Vascular Endothelial Growth Factor Protein Expression Research Articles

Effect of moxibustion on the expressions of hypoxia inducible factor-1α and vascular endothelial growth factor in ankle synovial tissue of rats with adjuvant arthritis

To observe the effect of moxibustion on the expressions of hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in ankle synovial tissue of rats with adjuvant arthritis(AA), so as to explore the mechanism of moxibustion in inhibiting synovial angiogenesis and improving joint symptoms of rheumatoid arthritis. Sixty healthy male SD rats were randomly divided into normal group, model group, moxibustion group and medication group, with 15 rats in each group. AA rat model was established by subcutaneous injection of Freund's complete adjuvant into the right hind paw. Rats in the moxibustion group were treated with "Zusanli" (ST36), "Guanyuan" (CV4) and "Ashi" point moxibustion, 20 min each time, once a day, for consecutive 3 weeks. Rats in the medication group were given methotrexate (0.35 mg/kg) intragastric administration, twice a week, for consecutive 3 weeks. Foot plantar volume of rats was measured by toe volume mea-suring instrument. HE staining was used to observe the histopathology of ankle synovium. The protein expressions of HIF-1α and VEGF in ankle synovial tissue were detected by immunohistochemistry and Western blot. Compared with the normal group, the foot plantar volume and the protein expressions of HIF-1α and VEGF in synovial tissue of ankle joint were significantly increased (P<0.01) in the model group, the synovial tissue showed obvious hyperplasia and a large number of neovasculogenesis. Following the interventions, the foot plantar volume and the protein expressions of HIF-1α and VEGF in synovial tissue of ankle joint were significantly decreased (P<0.05, P<0.01) in both moxibustion and medication groups in contrast to the model group, and there was no obvious proliferation of synovial tissue, and only a few neovascularization was observed. Compared with the medication group, the foot plantar volume was decreased (P<0.05) in the moxibustion group. Moxibustion can improve joint swelling and inhibit synovial angiogenesis in AA rats, and its mechanism may be related to down-regulating of HIF-1α and VEGF protein expressions.

Tumor Necrosis Factor-α Promotes the Tumorigenesis, Lymphangiogenesis, and Lymphatic Metastasis in Cervical Cancer via Activating VEGFC-Mediated AKT and ERK Pathways.

Lymphatic metastasis is a common phenomenon of cervical cancer. Tumor necrosis factor-α (TNF-α) was found to be closely associated with lymphatic cancer metastasis. However, the mechanism through which TNF-α regulates lymphatic metastasis in cervical cancer remains unclear. In this study, cervical cancer cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with or without TNF-α for 48 h, and then the corresponding conditional medium (CM-TNF-α or CM) was collected. The level of vascular endothelial growth factor (VEGFC) in the corresponding CM was then detected using an enzyme-linked immunosorbent assay (ELISA). Next, human lymphatic endothelial cells (HLECs) were cultured in CM-TNF-α or CM for 48 h. Cell viability was measured using the cell counting kit-8 (CCK-8) assay, and angiogenesis was detected using a tube formation assay. Subsequently, the expressions of AKT, p-AKT, ERK, and p-ERK in HLECs were detected using western blotting. In addition, to further investigate the effect of TNF-α on the progression of cervical cancer, a C33A subcutaneous xenograft model was established in vivo. We found that TNF-α significantly stimulated cervical cancer cells to secrete VEGFC. Additionally, the CM collected from the TNF-α-treated cervical cancer cells notably promoted the proliferation, migration, and angiogenesis of HLECs; however, these changes were reversed by MAZ51, a VEGFR3 inhibitor. Moreover, TNF-α obviously elevated D2-40 and VEGFC protein expressions in tumor tissues, promoting lymphangiogenesis and lymphatic metastasis in vivo. Meanwhile, TNF-α markedly upregulated p-AKT and p-ERK expressions in tumor tissues, whereas these changes were reversed by MAZ51. Collectively, TNF-α could promote tumorigenesis, lymphangiogenesis, and lymphatic metastasis in vitro and in vivo in cervical cancer via activating VEGFC-mediated AKT and ERK pathways. These results may provide new directions for the treatment of cervical cancer.

What is the Effect of Bevacizumab on Cartilage and Synovium in a Rabbit Model of Hemophilic Arthropathy?

Hemophilic arthropathy can cause recurrent hemarthroses and severe damage to the synovium and articular cartilage. Previous studies have shown that vascular endothelial growth factor (VEGF) plays an essential role in neoangiogenesis. Bevacizumab, a monoclonal VEGF inhibitor, is used clinically to prevent angiogenesis. However, its effects on hemophilic arthropathy are unknown. Using a hemophilic arthropathy rabbit model, we asked: Does an intra-articular injection of bevacizumab (1) inhibit VEGF, (2) decrease signal intensity in dynamic contrast-enhanced MRI (DCE-MRI) as an assessment of capillary permeability and neoangiogenesis, (3) reduce cartilage damage, (4) reduce synovial changes, and (5) affect macroscopic changes during the development of hemophilic arthropathy? Twenty-five male New Zealand rabbits were divided into four groups. Eight knees from four rabbits were used as the control group. We used an established animal model for hemophilic arthropathy in the remaining 21 rabbits. Animals were assigned randomly to three groups with seven rabbits in each group. One group was used to establish mild arthropathy, and the other two were used to establish severe arthropathy. Autologous blood from the rabbits' ears was injected into the right and left knees twice per week for 8 weeks to represent mild arthropathy and for 16 weeks to represent severe arthropathy. In the mild arthropathy group, bevacizumab was injected into the right knee once every 2 weeks. Bevacizumab was injected into the right knee of rabbits in one of the severe arthropathy groups once every 2 weeks for 16 weeks, and intra-articular bevacizumab injections were administered to the right knees of rabbits in the other severe arthropathy group once every 2 weeks after the eighth week. An equal volume of 0.9% saline was injected into the left knee of rabbits in all arthropathy groups. To explore the efficacy of bevacizumab, joint diameters were quantitatively measured, and cartilage and synovial changes were examined. Degeneration of articular cartilage was evaluated with the semiquantitative Osteoarthritis Research Society International grading system. Synovial damage was analyzed with a semiquantitative microscopic scoring system. In addition, we evaluated perfusion and angiogenesis using DCE-MRI (quantitative signal intensity changes). Immunohistochemical testing was used to measure VEGF levels (analyzed by Western blotting). Intra-articular bevacizumab treatment inhibited VEGF in our rabbit model of hemophilic arthropathy. VEGF protein expression levels were lower in the mild arthropathy group that received intra-articular bevacizumab (0.89 ± 0.45) than the mild arthropathy control group (1.41 ± 0.61) (mean difference -0.52 [95% CI -0.898 to -0.143]; p = 0.02). VEGF levels were lower in the severe arthropathy group that received treatment for 16 weeks (0.94 ± 0.27) than in the control knees (1.49 ± 0.36) (mean difference -0.55 [95% CI -0.935 to -0.161]; p = 0.01). In the severe arthropathy group, the Osteoarthritis Research Society International score indicating cartilage damage was lower in the group that received intra-articular bevacizumab treatment from the beginning than in the control group (median 17 [range 13 to 18] versus 18 [range 17 to 20]; difference of medians 1; p = 0.02). Additionally, the scores indicated synovial damage was lower in the group that received intra-articular bevacizumab treatment from the beginning than the control group (median 5 [range 4 to 9] versus 9 [range 8 to 12]; difference of medians 4; p = 0.02). The mean of mean values for signal intensity changes was higher in the nontreated severe groups than in the group of healthy knees. The signal intensity changes were higher in the severe arthropathy control groups (Groups BC and CC) (median 311.6 [range 301.4 to 361.2] and 315.1 [range 269.7 to 460.4]) than in the mild arthropathy control group (Group AC) (median 234.1 [range 212.5 to 304.2]; difference of medians 77.5 and 81, respectively; p = 0.02 and p = 0.04, respectively). In the severe arthropathy group, discoloration caused by hemosiderin deposition in the cartilage and synovium was more pronounced than in the mild arthropathy group. In the severe arthropathy group treated with intra-articular bevacizumab, joint diameters were smaller than in the control group (Group BT median 12.7 mm [range 12.3 to 14.0] versus Group BC median 14.0 mm [range 13.1 to 14.5]; difference of medians 1.3 mm; p = 0.02). Hemarthrosis damages the synovial tissues and cartilage in the knees of rabbits, regardless of whether they are treated with intra-articular bevacizumab. However, intra-articular injection of bevacizumab may reduce cartilage and synovial damage in rabbits when treatment is initiated early during the development of hemophilic arthropathy. If the findings in this study are replicated in larger-animal models that consider the limitations of our work, then a trial in humans might be appropriate to ascertain whether intra-articular injection of bevacizumab could reduce cartilage damage and synovial changes in patients with hemophilia whose hemarthroses cannot otherwise be controlled.

Placental micro blood perfusion for isolated single umbilical artery foetuses and VEGF protein expression in the placenta

Microblood perfusion of isolated single umbilical artery (ISUA) foetus placenta was evaluated using three-dimensional power Doppler ultrasound (3D-PDU). Vascular endothelial growth factor (VEGF) protein expression in the placenta was also semi-quantitative and qualitatively analysed. Differences between ISUA and control groups were compared. 3D-PDU was used to detect placental blood flow parameters, including vascularity index (VI), flow index, and vascularity flow index (VFI), in 58 foetuses in the ISUA group and 77 normal foetuses in the control group. Immunohistochemistry and polymerase chain reaction were employed to analyse the VEGF expression in placental tissues of 26 foetuses in the ISUA group and 26 foetuses in the control group. The control group exhibited higher VI and VFI than the ISUA group (p < 0.05). Meanwhile, the ISUA group showed a higher positivity rate of VEGF protein expression than the control group (χ 2=28.013, p˂0.001). The ISUA group also presented a higher VEGF mRNA protein expression than the control group (p˂0.001). 3D-PDU can be used to quantitatively analyse microblood perfusion of the placenta and provide an objective assessment of ISUA foetuses. Impact statement What is already known on this subject? Colour Doppler flow can be used to evaluate placental and maternal circulation and remains an ideal method for evaluating high-risk placental function. Three-dimensional power Doppler ultrasound (3D-PDU) can be used to quantify blood vessels and blood flow in placental parenchyma via the measurement of the amplitude of blood vessels and blood flow in normal foetuses, respectively. What do the results of this study add? 3D-PDU can be used to quantitatively analyse micro blood perfusion of the placenta and conduct an objective assessment of isolated single umbilical artery foetuses. The isolated single umbilical artery foetuses exhibited a higher positivity rate of vascular endothelial growth factor (VEGF) protein expression and higher VEGF mRNA protein expression than the normal foetuses. What are the implication of these findings for clinical practice and/or further research? The study provides a reliable basis for maternal-foetal monitoring during pregnancy in the isolated single umbilical artery foetuses. Objective assessment of the occurrence and development of foetuses with isolated single umbilical artery was performed.

Establishment of a Two-stage Limb Ischemia in Diabetic Rats

Abstract Background: This study aimed to establish a clinically relevant animal model for peripheral arterial disease (PAD) that better replicates the complexity observed in human patients. Materials and Methods: Thirty male rats were randomly assigned into the sham (SM), femoral artery resection (FE), constrictor-induced ischemia (CI), two-stage ischemia (TS), or diabetic two-stage ischemia (DT) groups. In the FE group, rats underwent femoral artery resection, whereas the SM group had sham surgery. The CI group received progressive ischemia using two ameroid constrictors, and the TS and DT groups underwent a two-stage ischemia procedure involving initial gradual narrowing with two ameroid constrictors and subsequent femoral artery resection in healthy and diabetic rats, respectively. Perfusion evaluation and functional assessment were conducted at postoperative days 14, 28, and 42. On day 42, hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) protein expression were measured, along with histological examination and immunofluorescence analysis. Results: Motor function deficits and reduced limb reperfusion were most prominent in the TS and DT groups on days 28 and 42 (P &lt; 0.05), exacerbated by type 2 diabetes. Gastrocnemius exhibited upregulated HIF-1α and VEGF protein expression, as well as increased capillary density in response to ischemia. However, the DT group showed significantly lower protein expression and capillary density, along with more severe structural damage compared to other groups (P &lt; 0.05). Conclusion: A clinically relevant rat model of PAD was established by implementing a two-stage ischemia procedure involving initial progressive narrowing and subsequent femoral artery excision in the context of diabetes.

Comparative analysis of clinicopathological characteristics of central necrotizing breast cancer and basal cell-like breast cancer.

This study aims to compare the clinicopathological and immunohistochemical characteristics of centrally necrotizing carcinoma of the breast (CNC) and basal-like breast cancer (BLBC), as well as to analyze the characteristics of the molecular typing of the CNC. The clinicopathological features of 69 cases of CNC and 48 cases of BLBC were observed and compared. EnVision immunohistochemical staining was performed to detect the expressions of hypoxia-inducible factor 1α (HIF-1α), breast cancer susceptibility gene 1 (BRCA1), and vascular endothelial growth factor (VEGF) in CNC and BLBC. The age of the 69 patients ranged from 32 to 80 years, with an average of 54.55 years. Gross examination showed that most tumors were well-defined single central nodules with a diameter of 1.2~5.0 cm. Microscopically, there is a large necrotic or acellular area in the center of the tumor, mainly composed of tumor coagulative necrosis with varying degrees of fibrosis or hyaline degeneration. A small amount of cancer tissue remained in the form of a ribbon or small nest around the necrotic focus. Among 69 cases of CNC, the proportion of basal cell type (56.5%) was significantly higher than that of lumen type A (18.84%), lumen type B (13.04%), HER2 overexpression (5.8%), and nonexpression (5.8%). A total of 31 cases were followed up for 8~50 months, with an average of 33.94 months. There have been nine cases of disease progression. When compared to BLBC, there were no significant differences in BRCA1 and VEGF protein expression in response to CNC (p > 0.05), but there were significant differences in protein expression in HIF-1α (p < 0.05). The molecular typing of CNC showed that over half of those were BLBC. No statistically significant difference in the expression of BRCA1 was observed between CNC and BLBC; thus, we predict that targeted therapy for BRCA1 in BLBC may also have considerable effects in CNC patients. The expression of HIF-1α is significantly different in CNC and BLBC, and perhaps HIF-1α can be used as a new entry point to distinguish between the two. There is a significant correlation between the expression of VEGF and HIF-1α in BLBC, and there was no significant correlation between the expression levels of the two proteins in CNC.

Protective effects of pentoxifylline against chlorine-induced acute lung injury in rats

ObjectiveChlorine is a chemical threat agent that can be harmful to humans. Inhalation of high levels of chlorine can lead to acute lung injury (ALI). Currently, there is no satisfactory treatment, and effective antidote is urgently needed. Pentoxifylline (PTX), a methylxanthine derivative and nonspecific phosphodiesterase inhibitor, is widely used for the treatment of vascular disorders. The present study was aimed to investigate the inhibitory effects of PTX on chlorine-induced ALI in rats.MethodsAdult male Sprague-Dawley rats were exposed to 400 ppm Cl2 for 5 min. The histopathological examination was carried out and intracellular reactive oxygen species (ROS) levels were measured by the confocal laser scanning system. Subsequently, to evaluate the effect of PTX, a dose of 100 mg/kg was administered. The activities of superoxide dismutase (SOD) and the contents of malondialdehyde (MDA), glutathione (GSH), oxidized glutathione (GSSG) and lactate dehydrogenase (LDH) were determined by using commercial kits according to the manufacturer’s instructions. Western blot assay was used to detect the protein expressions of SOD1, SOD2, catalase (CAT), hypoxia-inducible factor (HIF)-1α, vascular endothelial growth factor (VEGF), occludin, E-cadherin, bcl-xl, LC 3, Beclin 1, PTEN-induced putative kinase 1 (PINK 1) and Parkin.ResultsThe histopathological examination demonstrated that chlorine could destroy the lung structure with hemorrhage, alveolar collapse, and inflammatory infiltration. ROS accumulation was significantly higher in the lungs of rats suffering from inhaling chlorine (P<0.05). PTX markedly reduced concentrations of MAD and GSSG, while increased GSH (P<0.05). The protein expression levels of SOD1 and CAT also decreased (P<0.05). Furthermore, the activity of LDH in rats treated with PTX was significantly decreased compared to those of non-treated group (P<0.05). Additionally, the results also showed that PTX exerted an inhibition effect on protein expressions of HIF-1α, VEGF and occludin, and increased the level of E-cadherin (P<0.05). While the up-regulation of Beclin 1, LC 3II/I, Bcl-xl, and Parkin both in the lung tissues and mitochondria, were found in PTX treated rats (P<0.05). The other protein levels were decreased when treated with PTX (P<0.05).ConclusionPTX could ameliorate chlorine-induced lung injury via inhibition effects on oxidative stress, hypoxia and autophagy, thus suggesting that PTX could serve as a potential therapeutic approach for ALI.

Ginsenoside Rg3 alleviates the migration, invasion, and angiogenesis of lung cancer cells by inhibiting the expressions of cyclooxygenase-2 and vascular endothelial growth factor.

Lung cancer (LC) is a common cancer with high incidence and mortality rates. In recent years, ginsenoside Rg3 (Rg3), a traditional medicine, is widely used for the treatment of LC. Herein, we concentrate on assessing the effect of Rg3 on LC cell migration and invasion. The effects of Rg3 (0, 25, 50, and 100 μg/ml) on the viability, migration, invasion, angiogenesis, and expressions of epithelial-mesenchymal transition (EMT)-related proteins, cyclooxygenase-2 (COX2), and vascular endothelial growth factor (VEGF) of LC cell lines were evaluated by cell counting kit-8 (CCK-8), scratch, transwell, tube formation, and western blot assays. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to assess transfection efficiency. COX2 overexpression plasmid and short hairpin RNA for VEGF (shVEGF) were applied to evaluate whether the effect of Rg3 is related to COX2 and VEGF through rescue assay. In this study, Rg3 significantly dose-dependently suppressed the viability, migration, invasion, angiogenesis, and protein expressions of N-cadherin, vimentin, COX2, and VEGF in H1299 and A549 cells, while promoting the expression of E-cadherin protein. COX2 overexpression markedly reversed the effects of Rg3 on the viability, migration, invasion, angiogenesis, and EMT-related protein expression levels in LC cells; however, such effects of COX2 overexpression were offset by VEGF knockdown. In sum, Rg3 alleviates the migration, invasion, and angiogenesis of LC cells by inhibiting the expressions of COX2 and VEGF.

Wound healing properties of a new formulated flavonoid-rich fraction from Dodonaea viscosa Jacq. leaves extract.

Background: Dodonaea viscosa Jacq. (D. viscosa) belongs to the family of Sapindaceae, commonly known as "Sinatha," and is used as a traditional medicine for treating wounds due to its high flavonoids content. However, to date there is no experimental evidence on its flavonoid-rich fraction of D. viscosa formulation as an agent for healing wounds. Objective: The present study aimed to evaluate the wound healing effect of ethyl acetate fraction of D. viscosa leaves on dermal wounds. Methods: The ethyl acetate fraction was produced from a water-ethanol extract of D. viscosa leaves and was quantitatively evaluated using the HPLC technique. The in-vivo wound healing ability of the ethyl acetate fraction of D. viscosa ointment (DVFO, 2.5%w/w and 5%w/w) was investigated in Sprague-Dawley rats utilizing an incision and excision paradigm with povidone-iodine ointment (5% w/w) as a control. The percentage of wound closure, hydroxyproline and hexosamine concentrations, tensile strength and epithelialization duration were measured. Subsequently, histopathology analysis of skin samples as well as western blots were performed for collagen type 3 (COL3A1), basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). Results: The ethyl acetate fraction of D. viscosa revealed flavonoids with high concentrations of quercetin (6.46% w/w) and kaempferol (0.132% w/w). Compared to the control group, the DVFO (2.5% and 5.0% w/w) significantly accelerated wound healing in both models, as demonstrated by quicker wound contraction, epithelialization, elevated hydroxyproline levels and increased tensile strength. Histopathological investigations also revealed that DVFO treatment improved wound healing by re-epithelialization, collagen formation and vascularization of damaged skin samples. Western blot analysis further demonstrated an up-regulation of COL3A, vascular endothelial growth factor and bFGF protein in wound granulation tissue of the DVFO-treated group (p < 0.01). Conclusion: It is concluded that flavonoid-rich D. viscosa ethyl acetate fraction promotes wound healing by up-regulating the expressions of COL3A, VEGF and bFGF protein in wound granulation tissue. However, extensive clinical and pre-clinical research on the flavonoid-rich fraction of D. viscosa is needed to determine its significant impact in the healing of human wounds.

Liposome-polyethylenimine complexes for the effective delivery of HuR siRNA in the treatment of diabetic retinopathy.

Diabetic retinopathy (DR) is a vision-impairing complication of diabetes, damaging the retinal microcirculatory system. Overexpression of VEGF (vascular endothelial growth factor) is implicated in the pathogenesis of DR. Human antigen R (HuR) is an RNA-binding protein that favorably regulates VEGF protein expression by binding to VEGF-encoding mRNA. Downregulating HuR via RNA interference strategies using small interfering RNAs (siRNAs) may constitute a novel therapeutic method for preventing VEGF protein overexpression in DR. Delivery of siRNAs to the cellular cytoplasm can be facilitated by cationic peptides or polymers and lipids. In this study, a cationic polymer (polyethylenimine (PEI)) and lipid nanoparticles (liposomes) were co-formulated with siRNA to form lipopolyplexes (LPPs) for the delivery of HuR siRNA. LPPs-siRNA were analyzed for size, zeta potential, serum stability, RNase stability, heparin stability, toxicity, and siRNA encapsulation efficiency. Cellular uptake, downregulation of the target HuR (mRNA and protein), and associated VEGF protein were used to demonstrate the biological efficacy of the LPPs-HuR siRNA, in vitro (human ARPE-19 cells), and in vivo (Wistar rats). In vivo efficacy study was performed by injecting LPPs-HuR siRNA formulations into the eye of streptozotocin (STZ)-induced diabetic rats after the development of retinopathy. Our findings demonstrated that high retinal HuR and VEGF levels observed in the eyes of untreated STZ rats were lowered after LPPs-HuR siRNA administration. Our observations indicate that intravitreal treatment with HuR siRNA is a promising option for DR using LPPs as delivery agents.

Effects of Early Weight-Bearing Treadmill Training Combined with Pre-Emptive Analgesia on Femoral Fracture Recovery.

The effect of pre-emptive analgesia plus early weight-bearing treadmill training (EWBTT) on healing and motor function recovery of femoral shaft fracture is not clear. A total of 60 SD male rats were randomly allocated into 4 groups: group A (pre-emptive analgesia with EWBTT), group B (pre-emptive analgesia with delayed weight-bearing treadmill training, DWBTT), group C (pre-emptive analgesia with no weight-bearing), and group D (EWBTT with no pre-emptive analgesia). All rats were molded by internal fixation with Kirschner wire after right femoral shaft fracture. In groups A, B, and C, tramadol was intramuscularly injected 15 minutes before surgery. EWBTT was performed at day 1 postoperatively in groups A and D, and DWBTT was performed at day 14 postoperatively in group B. Oblique plate test was accomplished to assess hindlimb motor function recovery of rats in each group. Status of fracture healing was assessed through digital radiography (DR). Hematoxylin-eosin (HE) staining and immunohistochemistry of bone morphogenetic protein-2 (MBP-2) and vascular endothelial growth factor (VEGF) in callus were performed to explore fracture healing. The expression of BMP-2 and VEGF protein in quadriceps femoris muscle was detected by Western blot technique and mRNA expression of BMP-2 and VEGF in callus ascertained via reverse transcription-polymerase chain reaction (RT-PCR) technique. For oblique plate test, rats in group A outperformed those in groups B and C at all time points after operation. DR image revealed that large numbers of callus growth, blurred fracture line, and obvious continuous callus passing through the fracture line can be found in group A at day 28 postoperatively, which is the best healing status among all groups. HE staining of callus confirmed the optimal effect of healing for rats in group A. VEGF and BMP-2 expression by immunohistochemistry showed a significantly higher positive score for callus in group A while those in group C being the lowest at all time points postoperatively. Significantly higher expression level of VEGF and BMP-2 protein was detected in quadriceps femoris muscle from group A, which exceeded those in all other groups at all time points. RT-PCR testing proved the highest expression of BMP-2 and VEGF mRNA in callus of rats from group A, significantly higher than those of other groups. Both pre-emptive analgesia and EWBTT can effectively invoke the expression of VEGF and BMP-2 and promote recovery of hindlimb locomotor function in rats with femoral fracture, and the combination of them leads to more superior results.

Pirfenidone Attenuates the EMT Process and the Secretion of VEGF in TGF-β2-Induced ARPE-19 Cells via Inhibiting the Activation of the NF-κB/Snail Signaling Pathway.

Pirfenidone (PFD), an antifibrotic drug, has various beneficial functions such as antioxidant, antifibrotic, and anti-inflammatory effects. This study aimed to explore the molecular mechanisms underlying how PFD modulates retinal pigment epithelial (RPE) cells involved in neovascularization and subretinal fibrosis. ARPE-19 cell lines were treated with transforming growth factor-beta 2 (TGF-β2) alone or in combination with PFD. RPE cell viability, as a consequence of PFD use, was determined by the CCK-8 assay. Cell migration was assessed by the wound closure assay and quantified by the Image J software. Protein expression of the following markers was measured by the western blot analysis: an epithelial cell marker and E-cadherin; mesenchymal cell markers, fibronectin, matrix metalloprotein-9 (MMP-9), and alpha-smooth muscle actin (α-SMA); a fibrotic marker and connective tissue growth factor (CTGF); an angiogenesis marker and vascular endothelial growth factor (VEGF); NF-κB/Snail. The mRNA levels of fibronectin and α-SMA were determined by quantitative real-time PCR. VEGF was quantitatively measured by the enzyme-linked immunosorbent assay. The cell viability assay revealed that PFD had no significant cytotoxic effect on RPE cells at concentrations of less than 1 mg/mL. The cell scratch assay showed that TGF-β2 stimulation significantly improved the migration of RPE cells and that PFD attenuated this effect. PFD significantly inhibited the TGF-β2-induced protein expression of E-cadherin and increased the TGF-β2-induced protein expression of fibronectin, MMP-9, α-SMA, CTGF, and VEGF in ARPE-19 cells. The mRNA expression of fibronectin and α-SMA was inhibited by PFD in TGF-β2-inducedARPE-19 cells. Additionally, the increased intracellular and supernatant expression of VEGF protein was suppressed by PFD. Mechanistically, RPE cells treated with PFD + TGF-β2 exhibited a decrease in phosphorylation of the NF-κB P65 subunit and activation of Snail, compared with the RPE cells treated with TGF-β2 alone. PFD ameliorated TGF-β2-induced neovascularization and fibrosis by suppressing the NF-κB/Snail signaling pathway. Therefore, PFD may be a potential drug in the treatment of age-related macular degeneration.

Efficient delivery of VEGF-A mRNA for promoting diabetic wound healing via ionizable lipid nanoparticles

Diabetes is often accompanied by chronic non-healing wounds, and vascularendothelial growth factor A (VEGF-A) is crucial in the treatment of chronic diabetic wounds. However, the application of VEGF-A protein in clinic is limited due to poor absorption and short half-life of protein macromolecule. Herein, we employed an emerging protein replacement therapy by delivering VEGF-A mRNA into the body to express the desired protein to accelerate diabetic wound healing. Primarily, VEGF-A mRNA was synthesized by an in vitro transcription (IVT) method and encapsulated with an ionizable lipid-mediated nanoparticles (LNP) delivery system via a microfluidic method. The resultant LNP/VEGF-A mRNA were characterized by using dynamic light scattering (DLS) and transmission electron microscope(TEM). The nanoparticles have regular spherical morphology with an average particle size of 101.17 nm, a narrow polydispersity (PDI) of 0.17 and negative Zeta potential of −3.05 mV. The bioactivities of the nanoparticles formulation were evaluated against HUVEC cells through cell proliferation, migration and tube formation assays. It was found that the LNP/VEGF-A mRNA nanoparticles could promote endothelial cell proliferation. In addition, they exhibited successful mRNA delivery and high VEGF-A protein expression in vitro and in vivo by means of Western Blot assay and in vivo imaging system (IVIS). Finally, C57BL/6 diabetic mice model was established and intradermally treated with the LNP/VEGF-A mRNA nanoparticles. It was found that the LNP/VEGF-A mRNA treated wounds were almost healed after 14 days with an average wound area of 2.4 %, compared with the PBS group of 21.4 %. Apparently, the nanoparticles formulation was able to significantly expedite diabetic wound healing. The histological analysis containing H&E, Masson’s trichrome staining and CD31 further confirmed the healing efficacy and low toxicity of the formulation. Taken together, the LNP/VEGF-A mRNA nanoparticles can be taken up by cells to express protein effectively and improve diabetic wound healing, which might have potential application in the treatment of chronic diabetic wounds as a protein replacement therapy.

Treatment of Ovarian Hyperstimulation Syndrome in a Mouse Model by Cannabidiol, an Angiogenesis Pathway Inhibitor.

Studies suggest that ovarian hyperstimulation syndrome (OHSS) can be treated by reducing the level of vascular endothelial growth factor (VEGF). However, due to the side effects of commercially available VEGF-reducing drugs, they can be ruled out as a suitable treatment for OHSS; therefore, researchers are looking for new medications to treat OHSS. This study is aimed at investigating the effects of cannabidiol (CBD) in an OHSS model and to evaluate its efficacy in modulating the angiogenesis pathway and VEGF gene expression. For this purpose, 32 female mice were randomly divided into four groups (eight mice per group): control group, group 2 with OHSS induction, group 3 receiving 32 nmol of dimethyl sulfoxide after OHSS induction, and group 4 receiving 30 mg/kg of CBD after OHSS induction. The animals' body weight, ovarian weight, vascular permeability (VP), and ovarian follicle count were measured, and the levels of VEGF gene and protein expression in the peritoneal fluid were assessed. Based on the results, CBD decreased the body and ovarian weights, VP, and corpus luteum number compared to the OHSS group (p < 0.05). The peritoneal VEGF gene and protein expression levels reduced in the CBD group compared to the OHSS group (p < 0.05). Also, CBD caused OHSS alleviation by suppressing VEGF expression and VP. Overall, CBD downregulated VEGF gene expression and improved VP in OHSS.

CircHIPK3 promotes bone microvascular endothelial cell proliferation, migration and angiogenesis by targeting miR-7 and KLF4/VEGF signaling in steroid-induced osteonecrosis of the femoral head.

Circular RNA homeodomain interacting protein kinase 3 (circHIPK3) has been implicated in facilitating angiogenesis in various conditions. However, its role in steroid-induced osteonecrosis of the femoral head (ONFH) remains unclear. To investigate whether circHIPK3 promotes bone microvascular activity and angiogenesis by targeting miR-7 and Krüppel-like factor 4 (KLF4)/vascular endothelial growth factor (VEGF) signaling in ONFH. Fifty patients with steroid-induced ONFH undergoing hip-preserving surgery or total hip arthroplasty were included in this study. The expression of circHIPK3, miR-7 and KLF4 was evaluated using reverse transcription polymerase chain reaction (RT-PCR) in necrotic and healthy samples of the femoral head. Bone microvascular endothelial cells (BMECs) were extracted and cultured with 0.1 mg/mL hydrocortisone to create a hormonally deficient cell model. These BMECs were then transfected with either circHIPK3 overexpressing or silencing plasmids with or without miR-7 mimics. The MTT assays were used to detect cell proliferation. Scratch assays were used to assess the migration ability of the BMECs. The tube formation was carried out using an in vitro Matrigel angiogenesis assay. Annexin V-FITC/PI and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were used to assess the degree of apoptosis. Western blot assays were carried out to discern KLF4 and VEGF expression. The interactions of circHIPK3, miR-7 and KLF4 were confirmed using luciferase, RNA-binding protein immunoprecipitation (RIP), RNA pull-down, and fluorescence in situ hybridization (FISH) assays. The circHIPK3 and KLF4 expression was decreased, whereas miR-7 expression was increased in necrotic tissues compared to non-necrotic samples. Both circHIPK3 and KLF4 expression correlated negatively with miR-7. The overexpression of circHIPK3 promoted the proliferative, migratory and angiogenic capabilities of the BMECs, while adding an miR-7 mimic reversed these effects. At the same time, the overexpression of circHIPK3 reduced the apoptosis rate of the BMECs and increased KLF4 and VEGF protein expression, but adding an miR-7 mimic reversed these effects. The FISH, RNA pull-down, RIP, and luciferase assays revealed an interaction between circHIPK3, miR-7 and KLF4. The circHIPK3 promotes BMEC proliferation, migration and angiogenesis by targeting miR-7 and KLF4/VEGF signaling.

Aidi injection enhances the anti-tumor impact of doxorubicin in H22 tumor-containing mice

Ethnopharmacological relevanceAidi injection (AD) is a traditional medical preparation that has a Chinese origin. It is extensively used particularly in combination with doxorubicin (DOX) for the management of hepatocellular carcinoma (HCC). However, the combination's synergistic mechanism has not yet been clarified. Aim of the studyTo investigate the anti-tumor impact of AD in combination with DOX and their synergistic mechanism in HCC. Materials and methodsAn H22 mouse xenograft model was utilized to study the impact of AD, DOX, and their combination on HCC in vivo. Their effects on cell vitality, apoptosis, mitochondrial membrane potential, reactive oxygen species (ROS) production, caspase-3, and cleaved caspase-3 protein expression were also investigated in H22 cells in vitro. Subsequently, human umbilical vein endothelial cells (HUVECs) were utilized to investigate the impacts of AD, DOX, and their combination on cell viability, migration, invasion, tube formation, and vascular endothelial growth factor (VEGF) protein expression. ResultsThe study established that the tumor inhibition rate of AD combined with DOX reached 79.51%, which was significantly higher than that of AD (25.14%) or DOX (49.48%) alone. Additionally, the Q-value characterizing the synergy between AD and DOX was 1.72, demonstrating a strong synergistic effect. Furthermore, compared to AD or DOX administration alone, the combined administration group significantly decreased the alpha-fetoprotein (AFP) level in the serum, increased the tumor necrosis area, increased the Bax/Bcl-2, Cyt-c, caspase-9, Fas, Fasl, caspase-8, and caspase-3 protein expression, and significantly increased the CD31 and Ki67 protein expression in tumor tissue. Compared to AD or DOX alone, AD combined with DOX treatment had a synergistic effect on H22 cells (combination index values < 0.9), which inhibited cell viability, reduced mitochondrial membrane potential (MMP), induced apoptosis, promoted MMP loss, and increased ROS generation, cleaved caspase-3/caspase-3 levels, and caspase-3 activity. Moreover, combined administration showed a more pronounced inhibition of cell viability, migration, invasion, tube formation, and VEGF protein expression in HUVECs. ConclusionsAD enhances the anti-tumor effect of DOX by promoting apoptosis and inhibiting angiogenesis and cell proliferation. The findings of this study lay experimental foundations for the clinical combination of AD and DOX.

The Mechanism of Nemo-Like Kinase (NLK) in Non-Small Cell Lung Cancer (NSCLC) Cells by Regulating Vascular Endothelial Growth Factor (VEGF)

Nemo-like kinase (NLK) is abnormally expressed in several tumors, but its role in NSCLC have not been reported. Real time PCR and Western blot were used to assess NLK level in tumor tissues and adjacent tissues of NSCLC. NSCLC cell line A549 cells were divided into three groups; NC group and si-NLK group which was transfected with NLK negative control or NLK siRNA respectively followed by analysis of NLK expression by real time PCR and Western blot, cell proliferation by MTT assay, cell migration by cell wound healing assay, cell invasion by transwell chamber and MMP-9 and VEGF expression by Western blot. The expression of NLK in NSCLC tumor tissue was increased, and the difference was statistically significant compared with adjacent tissues (P &lt;0.05), and it was related to tumor size, degree of differentiation, metastasis and survival time (P &lt;0.05). A549 cells showed significantly increased NLK. Transfection of NLK siRNA could significantly inhibit tumor cell proliferation, migration and invasion, and decrease the expression of MMP-9 and VEGF proteins (P &lt;0.05). Elevated NLK level in NSCLC tumor tissues is related to clinicopathological characteristics. Decreased the expression of NLK can inhibit VEGF and MMP-9 expression, and inhibit cell function.

Correlation and significance of COX-2, Ki67, VEGF and other immune indexes with the growth of malignant pulmonary nodules

ObjectiveThis study intends to explore the factors affecting the growth of pulmonary nodules in the natural process by immunohistochemical method.Methods40 cases of pulmonary nodules followed up for more than 3 years were divided into growth group (n = 20) and stable group (n = 20). The expressions of cyclooxygenase-2 (COX-2), Ki67, vascular endothelial growth factor (VEGF), CD44V6, epidermal growth factor receptor (EGFR), double microsome 2 (MDM2) and transforming growth factor (TGF)-β1 in pulmonary nodules were detected by immunohistochemical method so as to explore the relationship between it and the growth of pulmonary nodules.ResultsCompared with stable pulmonary nodules, the positive rates of COX-2, Ki67 and VEGF in the growth group were 85%, 80% and 55%, respectively. There was significant difference between the stable group and the growth group (P < 0.05). The correlation between other indexes and the growth of pulmonary nodules was not statistically significant (Pcd44v6 = 0.104;PEGFR = 0.337; PMDM2 = 0.49; PTGF-β1 = 0.141). In the subgroup of patients with non-invasive lung cancer, there was a correlation between VEGF and the growth of pulmonary nodules (P < 0.05).ConclusionThe high expression of COX-2, Ki67 and VEGF proteins may be significantly related to the growth of pulmonary nodules, and VEGF may be an important factor affecting the growth of malignant pulmonary nodules. This study intends to provide a research direction for further searching for the essential causes of the growth of pulmonary nodules.

Effect of Biantie pretreatment on serum level of PHD2/HIF-1α and brain tissue damage in rats during acute hypobaric hypoxia exposure

To observe the effect of Biantie (bian stone plaste) pretreatment on serum level of prolyl hydroxylase domain 2 (PHD2) and hypoxia-inducible factor-1α (HIF-1α) in rats with acute hypobaric hypoxia induced-brain injury, and to explore the possible mechanism of Biantie on preventing brain injury at high altitude. Forty-five male SD rats were randomly divided into a blank group, a model group, a Biantie group, a medication group and a Biantie+inhibitor group, 9 rats in each group. The rats in the Biantie group the and the Biantie+inhibitor group were pretreated with Biantie at "Taiyuan" (LU 9), "Neiguan" (PC 6) and "Renying" (ST 9), 2 h each time, once a day; the rats in the medication group were treated with intragastric administration of rhodiola capsule solution (280 mg/kg) for 14 d; the rats in the Biantie+inhibitor group were intraperitoneally injected with the PHD inhibitor dimethyloxalyl glycine (DMOG) at a dose of 40 mg/kg 24 h before the establishment of the model. After the intervention, except for the blank group, the rats in the remaining 4 groups were placed in the oxygen chamber to simulate a high-altitude environment to establish the acute hypobaric hypoxia brain injury model. The arterial blood-gas analysis indexes [blood oxygen saturation (SaO2), lactic acid (Lac), blood sodium (Na+), blood potassium (K+)] and brain water content were detected in each group; the histomorphology of cerebral cortex was observed by HE staining; the serum levels of PHD2 and HIF-1α as well as vascular endothelial growth factor (VEGF) were detected by ELISA; the VEGF protein expression in brain tissue was detected by Western blot; the VEGF mRNA expression in brain tissue was detected by real-time fluorescent quantitative PCR. Compared with the blank group, the levels of SaO2 and Na+ in the model group were decreased (P<0.05), while the levels of Lac and K+ as well as the water content of brain tissue were increased (P<0.05). Compared with the model group, the level of SaO2 in the Biantie group and the medication group was increased (P<0.05), while the levels of Lac, K+ and the water content of brain tissue were decreased (P<0.05); the level of Na+ in the Biantie group was increased (P<0.05). Compared with the Biantie group, the level of SaO2 in the Biantie+inhibitor group was decreased (P<0.05), and the level of Lac and the water content of brain tissue were increased (P<0.05). In the model group, the cortical tissue cells were loose and disordered, the cortical blood vessels were dilated, and the cells were obviously swollen; the anoxic injury in the Biantie group and the medication group was lighter, and the anoxic injury in the Biantie+inhibitor group was more obvious than that in the Biantie group. Compared with the blank group, the serum PHD2 content in the model group was decreased and the HIF-1α content was increased (P<0.05), and the content of VEGF in serum and VEGF protein and mRNA expressions in brain were increased (P<0.05). Compared with the model group, the content of PHD2 in serum in the Biantie group and the medication group was increased (P<0.05), and the level of HIF-1α was decreased (P<0.05), and the content of VEGF in serum as well as VEGF protein and mRNA expressions in brain were decreased (P<0.05). Compared with the Biantie group, the serum PHD2 content in the Biantie+inhibitor group was decreased and HIF-1α level were increased (P<0.05), and the content of VEGF in serum as well as VEGF mRNA expression in brain were increased (P<0.05). Biantie at "Taiyuan" (LU 9), "Neiguan" (PC 6) and "Renying" (ST 9) could regulate serum PHD2/HIF-1α to down-regulate VEGF expression, reduce brain edema and enhance anti-hypoxia ability, so as to achieve the purpose of preventing brain injury at high altitude.

Cardiac epigenetic changes in VEGF signaling genes associate with myocardial microvascular rarefaction in experimental chronic kidney disease.

Chronic kidney disease (CKD) is common in patients with heart failure and often results in left ventricular diastolic dysfunction (LVDD). However, the mechanisms responsible for cardiac damage in CKD-LVDD remain to be elucidated. Epigenetic alterations may impose long-lasting effects on cellular transcription and function, but their exact role in CKD-LVDD is unknown. We investigate whether changes in cardiac site-specific DNA methylation profiles might be implicated in cardiac abnormalities in CKD-LVDD. CKD-LVDD and normal control pigs (n = 6 each) were studied for 14 wk. Renal and cardiac hemodynamics were quantified by multidetector CT and echocardiography. In randomly selected pigs (n = 3/group), cardiac site-specific 5-methylcytosine (5mC) immunoprecipitation (MeDIP)- and mRNA-sequencing (seq) were performed, followed by integrated (MeDiP-seq/mRNA-seq analysis), and confirmatory ex vivo studies. MeDIP-seq analysis revealed 261 genes with higher (fold change > 1.4; P < 0.05) and 162 genes with lower (fold change < 0.7; P < 0.05) 5mC levels in CKD-LVDD versus normal pigs, which were primarily implicated in vascular endothelial growth factor (VEGF)-related signaling and angiogenesis. Integrated MeDiP-seq/mRNA-seq analysis identified a select group of VEGF-related genes in which 5mC levels were higher, but mRNA expression was lower in CKD-LVDD versus normal pigs. Cardiac VEGF signaling gene and VEGF protein expression were blunted in CKD-LVDD compared with controls and were associated with decreased subendocardial microvascular density. Cardiac epigenetic changes in VEGF-related genes are associated with impaired angiogenesis and cardiac microvascular rarefaction in swine CKD-LVDD. These observations may assist in developing novel therapies to ameliorate cardiac damage in CKD-LVDD.NEW & NOTEWORTHY Chronic kidney disease (CKD) often leads to left ventricular diastolic dysfunction (LVDD) and heart failure. Using a novel translational swine model of CKD-LVDD, we characterize the cardiac epigenetic landscape, identifying site-specific 5-methylcytosine changes in vascular endothelial growth factor (VEGF)-related genes associated with impaired angiogenesis and cardiac microvascular rarefaction. These observations shed light on the mechanisms of cardiac microvascular damage in CKD-LVDD and may assist in developing novel therapies for these patients.