VEGF mRNA Levels Research Articles

Expression of Five Important Biomarkers in Non-small Cell Lung Cancer: MicroRNAs-128, miR-335-5p, miR-1254, VEGF, and K-RAS

Background: Lung cancers, such as non-small cell lung cancer (NSCLC), are the most common malignancy and leading cause of cancer-related death worldwide. MicroRNAs (miRNAs) play a role in the occurrence of lung cancer by targeting both tumor suppressor genes and oncogenes. Among them, miR-128, miR-335-5p, and miR-1254 are mainly reported to function in regulating lung cancer cell behavior. Objectives: The aim of the study was to investigate the existence and expression levels of miRNA-128, miR-335-5p, and miR-1254 as well as mRNA and protein expression levels of VEGF and mRNA expression levels of K-RAS in both peripheral blood and cancerous tissue of NSCLC patients compared to the healthy subjects. Methods: Blood and tissue samples were collected from 50 healthy donors and 50 NSCLS patients. The serum level of VEGF was measured using the commercial enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of K-RAS and VEGF, as well as the expression of miRNA-128, miR-335-5p, and miR-1254, were determined, using real-time PCR. Results: Lower mRNA expression levels of miRNA-128, miR-335-5p, and miR-1254, as well as higher mRNA expression levels of VEGF and K-RAS, were detected in both peripheral blood and tissue samples from NSCLC patients compared to those of the healthy subjects. The VEGF protein levels in the peripheral blood of the patients were also found to be significantly lower than those in the healthy subjects. Conclusions: The findings from this study suggest that miR-335-5p, miR-1254, and miR-128, may contribute to the pathogenesis and tumorigenesis of NSCLC at least in part by modulation of proliferation, migration, and angiogenesis via targeting VEGF and K-RAS signaling pathways. K-RAS and VEGF may be target genes for miR-335-5p, miR-1254, and miR-128 in NSCLC patients.

Association of Donor Vascular Endothelial Growth Factor Gene Polymorphism With Acute Renal Allograft Rejection.

Recipient's VEGF-A polymorphisms have been reported to be associated with the risk of acute allograft rejection. However, an association of the donor's VEGF-A gene polymorphism with rejection remained unelucidated till now. In this study, VEGF-A gene SNPs at nine loci were analyzed in 160 kidney donors and recipients with rejection (rejectors, n=80) and without rejection (non-rejectors, n=80). Blood VEGF-A mRNA, plasma VEGF level, and intragraft VEGF expression were also analyzed by RT-PCR, ELISA, and immunohistochemistry, respectively. On comparing between the donor and rejectors, the polymorphic genotypes of VEGF -634 C>G [GG genotype, p<0.0001; OR (95% CI) = 17.74 (5.16-60.96)]; VEGF -1154 G>A [AG genotype, p<0.0001, OR (95% CI)=16.07 (3.68-70.15)]; VEGF +936 C>T [CT genotype, p<0.0001, OR (95% CI)=178.64 (23.28-1370.9), and TT genotype, p<0.0001; OR (95% CI)=3149 (278.91-35553)]; VEGF -1455 T>C [CC genotype, p value=0.0464, OR (95% CI) = 3.13 (1.07-9.10)]; VEGF -2578 C>A [CA genotype, p=0.0426, OR (95% CI)=4.62 (1.03-20.59), and AA genotype, p value<0.0001, OR (95% CI) = 21.89 (4.94-97.04)]; VEGF -2549 18bp Insertion/Deletion [ID genotype, p value<0.0001, OR (95% CI)=27.27 (3.61-205.73) and DD genotype, p value<0.0001, OR (95% CI)=25.18 (3.30-191.89) were significantly associated with acute rejection risk. On comparing rejectors versus non-rejectors, GA genotype of VEGF -1190 G>A and TC genotype of VEGF -1455 T>C were associated with the risk of rejection. On comparing donor VEGF between rejectors and non-rejectors, CG genotype of VEGF -634 C>G, AG of VEGF -1154 G>A; GA of VEGF -1190 G>A were associated with rejection. The blood VEGF-A mRNA and plasma VEGF levels were higher in the rejectors group compared to non-rejectors. Besides the recipient's VEGF SNPS, the donor's VEGF SNPs are also associated with the risk of acute rejection and may be closely monitored in evaluation to determine the risk of rejection.

The role of MDM2 in angiogenesis: implications for endothelial tip cell formation.

In the present study, we examined the role of MDM2 in the angiogenesis process and its potential association with the sprouting of endothelial tip cells. To address this, we performed hypoxia-treated gastric cancer cells (HGC-27) to quantitative RT-PCR and Western blot analysis to measure the levels of MDM2 and VEGF-A mRNA and protein expression. Subsequently, we employed siRNA to disrupt MDM2 expression, followed by hypoxia treatment. The expression levels of MDM2 and VEGF-A mRNA and protein were subsequently reassessed. Additionally, ELISA was utilized to quantify the secretion levels of VEGF-A in each experimental group. A conditioned medium derived from HGC-27 cells treated with different agents was employed to assess its influence on the formation of EA.hy926 endothelial tip cells, using various techniques including Transwell plates migration assays, wound healing experiments, vascular formation assays, scanning electron microscopy, and immunofluorescence staining. These findings demonstrated that the in vitro knockdown of MDM2 in the conditioned medium exhibited significant inhibitory effects on endothelial cell migration, wound healing, and vascular formation. Additionally, the intervention led to a reduction in the presence of CD34+ tip cells and the formation of filopodia in endothelial cells, while partially restoring the integrity of tight junctions. Subsequent examination utilizing RNA-seq revealed that the suppression of MDM2 in HGC-27 cells resulted in the downregulation of the PI3K/AKT signaling pathway. Consequently, this downregulation led to an elevation in angiogenic effects induced by hypoxia.

Integration of physiological, miRNA-mRNA interaction and functional analysis reveals the molecular mechanism underlying hypoxia stress tolerance in crucian carp (Carassius auratus).

Hypoxia has become one of the most critical factors limiting the development of aquaculture. Crucian carp (Carassius auratus) is widely consumed fish in China, with excellent tolerance to hypoxic environment. However, the molecular mechanisms underlying hypoxia adaptation and tolerance in crucian carp remain unclear. Compared with the control, increased T-SOD, CAT, GSH-Px, T-AOC, ALT, and AST activities and MDA, TCHO, and TG contents, and decreased TP and ATP contents were observed after hypoxia stress. Based on RNA-seq, 2479 differentially expressed (DE) mRNAs and 60 DE miRNAs were identified, and numerous DE mRNAs involved in HIF signaling pathway (hif-1α, epo, vegfa, and ho), anaerobic metabolism (hk1/hk2, pfk, gapdh, pk, and ldh) and immune response (nlrp12, cxcr1, cxcr4, ccr9, and cxcl12) were significantly upregulated after hypoxia exposure. Integrated analysis found that ho, igfbp1, hsp70, and hk2 were predicted to be regulated by novel_867, dre-miR-125c-3p/novel_173, dre-miR-181b-5p, and dre-miR-338-5p/dre-miR-17a-3p, respectively, and targets of DE miRNAs were significantly enriched in MAPK signaling pathway, FoxO signaling pathway, and glycolysis/gluconeogenesis. Expression analysis showed that the mRNA levels of vegfa, epo, ho, hsp70, hsp90aa.1, igfbp1, ldh, hk1, pfk, pk, and gapdh exhibited a remarkable increase, whereas sdh and mdh were downregulated in the H3h, H12h, and H24h groups compared with the control. Furthermore, research found that hk2 is a target of dre-miR-17a-3p, overexpression of dre-miR-17a-3p significantly decreased the expression level of hk2, while the opposite results were obtained after dre-miR-17a-3p silencing. These results contribute to our understanding of the molecular mechanisms of hypoxia tolerance in crucian carp.

Ornidazole Inhibits the Angiogenesis and Migration Abilities of Non-small Cell Lung Cancer (NSCLC) via Downregulation of VEGFA/VEGFR2/NRP-1 and PI3K/AKT/mTOR Pathways.

Around the world, non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths among all cancers. Despite advancements in new therapeutic approaches over the past few decades, the five-year survival rate still remains disappointing. The lack of effective anti-angiogenic and anti-migration drugs is the biggest obstacle to the treatment of metastatic lung cancer. Therefore, there is a need to develop new and effective therapeutic compounds targeting anti-angiogenic and anti-migration pathways for the treatment of lung cancer. Ornidazole is a nitroimidazole agent widely used in the treatment of parasitic infections such as trichomonas vaginalis, amebiasis and giardiasis. This study aimed to investigate the anti-proliferative, anti-angiogenic and anti-mitotic activities of the anti-parasitic drug Ornidazole in two human lung cancer cell lines (A549, H1299). We determined the effects of Ornidazole, on cell viability, apoptosis, migration, angiogenesis and metastatic ability against NSCLC in lung cancer cell lines. Its action on the mRNA and protein expression levels of VEGFA, VEGFR2, NRP1, Casp9, Casp3, Bax, Bcl-2, PIK3CA, AKT, MTOR, PTEN and FOX3A was assessed. Furthermore, in this study the effects on cell migration, cell viability and proliferation was evaluated through wound healing, MTT and Crystal violet assays. This study demonstrated that Ornidazole effectively reduces cell viability and migration ability, inhibits angiogenesis and metastatic abilities in NSCLC cells. In conclusion, these results may shed light on the treatment of NSCLC, and we suggest the anti-parasitic drug Ornidazole as a new agent with potential anti-angiogenic and anti-mitotic activity by interfering with the molecular pathways that trigger tumor angiogenesis and migration.

Cannabidivarin and cannabigerol induce unfolded protein response and angiogenesis dysregulation in placental trophoblast HTR-8/SVneo cells

Cannabidivarin (CBDV) and cannabigerol (CBG) are minor phytocannabinoids from Cannabis sativa, whose health benefits have been reported. However, studies about the impact of these cannabinoids on fundamental cellular processes in placentation are scarce. Placental development involves physiological endoplasmic reticulum (ER) stress, however when exacerbated it can lead to altered angiogenesis and pregnancy disorders, such as intrauterine growth restriction and preeclampsia. In this work, the effects of CBDV and CBG (1–10 µM) on placental extravillous trophoblasts were studied, using the in vitro model HTR-8/SVneo cells. Both cannabinoids induced anti-proliferative effects and reactive oxygen/nitrogen species generation, which was dependent on transient receptor potential vanilloid 1 (TRPV1) activation. Moreover, CBDV and CBG significantly upregulated, in a TRPV-1 dependent manner, the gene expression of HSPA5/Glucose-regulated protein 78 (GRP78/BiP), a critical chaperone involved in ER stress and unfolded protein response (UPR) activation. Nevertheless, the UPR pathways were differentially activated. Both cannabinoids were able to recruit the IRE branch, while only CBDV enhanced the expression of downstream effectors of the PERK pathway, namely p-eIF2α, ATF4 and CHOP. It also augmented the activity of the apoptotic initiator caspases-8 and -9, though the effector caspases-3/-7 were not activated. TRB3 expression was increased by CBDV, which may hinder apoptosis termination. Moreover, both compounds upregulated the mRNA levels of the angiogenic factors VEGFA, PGF and sFLT1, and disrupted the endothelial-like behavior of HTR-8/SVneo cells, by reducing tube formation. Thus, CBDV and CBG treatment interferes with EVTs functions and may have a negative impact in placentation and in pregnancy outcome.

Poly(ethylenimine)-Cyclodextrin-Based Cationic Polymer Mediated HIF-1α Gene Delivery for Hindlimb Ischemia Treatment.

Hindlimb ischemia is a common disease worldwide featured by the sudden decrease in limb perfusion, which usually causes a potential threat to limb viability and even amputation or death. Revascularization has been defined as the gold-standard therapy for hindlimb ischemia. Considering that vascular injury recovery requires cellular adaptation to the hypoxia, hypoxia-inducible factor 1 α (HIF-1α) is a potential gene for tissue restoration and angiogenesis. In this manuscript, effective gene delivery vector PEI-β-CD (PC) was reported for the first application in the hindlimb ischemia treatment to deliver HIF-1α plasmid in vitro and in vivo. Our in vitro finding demonstrated that PC/HIF-1α-pDNA could be successfully entered into the cells and mediated efficient gene transfection with good biocompatibility. More importantly, under hypoxic conditions, PC/HIF-1α-pDNA could up-regulate the HUEVC cell viability. In addition, the mRNA levels of VEGF, Ang-1, and PDGF were upregulated, and transcriptome results also demonstrated that the cell-related function of response to hypoxia was enhanced. The therapeutic effect of PC/HIF-1α-pDNA was further estimated in a murine acute hindlimb ischemia model, which demonstrated that intramuscular injection of PC/HIF-1α-pDNA resulted in significantly increased blood perfusion and alleviation in tissue damage, such as tissue fibrosis and inflammation. The results provide a rationale that HIF-1α-mediated gene therapy might be a practical strategy for the treatment of limb ischemia.

Chinese Herbal Compound Xiaoliu Pingyi Recipe Inhibits the Growth of Lung Adenocarcinoma by Regulating the Tumor Vascular Microenvironment.

The traditional Chinese medicine (TCM) Xiaoliu Pingyi recipe (XLPYR) has been clinically used for several decades, demonstrating favorable therapeutic effects. However, the underlying regulatory mechanisms remain unclear. The aim of this study was to explore the anti-tumor effects of XLPYR and its regulatory role in the vascular microenvironment through in vivo and in vitro experiment. In the in vivo study, a C57BL/6J mouse model of lung adenocarcinoma (LUAD) allografts was established, and various interventions were administered for 14 days (Model group: administered normal saline via oral gavage; Pemetrexed (PEM) group: intraperitoneally injected with a solution of pemetrexed, once every 3d; XLPYR group: administered XLPYR via oral gavage; Combination (COMBI) group: received XLPYR via oral gavage simultaneously with intraperitoneal injection of pemetrexed solution). Tumor volume and weight were then compared among the groups. The impact of XLPYR on the tumor vascular microenvironment was assessed using immunohistochemistry staining. In the in vitro study, XLPYR-containing serum was prepared by oral administration to SD rats. The CCK-8 assay evaluated the effect of the serum on the proliferation of normal lung epithelial BEAS-2B cells and LUAD A549 cells, determining the optimal intervention concentrations. The cell migration and invasion abilities were evaluated using the wound-healing assay and Transwell assay, respectively. Finally, ELISA assay measured VEGF secretion levels in the LUAD cell supernatant, and RT-qPCR and Western Blot were employed to detect differences in HIF-1α, VEGFA, Ang-2, and PI3K/Akt mRNA and protein expression levels in both in vivo and in vitro experiments. In the in vivo study, XLPYR significantly inhibited the growth of mice LUAD allografts, with enhanced anti-tumor effects observed with prolonged drug intervention. Immunohistochemistry staining revealed reduced MVD and increased pericyte coverage in all intervention groups. Regarding vascular function, FITC-Dextran extravasation in the tumor tissues of the Model group was significantly higher than in the intervention groups, particularly with lower extravasation in the COMBI group compared to the PEM group. In the in vitro study, XLPYR demonstrated a time- and concentration-dependent inhibitory effect on LUAD cells, and with greater sensitivity in inhibiting LUAD cells compared to BEAS-2B cells. The wound-healing assay and Transwell assay confirmed that XLPYR significantly suppressed the migration and invasion abilities of LUAD cells. ELISA experiments further revealed a significant decrease in VEGF expression in the supernatant of each intervention group. RT-qPCR and Western Blot results showed consistent findings between the in vivo and in vitro experiments. HIF-1α, VEGFA, and Ang-2 mRNA and protein expression levels were significantly downregulated in the PEM group, XLPYR group, and COMBI group. There were no significant differences in the expression of PI3K and Akt mRNA and total protein, but the expression levels of phosphorylated p-PI3K and p-Akt were notably downregulated. XLPYR significantly inhibited C57BL/6J mouse LUAD allograft growth and improved the vascular microenvironment, thereby intervening in tumor angiogenesis and inducing vascular normalization. It suppressed LUAD cell proliferation, migration, and invasion, while reducing VEGF concentration in the cell supernatant. The regulatory mechanism may involve inhibiting PI3K/Akt protein phosphorylation and downregulating angiogenesis-related factors, such as HIF-1α, VEGF, and Ang-2.

Melittin inactivates YAP/HIF-1α pathway via up-regulation of LATS2 to inhibit hypoxia-induced proliferation, glycolysis and angiogenesis in NSCLC

BackgroundNSCLC is one of the most common causes of death. The hypoxia microenvironment contributes to cancer progression. The purpose was to explore the effects and mechanism of melittin on NSCLC cells in the hypoxic microenvironment. MethodsNSCLC cell lines (A549 and H1299) were cultured in normoxia or hypoxia conditions with or without melittin treatment. The viability of the cells was detected via MTT assay and the proliferation ability was evaluated by EdU assay. QRT-PCR was performed to evaluate GLUT1, LDHA, HK2, VEGF and LATS2 mRNA levels. Glucose transport was assessed by the 2-NBDG uptake assay. The angiogenesis was determined by the tubule formation assay. The protein expressions of GLUT1, LDHA, HK2, VEGF, LATS2, YAP, p-YAP and HIF-1α were detected via western blotting assay. The tumor formation assay was conducted to examine the roles of melittin and LATS2 in vivo. ResultsMelittin inhibited hypoxia-induced cell viability, proliferation, glycolysis and angiogenesis as well as suppressed YAP binding to HIF-1α in NSCLC. Melittin inactivated the YAP/HIF-1α pathway via up-regulation of LATS2, ultimately inhibiting cancer progression of NSCLC. Moreover, melittin suppressed tumor growth via up-regulation of LATS2 in vivo. ConclusionMelittin inactivated the YAP/HIF-1α pathway via up-regulation of LATS2 to contribute to the development of NSCLC. Therefore, melittin is expected to become a potential prognostic drug for the therapy of NSCLC.

Effect of RNA interference with HIF-1α on the growth of pulmonary artery endothelial cells in broiler chickens

Pulmonary artery remodeling is a characteristic feature of broiler ascites syndrome (BAS). Pulmonary artery endothelial cells (PAECs) regulated by HIF-1α play a critical role in pulmonary artery remodeling, but the underlying mechanisms of HIF-1α in BAS remain unclear. In this experiment, primary PAECs were cultured in vitro and were identified by coagulation factor VIII. After hypoxia and RNA interference, the mRNA and protein expression levels of HIF-1α and VEGF were determined by qPCR and Western blotting. The transcriptome profiles of PAECs were obtained by RNA sequencing. Our results showed that the positive rate of PAECs was more than 90%, hypoxia-induced promoted the proliferation and apoptosis of PAECs, and RNA interference significantly downregulated the expression of HIF-1α, inhibited the proliferation of PAECs, and promoted the apoptosis of PAECs. In addition, transcriptome sequencing analysis indicated that HIF-1α may regulate broiler ascites syndrome by mediating COL4A, vitronectin, vWF, ITGα8, and MKP-5 in the ECM, CAMs and MAPK pathways in PAECs. These studies lay the foundation for further exploration of the mechanisms of pulmonary artery remodeling, and HIF-1α may be a potentially effective gene for the prevention and treatment of BAS.

Involvement of Angiopoietin 2 and vascular endothelial growth factor in uveitis.

Angiopoietin (Ang) 2 is released from vascular endothelial cells by the stimulation of vascular endothelial growth factor (VEGF)A. Ang2 increases the expression of leukocyte adhesion molecules on endothelial cells via nuclear factor κB. The aim of this study was to evaluate the effects of Ang2 and VEGFA on ocular autoimmune inflammation. We measured the concentrations of Ang2 and VEGFA in vitreous samples among patients with uveitis. Vitreous samples were collected from 16 patients with idiopathic uveitis (uveitis group) and 16 patients with non-inflammatory eye disease (control group). Experimental autoimmune uveoretinitis (EAU) was induced in B10.BR mice with a human interphotoreceptor retinoid-binding protein-derived peptide. The retinochoroidal tissues of the EAU mice were removed, and the mRNA levels of Ang2 and VEGFA were examined. EAU mice treated with anti-Ang2, anti-VEGFA, a combination of anti-Ang2 and anti-VEGFA, anti-Ang2/VEGFA bispecific, or IgG control antibodies were clinically and histopathologically evaluated. The protein levels of Ang2 and VEGFA were significantly higher in the vitreous samples of patients with uveitis than in controls (P<0.05). The retinochoroidal mRNA levels of Ang2 and VEGFA were significantly upregulated in EAU mice compared to controls (n = 6, P<0.05). Although there was no significant difference, treatment with anti-VEGFA antibody reduced the clinical and histopathological scores. However, treatment with anti-Ang2 antibody reduced the clinical and histopathological scores (n = 18-20, P<0.05). Furthermore, these scores were further decreased when treated by inhibiting both Ang2 and VEGFA. Based on these results, VEGFA and Ang2 were shown to be upregulated locally in the eye of both uveitis patients and models of uveitis. Dual inhibition of Ang2 and VEGFA is suggested to be a new therapeutic strategy for uveitis.

EVs derived from physiologically loaded myocardial slices affect remodelling of overloaded myocardial slices

Abstract Introduction Mechanical load is an important determinant of cardiac output, and excessive mechanical load can lead to heart remodelling, and ultimately failure. Cardiac intercellular communication is partially mediated by extracellular vesicles (EVs), which are 30-200 nm sized nanoparticles acting as shuttles of many bioactive cargoes that modulate cell behaviour, such as microRNAs. Living myocardial slices are an ideal platform to study how mechanical load affect intercellular communication through EVs, as they can be manipulated to obtain different degrees of preload and are an organotypic multicellular platform that retains the electrophysiology and extracellular matrix of the adult myocardial tissue. Purpose The aim of the study is to investigate how EVs derived from physiologically-loaded slices modulate the myocardial remodelling of overloaded slices. Methods 300 µm slices were obtained from the left ventricle of 300-350 g male Sprague Dawley rats. Mechanical load was applied by stretching the slices to either 2.2 or 2.4 µm of their sarcomere length, to recapitulate physiological and overloaded condition, respectively. Following 48-hours of biomimetic culture, force-stretch relationship of the slices was assessed, the tissue collected for RNA and protein extraction and the media of physiological slices was harvested for EVs isolation by size exclusion chromatography. Physiological EVs were then applied onto overloaded slices (1x108 EVs/slice) and cultured for 48-hours to assess force-stretch relationship, and protein and mRNA expression associated with cardiac remodelling. Physiological and overloaded EVs content was analysed through a microRNA array. Results Physiological slices had a significantly higher contractility (P &amp;lt; 0.05), higher blood vessel density (P &amp;lt; 0.0001) and lower apoptosis (P&amp;lt;0.01) compared to overloaded slices. Application of physiological EVs on overloaded slices restored their contractility (P&amp;lt;0.05), attenuated apoptosis by reducing protein PARP-1 expression (P&amp;lt;0.05) and reduced blood vessels density loss (P&amp;lt; 0.01), as shown by Isolectin B4 staining and increased mRNA levels of VEGF. microRNAs analysis showed significant downregulation of miR-346 and miR-16-5p and upregulation of miR-143-3p miR-378a-3p in physiological EVs, compared to overloaded. The upregulation or downregulation of these microRNAs was positively associated with cardiac apoptosis and migration of vascular smooth muscle cells. Conclusions Physiological EVs affect remodelling in overloaded slices. This is likely due to an effect on apoptosis or to an indirect effect on endothelial cells. Further validation and mechanistic studies with miRNAs selected candidates may reveal interesting therapeutic targets for heart failure.

Morphological and Molecular Biological Features of the Systemic Inflammatory Response in Old Wistar Rats with High and Low Resistance to Hypoxia.

Morphological and molecular biological features of LPS-induced systemic inflammatory response were assessed in old male Wistar rats with high (HR) and low (LR) resistance to hypoxia. The systemic inflammatory response was modeled by intraperitoneal injection of E. coli O26:B6 LPS; the animals were sacrificed after 6 h. In histological sections, the number of neutrophils in the interalveolar septa and the area of necrosis in the liver were determined. The expression levels of Hif1a, Hif2a, Nfkb, Vegf, Il1b, Il6, Il10, and Tgfb mRNA in the liver and serum concentrations of HIF-1α and IL-1β were determined. In 4-6 h after LPS injection, 3 (43%) of 7 HR rats died, whereas no deaths were observed among LR rats. At 6 h after LPS injection, the number of neutrophils in the interalveolar septa of the lungs in LR rats was significantly higher than in HR rats, while the area of necrosis in the liver did not differ. At the same time, the mRNA expression levels of the proinflammatory cytokine genes Il1b and Il6 increased in the liver of both HR and LR rats, whereas the expression of Il10 increased only in HR rats. The expression of the Hif1a gene in the liver was higher in LR rats, but the content of HIF-1α protein in blood serum was higher in HR animals. These data should be taken into account when developing new approaches to the therapy of systemic inflammatory response in infectious and inflammatory diseases in the elderly.

Cellular and Molecular Mechanisms of Toxic Liver Fibrosis in Rats Depending on the Stages of Its Development.

Liver fibrogenesis in male Wistar rats was induced with the thioacetamide solution by introducing into the stomach with a probe at a dose of 200 mg/kg of animal body weight 2 times per week. The process dynamics was studied at 5 time points (control, week 3, week 5, week 7, and week 9). The mRNA levels of tweak, fn14, ang, vegfa, cxcl12, and mmp-9 genes in liver were detected by real-time polymerase chain reaction. Immunohistochemical study was performed on paraffin sections. The CD31, CD34, CK19, α-SMA, FAP, CD68, CD206, CX3CR1, and CD45 cells were used as markers. Fibrosis degree was determined in histological sections, stained in line with the Mallory technique, according to the Ishak's semi-quantitative scale. Two simultaneously existing morphologically heterogeneous populations of myofibroblasts expressing different types of markers (FAP, α-SMA) were identified in rat liver. Prior to the onset of transformation of fibrosis into cirrhosis (F1-F4, weeks 3-7), FAP+ and SMA+ cells were localized in different places on histological specimens. All stages of liver fibrosis development were accompanied by an increase in the number (p=0.0000), a change in the phenotypic structure and functional properties of macrophages. The CK19+ cells of the portal areas differentiated into cholangiocytes that formed interlobular bile ducts and ductules, as well as hepatocytes that formed rudiments of new hepatic microlobules. Pathological venous angiogenesis and heterogeneity of endotheliocytes of the intrahepatic vascular bed were detected. Two options for changes in mRNA expression of the selected genes were identified. The level of the fn14 and mmp-9 mRNAs at all stages of fibrosis was higher (p=0.0000) than in control rats. For tweak, ang, vegfa, and cxcl12 mRNAs, the situation was the opposite - the level of genes decreased (p=0.0000). There were strong and moderate correlations between the studied target genes (p<0.05). It was established that the stages of toxic fibrosis had morphological and molecular genetic features. The FAP+ cells make the main contribution to development of portal and initial stage of bridging fibrosis. The stellate macrophages and infiltrating monocytes/ macrophages can potentially be used for development of new therapeutic strategies for liver pathology treatment. One should take into account the features of the markers' expression by endothelial cells during the study of the intrahepatic vascular bed. Joint study of genes is a necessary ad-hoc parameter in fundamental and preclinical research.

Comparative study of Curcumin and Lucentis on retinal neovascularization

Introduction To investigate the effect of Curcumin on retinal neovascularization in mice with oxygen-induced lesions. Methods 7-day-old (P7) C57/BL6J mice were randomly divided into control group, OIR group, DMSO group, 100 mg/kg, 50 mg/kg and 25 mg/kg curcumin group and Lucentis group (15 mice per group). Mice in the experimental group were fed for 5 days in high oxygen partial pressure environment, and then in normal oxygen air environment for another 5 days. Corresponding interventions were given at 12-16 days of age (P12-16). At 17 days of age (P17), the eyeball was removed and the retina was paved with Isolectin GS-IB4 fluorescence staining. Real-time PCR was used to detect VEGF mRNA levels in tissues and cells. The protein expression level of VEGF was detected by Western blot. Results Immunofluorescence showed that curcumin injection could significantly reduce the formation of retinal neovascularization and astrocyte injury in OIR, and 100 mg/kg curcumin group had the best effect. Compared with the control group, mRNA and protein expression of VEGF in retina of mice in OIR and DMSO groups were significantly up-regulated (P<0.05); Compared with OIR group, curcumin group and Lucentis group were down-regulated (P<0.05). The protein expression and mRNA level of VEGF in HRCECs of curcumin group decreased with the increase of curcumin concentration, and the effect of curcumin group at 80μmol/L was similar to that of Lucentis group. In the HRCECs cultured with the same concentration of curcumin, the protein expression and mRNA level of VEGF decreased with the prolongation of drug intervention time. Conclusion Curcumin can down-regulate the expression of VEGF in retinal tissues and cells, thereby inhibiting retinal neovascularization and HRCECs cell proliferation.

Cytotoxic Flavokawain B Inhibits the Growth and Metastasis of Hepatocellular Carcinoma through UCK2 Modulation of the STAT3/Hif-1α/VEGF Signalling Pathway.

Hepatocellular carcinoma (HCC) is associated with a high mortality rate due to early recurrence and its metastasis features. To this day, effective treatment options for metastatic HCC remain a major challenge to patient treatment. Flavokawain B (FKB) is a naturally occurring chalcone molecule capable of providing effective therapy against this life-threatening disease. This study investigated the anti-metastatic effects of FKB on the growth and development of metastatic HCC. HepG2 cells were used in this study and a neutral red assay was performed to determine the IC50 value of FKB. Cell scratch and exclusion zone assays were performed to assess the rate of cell migration and invasion. Relative mRNA levels of UCK2, STAT3, VEGF and HIF-1α genes were quantified using RT-qPCR. FKB inhibited the proliferation of HepG2 cells at an IC50 value of 28 μM after 72 h of incubation. Its cytotoxic effect was confirmed to induce apoptosis through the phase-contrast inverted microscope. Cell migration and invasion were significantly inhibited at 7, 14, and 28 μM of FKB as compared to untreated cells. The inhibition in the cell migration significantly increased with the increasing concentrations of the bioactive compound. The relative expression levels of the UCK2 gene and its downstream genes, STAT3, VEGF and HIF-1α, were significantly downregulated after 72 h exposure to FKB treatment. Our data suggest that FKB inhibited HepG2 proliferation and further suppressed its metastasis partly by regulating the STAT3/Hif-1α/VEGF signalling pathway. FKB could be a potential alternative and viable strategy against HCC.

Gentiopicroside-Loaded Chitosan Nanoparticles Inhibit TNF-α-Induced Proliferation and Inflammatory Response in HaCaT Keratinocytes and Ameliorate Imiquimod-Induced Dermatitis Lesions in Mice.

In this study, we aimed to report the biological characteristics of the first successful synthesis of gentiopicroside-loaded chitosan nanoparticles and to evaluate the therapeutic effects and preliminary mechanisms of gentiopicrin-loaded chitosan on psoriasis-like cell and mouse models. Gentiopicroside-loaded chitosan nanoparticles (CHI-GEN) were prepared, and their biological characteristics were evaluated. HaCaT keratinocytes were stimulated with TNF-α to establish a psoriatic keratinocyte model. MTT assay and flow cytometry were used to measure cell viability and apoptosis, respectively. mRNA levels of K17, VEGF A, and IL-6 and IL-23A were detected using qRT-PCR. These tests were used to preliminarily assess the effects of CHI-GEN on keratinocyte proliferation and inflammation. Imiquimod was used to construct a psoriasis-like mice model. The severity of psoriasis was scored based on the psoriasis area severity index (PASI), H&E staining was used to observe the histological changes and the level of inflammation and cell proliferation of skin lesions was evaluated by measuring the mRNA levels of K17, IL-23A, and IL-17A using qRT-PCR. The average particle size of CHI-GEN nanoparticles was approximately 100 nm, and the zeta potential was 2.69 ± 0.87 mV. The cumulative release was 67.2% in solutions of pH 5.5 at 24 h. GEN reduced TNF-α-induced excessive proliferation of HaCaT keratinocytes and downregulated mRNA levels of K17, VEGF A, and inflammatory cytokines IL-6 and IL-23A, which was more obvious in the CHI-GEN treatment group. Additionally, CHI-GEN significantly improved the severity of skin lesions in psoriasis-like mice and downregulated the mRNA expressions of IL-6, IL-23A, and IL-17A in mice skin lesions. In conclusion, we successfully prepared gentiopicrin-chitosan nanoparticles. Our results show that these nanoparticles have anti-psoriasis activity, inhibits keratinocyte proliferation and improves symptoms in psoriasis model mice and can be used to develop an effective strategy for the treatment of psoriasis.

Mechanism of the HIF-1α/VEGF/VEGFR-2 pathway in the proliferation and apoptosis of human haemangioma endothelial cells.

Haemangiomas (HAs) are prevalent vascular endothelial cell tumours. With respect to the possible involvement of HIF-1α in HAs, we have explored its role in haemangioma endothelial cell (HemEC) proliferation and apoptosis. shRNA HIF-1α and pcDNA3.1 HIF-α were manipulated into HemECs. HIF-α, VEGF, and VEGFR-2 mRNA and protein levels were assessed by qRT-PCR and Western blotting. Cell proliferation and viability, cell cycle and apoptosis, migration and invasion, and ability to form tubular structures were assessed by colony formation assay, CCK-8, flow cytometry, Transwell assay, and tube formation assay. Cell cycle-related protein levels, and VEGF and VEGFR-2 protein interaction were detected by Western blot and immunoprecipitation assays. An Haemangioma nude mouse model was established by subcutaneous injection of HemECs. Ki67 expression was determined by immunohistochemical staining. HIF-1α silencing suppressed HemEC neoplastic behaviour and promoted apoptosis. HIF-1α facilitated VEGF/VEGFR-2 expression and the VEGF had interacted with VEGFR-2 at protein - protein level. HIF-1α silencing arrested HemECs at G0/G1 phase, diminished Cyclin D1 protein level, and elevated p53 protein level. VEGF overexpression partially abrogated the effects of HIF-1α knockdown on inhibiting HemEC malignant behaviours. Inhibiting HIF-1α in nude mice with HAs repressed tumour growth and Ki67-positive cells. Briefly, HIF-1α regulated HemEC cell cycle through VEGF/VEGFR-2, thus promoting cell proliferation and inhibiting apoptosis.

Combined treatment with Alhagi maurorum and docetaxel inhibits breast cancer progression via targeting HIF-1α/VEGF mediated tumor angiogenesis in vivo

Breast cancer is a challenging disease and leading cause of cancer death in women. There is no effective agent for metastatic breast cancer after surgery and chemotherapy. Alhagi maurorum (A.m) has been reported to exhibit an anticancer effect on various types of cancer cells in vitro. This study aimed to examine the suppressive effect of A.m alone and combined with docetaxel (DTX) on the breast cancer growth in mice models and the possible underlying mechanisms. In the present study, the mice were inoculated subcutaneously with the injections of 4T1 cells. Then, A.m, DTX, and their combination were administered intraperitoneally. The expressions of β-catenin (β-cat), FZD7, MMP2, HIF1-α, and VEGF A (vascular endothelial growth factor A) were investigated using RT-PCR method. Also, plasma alkaline phosphatase (ALP), alanine aminotransferase (GPT or ALT), aspartate transaminase (GOT or AST), serum creatinine, and urea were examined, and histological analyses of the tissues were conducted. The results demonstrated that A.m (500 mg/kg) combined with DTX significantly decreased the expression of β-cat, MMP2, and FZD7 as compared with the negative control group and monotherapies. Also, the mRNA levels of HIF1-α and VEGF A were suppressed significantly by DTX + A.m (500 mg/kg). Tumor weights and sizes were significantly lower and tumor inhibition rate was significantly higher in the DTX + A.m group. The A.m 500 mg/kg + DTX also suppressed the serum GPT level in tumor-bearing mice and decreased the serum urea level. Taken together, our findings suggest that DTX combined with A.m at an optimal dose of 500 mg/kg as the optimal dose can inhibit β-cat, FZD7, MMP2, and breast cancer growth via interrupting HIF-1α/VEGF signaling and might be used as a promising antiangiogenic agent for breast cancer treatment.

1α,25(OH)2D3 regulates pro-angiogenic factors in endothelial cells transformed by Kaposi's sarcoma-associated herpesvirus G protein coupled receptor

When tumoral cell expansion exceeds the vascular supply, regions of hypoxia or low oxygen concentration are generated promoting the formation of new vessels through cell proliferation and migration. Viral G protein-coupled receptor (vGPCR) is associated to Kaposi's sarcoma pathology and induces a paracrine transformation when is stably expressed in murine endothelial cells activating hypoxia-induced transcription factors. Previously, we reported the antiproliferative actions of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in endothelial cells transformed by the vGPCR (SVEC-vGPCR). Herein, we further investigated if pro-angiogenic factors as AP-1, HIF-1α and VEGF are modulated by 1α,25(OH)2D3. We found by qRT-PCR analysis that the mRNA level of JunB, a negative regulator of cell proliferation, was similarly increased at all-time points tested after 1α,25(OH)2D3 treatment in SVEC-vGPCR cells. Also, mRNA levels of the pro-angiogenic factor c-Fos, which induces tumor invasion, were only decreased during one short period treatment. In addition, Hif-1α mRNA and protein levels were significantly reduced after 1α,25(OH)2D3 treatment in a VDR dependent fashion. However, mRNA levels of the angiogenic activator Vegf, promoted in turn by Hif-1α expression, were surprisingly high depending on VDR expression as well. Moreover, Egr-1, which has been reported to induce VEGF expression independently of HIF-1α, diminished its expression with 1α,25(OH)2D3 treatment, fact that was related to the decline of p-ERK1/2. Altogether, these results suggest a negative modulation of some pro-angiogenic factors like AP-1 and HIF-1α, as part of the antiproliferative mechanism of 1α,25(OH)2D3 in SVEC-vGPCR endothelial cells.