylation of MAPK signalling pathway components in IL-17A-stimulated HUVECs. In vivo studies showed that PSORI-CM02 markedly reduced angiogenesis in the skin of mice with IMQ-induced psoriasis, while significantly rebalancing antioxidant/oxidant levels; inhibiting the production of IL-6, TNF-α, IL-17A, and IL-17F; and repressing the synthesis of angiogenic mediators. In addition, PSORI-CM02 markedly reduced the activation of the MAPK signalling pathway in psoriatic skin tissue. Taken together, our results demonstrated that PSORI-CM02 inhibited psoriatic angiogenesis by reducing the oxidative status and inflammation, suppressing the expression of angiogenesis-related molecules, and inhibiting the activation of the MAPK signalling pathway in vitro and in vivo.

Regulatory role of TIGAR on endothelial metabolism and angiogenesis.

Endothelial glycolytic metabolism plays an important role in the process of angiogenesis. TP53-induced glycolysis and apoptosis regulator (TIGAR) is a significant mediator of cellular energy homeostasis. However, the role of TIGAR in endothelial metabolism, angiogenesis, and coronary flow reserve (CFR) has not been studied. The present study investigated whether knockout (KO) of TIGAR improves endothelial glycolytic function and angiogenesis. In vitro, aortic endothelial cells (ECs) from TIGAR KO mice exhibited increased expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform-3 (PFKFB3) and increased glycolytic function. These were accompanied by increased mitochondrial basal/maximal respiration and ATP production. Furthermore, knockout of TIGAR in ECs enhanced endothelial proliferation, migration, and tube formation. Knockout of TIGAR also significantly increased aortic sprouting ex vivo. In vivo, knockout of TIGAR increased the expression of proangiogenic factor, angiopoietin-1 (Ang-1) in mouse hearts. Knockout of TIGAR also significantly increased coronary capillary density with enhanced CFR in these hearts. Furthermore, TIGAR KO mice subjected to pressure overload (PO), a common model to study angiogenesis and cardiac hypertrophy, exhibited elevated expression of Ang-1, VEGF, and PFKFB3 than that of the wild-type (WT) mice. WT mice subjected to PO exhibited a significant reduction of coronary capillary density and impaired CFR, but TIGAR KO mice did not. In addition, knockout of TIGAR blunted TAC-induced cardiac hypertrophy and dysfunction seen in the WT mice. In conclusion, knockout of TIGAR improves endothelial angiogenetic capabilities by enhancing the endothelial glycolytic function, mitochondrial respiration, and proangiogenic signaling, which leads to increased coronary capillary density and vascular function and protects against chronic stress.

Effect and Mechanism of Si-Miao-Yong-An on Vasa Vasorum Remodeling in ApoE-/- Mice with Atherosclerosis Vulnerable Plague.

Objective: To observe the effect of Si-Miao-Yong-An (SMYA) on atherosclerosis (AS) vulnerable plaques, and to further explore the mechanism by vasa vasorum (VV) angiogenesis and maturation as an entry point. Methods: SPF-class healthy male ApoE−/− mice were randomized into model group, simvastatin group and SMYA group, and C57BL/6 mice were used as the control group. After 8 weeks of intervention, the pathological morphology of plaque was observed by HE staining; the VV density in plaque and aortic adventitia were observed by immunohistochemistry; VV maturation was measured by double-labelling immunofluorescence; the critical proteins of HIF-1α-Apelin/APJ and Ang-1/Tie signal pathways were detected by western blotting. Results: SMYA decreased the plaque area and the ratio of plaque to lumen area; increased the minimum thickness of fibrous cap and its effect was greater than simvastatin. SMYA suppressed the VV neovascularization; promoted smooth muscle cells recruitment and VV maturation, which maintained plaque stability; its effect was obviously superior to simvastatin. SMYA deceased the expression of HIF-1α, Apelin, APJ, Phospho-MEK1/2 (Ser217/221), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), Phospho-p70 S6 Kinase (Thr421/Ser424), Ang-2 and Tie-2; it also increased the expression of Ang-1, Phospho-Akt (Ser473), Phospho-FOXO1 (Ser256) and Survivin. Conclusions: SMYA can decrease the AS plaque area in ApoE−/− mice, suppress the VV neovascularization and promote the VV maturation, and stabilize AS vulnerable plaque. The mechanism could be regulating the HIF-1α-Apelin/APJ and Ang-1/Tie signal pathways.

Interleukin-6 as an enhancer of anti-angiogenic therapy for ovarian clear cell carcinoma

Ovarian clear cell carcinoma (OCCC) is a subtype of epithelial ovarian cancer (EOC) that is associated with elevated interleukin-6 (IL-6) expression, resistance to chemotherapy, and increased mortality. Although bevacizumab (Bev) is a widely used anti-angiogenic agent for EOC, the efficacy of Bev and the role of IL-6 in modulating angiogenesis in OCCC are unknown. We performed tube formation assays using human umbilical vein endothelial cells (HUVEC) cultured in OCCC cell-conditioned medium and using cells directly co-cultured with OCCC cells. We observed that IL-6 inhibition significantly mitigated the ability of Bev to impede tube formation in both cases. Furthermore, IL-6 blockade disrupted the anti-angiogenic efficacy of Bev and its concomitant anti-tumor activity. In addition, IL-6 inhibition resulted in a significant increase in angiopoietin-1 (Ang1) secretion and decreased vascular endothelial growth factor (VEGF) expression. Clinical specimens also exhibited this reciprocal relationship between IL-6 and Ang1 expression. Finally, depletion of Ang1 abrogated the effects of IL-6 inhibition on Bev activity, demonstrating that IL-6 supports the anti-angiogenic activity of Bev by suppressing Ang1 expression and promoting dependence on VEGF for angiogenesis. Altogether, our data suggest that OCCC tumors with high IL-6 levels are candidates for Bev therapy.

Angiopoietin 2 stimulates trophoblast invasion via a mechanism associated with JNK signaling.

Extravillous trophoblast cell (EVT) invasion is tightly controlled, and its dysregulation can lead to altered spiral artery remodeling and contribute to a number of different pregnancy complications. Angiopoietin-2 (Ang-2) is expressed by trophoblast cells and various cells in the decidua, and trophoblast cells express its receptor, Tie2. Ang-2 has been shown to play roles in tumor progression and metastasis but it is not known if it also regulates EVT invasion. Here, we show that both the HTR-8/SVneo cell line and primary isolates of human EVT expressed various integrins and the Tie2 receptor, and Ang-2 stimulated their migration and/or invasion. Ang-2 increased expression of matrix metalloproteinase (MMP)2 and MMP9, altered the cytoskeleton of HTR-8/SVneo cells and also induced phosphorylation of Tie2, JNK and c-Jun. Inhibition of p-JNK (using SP600125) blocked the Ang-2 induced invasion of HTR-8/SVneo cells. In addition, inhibition of Tie2 (pexmetinib) and integrin signaling (RGDS and ATN-161) also blocked Ang-2-induced invasion. In conclusion, we demonstrate that Ang-2 can stimulate EVT invasion via a mechanism associated with activation of both the Tie2 receptor and integrins, which appear to work through different pathways; Tie2 through the JNK/c-JUN pathway and integrins through an as yet unidentified pathway(s). We therefore propose that any alterations in Ang-2 expression in the decidua would lead to an imbalance in pro- and anti-invasive factors, disrupting regulation of EVT invasion and spiral artery remodeling and thereby contribute to the etiology of several complications of pregnancy.

Sequential Exposure to Antenatal Microbial Triggers Attenuates Alveolar Growth and Pulmonary Vascular Development and Impacts Pulmonary Epithelial Stem/Progenitor Cells.

Perinatal inflammatory stress is strongly associated with adverse pulmonary outcomes after preterm birth. Antenatal infections are an essential perinatal stress factor and contribute to preterm delivery, induction of lung inflammation and injury, pre-disposing preterm infants to bronchopulmonary dysplasia. Considering the polymicrobial nature of antenatal infection, which was reported to result in diverse effects and outcomes in preterm lungs, the aim was to examine the consequences of sequential inflammatory stimuli on endogenous epithelial stem/progenitor cells and vascular maturation, which are crucial drivers of lung development. Therefore, a translational ovine model of antenatal infection/inflammation with consecutive exposures to chronic and acute stimuli was used. Ovine fetuses were exposed intra-amniotically to Ureaplasma parvum 42 days (chronic stimulus) and/or to lipopolysaccharide 2 or 7 days (acute stimulus) prior to preterm delivery at 125 days of gestation. Pulmonary inflammation, endogenous epithelial stem cell populations, vascular modulators and morphology were investigated in preterm lungs. Pre-exposure to UP attenuated neutrophil infiltration in 7d LPS-exposed lungs and prevented reduction of SOX-9 expression and increased SP-B expression, which could indicate protective responses induced by re-exposure. Sequential exposures did not markedly impact stem/progenitors of the proximal airways (P63+ basal cells) compared to single exposure to LPS. In contrast, the alveolar size was increased solely in the UP+7d LPS group. In line, the most pronounced reduction of AEC2 and proliferating cells (Ki67+) was detected in these sequentially UP + 7d LPS-exposed lambs. A similar sensitization effect of UP pre-exposure was reflected by the vessel density and expression of vascular markers VEGFR-2 and Ang-1 that were significantly reduced after UP exposure prior to 2d LPS, when compared to UP and LPS exposure alone. Strikingly, while morphological changes of alveoli and vessels were seen after sequential microbial exposure, improved lung function was observed in UP, 7d LPS, and UP+7d LPS-exposed lambs. In conclusion, although sequential exposures did not markedly further impact epithelial stem/progenitor cell populations, re-exposure to an inflammatory stimulus resulted in disturbed alveolarization and abnormal pulmonary vascular development. Whether these negative effects on lung development can be rescued by the potentially protective responses observed, should be examined at later time points.

Macrophages enhance lipopolysaccharide induced apoptosis via Ang1 and NF-\u03baB pathways in human umbilical vein endothelial cells

Lipopolysaccharide (LPS) could induce apoptosis and dysfunction of endothelial cells. We aimed to reveal the effects of macrophages on cell proliferation and apoptosis in LPS induced human umbilical vein endothelial cells (HUVECs). THP-1 derived macrophages and HUVECs were co-cultured in the presence of LPS. Cell viability was measured by Cell Counting Kit-8 and apoptosis was analyzed by flow cytometry. Expression of Ang1, the NF-κB component p65 was evaluated by western blot and quantitative PCR. Small interfering RNAs (siRNAs) were used to knockdown the expression of proinflammatory cytokines and p65 in HUVECs. Plasmid transfection-mediated overexpression of Ang1 was employed to see its effects on cell proliferation and apoptosis in HUVECs. Macrophages enhanced LPS-induced proliferation impairments and apoptosis in HUVECs, which could be attenuated by siRNA-mediated knockdown of cytokines TNF-α, IL-1β, IL-6 and IL-12p70 in macrophages. The dysfunction of HUVECs was tightly associated with reduced Ang1 expression and increased phosphorylated p65 (p-65). Overexpression of Ang1 in HUVECs significantly decreased p-p65, suggesting negatively regulation of p-p65 by Ang1. Overexpression of Ang1, adding recombinant Ang1 or silencing of p65 substantially attenuated the dysfunction of HUVECs in terms of cell proliferation and apoptosis. In conclusions, THP-1-derived macrophages enhance LPS induced dysfunction of HUVECs via Ang1 and NF-κB pathways, suggesting new therapeutic targets for sepsis.

LncRNA NEAT1 promotes proliferation of ovarian cancer cells and angiogenesis of co-incubated human umbilical vein endothelial cells by regulating FGF9 through sponging miR-365: An experimental study.

Objective:To uncover the function of lncRNA NEAT1 in ovarian cancer (OC) cells and its mechanism.Methods:The expression patterns of lncRNA NEAT1 and FGF9 in human OC cells and human ovarian epithelial cells was determined. OC cells were transfected with sh-NEAT1, pcDNA3.1-NEAT1, miR-365 mimic, miR-365 inhibitor or pcDNA3.1-NEAT1 + sh-NEAT1 before cell proliferation rate and cell clone formation rate were measured. After the transfected OC cells were co-cultivated with human umbilical vein endothelial cells (HUVECs), Matrigel angiogenesis assay tested angiogenesis of HUVECs; qRT-PCR and Western blot tested the expressions of vascular endothelial growth factor (VEGF), angiogenin 1 (Ang-1) and matrix metalloproteinase 2 (MMP2). Dual-luciferase reporter assay determined the targeted binding of NEAT1 and FGF9 to miR-365.Results:LncRNA NEAT1 and FGF9 are over-expressed in OC cells. Knockdown of NEAT1 or FGF9, or over-expression of miR-365 results in decreased proliferation rate and cell clones as well as inhibited angiogenesis and down-regulated expressions of VEGF, Ang-1 and MMP2. Over-expression of NEAT1 or knockdown of miR-365 can reverse the effect caused by FGF9 knockdown. NEAT1 can down-regulate the expression of miR-365 while up-regulating that of FGF9. Dual-luciferase reporter assay determined that NEAT1 competes with FGF9 for binding to miR-365.Conclusion:LncRNA NEAT1 up-regulates FGF9 by sponging miR-365, thus promoting OC cell proliferation and angiogenesis of HUVECs.

A Correlative Study Between IVIM-DWI Parameters and the Expression Levels of Ang-2 and TKT in Hepatocellular Carcinoma.

BackgroundNoninvasive evaluation of the expression of angiopoietin-2 (Ang-2) and transketolase (TKT) in hepatocellular carcinoma (HCC) is of great significance for the clinical development of individualized treatment plans. However, the correlation between intravoxel incoherent motion diffusion weighted imaging (IVIM-DWI) and the expression of Ang-2 and TKT has not been reported. We sought to investigate the correlations between IVIM-DWI parameters and Ang-2 and TKT expression levels in HCCs.MethodsConventional non-enhanced magnetic resonance imaging (MRI) and IVIM-DWI and dynamic contrast MRI were performed for 61 patients with HCC before surgical treatment. Various IVIM-DWI parameters, such as apparent diffusion coefficient (ADC), slow apparent diffusion coefficient (D), fast apparent diffusion coefficient (D*) and fraction of fast apparent diffusion coefficient (f), were calculated using Function-MADC software. Expression levels of Ang-2 and TKT in HCC were detected via immunohistochemical staining and classified into two grades. Independent sample t tests were used to compare differences in parameters between the two groups. The Spearman rank correlation test was used to analyze the correlations between IVIM-DWI parameters and Ang-2 and TKT expression levels in HCCs.ResultsThe D* and f values were significantly higher in the high Ang-2 group than in the low Ang-2 group; there were no obvious between-group differences in ADC and D. Ang-2 expression was positively correlated with D* and f but not with ADC and D. The ADC and D values were significantly lower in the high TKT group than in the low TKT group, whereas the between-group differences for D* and f were not significant. TKT expression was negatively correlated with ADC and D but not with D* and f.ConclusionsIVIM-DWI can be used to evaluate Ang-2 and TKT expression in HCC.

The Dual Role of Angiopoietin 2 in Pancreatic Cancer

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is well-known for its inflammatory nature. Inflammation affects the tumour in many ways, one of which is vascular remodeling. Angiopoietins are a family of proteins that interact in vascular remodeling. Angiopoietin-1 (Ang-1) is continuously secreted and works in vascular homeostasis. Inflammation causes endothelial secretion of angiopoietin-2 (Ang-2), which has more complex effects in vascular remodeling. Both endothelial and circulating Ang-2 is increased in various cancers and high circulating Ang-2 is a marker for shorter survival in PDAC. Different from other tumours, in addition to the endothelial expression, PDAC cells themselves express Ang-2. No correlation between tumour cell Ang-2 expression and survival has been published thus far. The aim of this study was to look into tumour cell Ang-2 expression and survival. Method: The study setting constituted of PDAC patients operated at Helsinki University Hospital between 2000 and 2013. Ang-2 immunohistochemistry (IHC) staining was done on 158 PDAC patient samples and staining intensity was scored (ranging from negative (0), weakly positive (1), moderately positive (2), to strongly positive (3)) by two examiners. We used ELISA assay to measure circulating Ang-2 levels in sera of 196 patients. IHC scores and circulating Ang-2 levels were assessed with respect to clinical data and patient outcomes. Results: Neither circulating nor tumour Ang-2 expression was affected by age or sex and had no relationship with tumour size, stage, grade, or lymph node metastasis number. There was a negative association between serum and tumour Ang-2 expession. Disease specific survival (DSS) was shorter in patients with a negative to weak tumour Ang-2 expression (0.95 years, IQR 1.99), when compared to those with an intermediate to high expression (1.84 years, IQR 2.73, p<0.017). In contrast, higher than median (≥2.72ng/ml) circulating Ang-2 associated with shorter DSS (1.49 years, IQR 2.02) when compared to patients with low circulating Ang-2 (2.17years, IQR 2.49, p = 0.002). Conclusion: We found that Ang-2 has a dual role in PDAC with respect to survival. Further studies need to be conducted to find out where the circulating Ang-2 originates and the mechanisms that cause tumour cell Ang-2 expression and its beneficial association with respect to survival.

An oxidation responsive nano-radiosensitizer increases radiotherapy efficacy by remolding tumor vasculature.

As an excellent candidate material for nano-sensitizers, gold nanostructures have shown great potential in radiotherapy. Nevertheless, severe hypoxia and low accumulation of nanomedicine caused by poor perfusion at the tumor site have significantly reduced radiotherapy efficacy. Vascular normalization has gained attention owing to its ability to relieve hypoxia and increase perfusion. The synergistic therapy of tumor vascular normalization and radiotherapy has become a new option to increase anti-cancer efficacy. However, the commonly used strategy of suppressing a single growth factor to induce vascular normalization is limited by tumor compensatory effects. In this work, we developed a strategy to inhibit oxidative stress in tumors by generating chelating agents in response to hydrogen peroxide, thereby inhibiting multi-angiogenic factors simultaneously to normalize blood vessels. Concretely, sodium alginate (SA) reacted with 8-quinoline boric acid (QBA) to form SA-QBA. Then gold nanoparticles (Au NPs) were modified with SA-QBA to obtain Au@SA-QBA. The system was simple in structure and could generate 8HQ in response to H2O2in vitro to inhibit oxidative stress and reduce the expression of VEGF, bFGF, and Ang-2. In vivo, the perfusion unit (PU) increased by 78% after Au@SA-QBA treatment, and the coverage of pericytes increased by 32%, which in turn induced vascular normalization. In addition, blood routine and blood biochemical tests confirmed its good biocompatibility and 8HQ was not detected in the supernatant after homogenization of major organs. More importantly, after the synergistic treatment of vascular normalization and radiotherapy (4 Gy), the tumor growth inhibition rate was increased by 38.6% compared to the Au@SA-treated group with negligible side effects to normal tissues.