Vascular Endothelial Growth Factor mRNA Expression Research Articles

KGRT peptide incorporated hydrogel with antibacterial activity for wound healing by optimizing cellular functions via ERK/eNOS signaling

Bacterial infected wounds, which is characterized by easy infection, multiple inflammation and slow healing, is a complex symptom, resulting from metabolic disorder of the wound microenvironment. In this study, a series of self-healing double-network hydrogels based on KGRT peptide (Lys-Gly-Arg-Thr) with antibacterial, anti-inflammatory and optimizing cellular functions were designed to promote the healing of infected wounds with full-thickness skin defects. Moreover, the dextran hydrogelintroduces a large number of side chains, which are entangled with each other in the Schiff base network to form an interpenetrating structure. The hydrogel might regulate cell metabolism, differentiation and vascular endothelial growth factor (VEGF) function. Importantly, both in vitro and in vivo data showed that hydrogel not only has good antibacterial properties (99.8 %), but also can eradicate bacterial biofilm, effectively reduce inflammation (down-regulated IL-1β, TNF-α and ROS) and accelerate chronic wound healing process by speeding-up wound closure, increasing granulation tissue thickness, collagen deposition, angiogenesis (up-regulated CD31). The hydrogel could up-regulate mRNA expression of PI3K, AKT, ERK, eNOS, HIF-1α and VEGF, which were correlated with wound healing. Consistently, the hydrogel could promote infected wounds healing and inhibit inflammation through ERK/eNOS signaling pathway. Collectively, hydrogel has excellent clinical application potential for promoting infected wound healing.

Peptidylarginine Deiminases Inhibitors Decrease Endothelial Cells Angiogenic Potential by Affecting Akt Signaling and the Expression and Secretion of Angiogenic Factors.

Endothelial cells (ECs) play a crucial role in various physiological processes, particularly those related to the cardiovascular system, but also those affecting the entire organism. The biology of ECs is regulated by multiple biochemical stimuli and epigenetic drivers that govern gene expression. We investigated the angiogenic potential of ECs from a protein citrullination perspective, regulated by peptidyl-arginine deiminases (PADs) that modify histone and non-histone proteins. Although the involvement of PADs has been demonstrated in several physiological processes, inflammation-related disorders and cancer, their role in angiogenesis remains unclear. To elucidate the role of PADs in endothelial angiogenesis, we used two human EC models: primary vein (HUVECs) and microvascular endothelial cells (HMEC-1). PADs activity was inhibited using irreversible inhibitors: BB-Cl-amidine, Cl-amidine and F-amidine. We analyzed all three steps of angiogenesis in vitro : proliferation, migration, and capillary-like tube formation, as well as secretory activities, gene expression and signaling in ECs. All used PAD inhibitors reduced the histone H3 citrullination (H3cit) mark, inhibited endothelial cell migration and capillary-like tube formation, and favored an angiostatic activity in HMEC-1 cells, by increasing PEDF (pigment epithelium-derived factor) and reducing VEGF (vascular endothelial growth factor) mRNA expression and protein secretion. Additionally, BB-Cl-amidine reduced the total activity of MMPs (Matrix metalloproteinases). The observed effects were underlined by the inhibition of Akt phosphorylation.>. Our findings suggest that pharmacological inhibitors of citrullination are promising therapeutic agents to target angiogenesis.

Polyurethane foam dressing with non-adherent membrane improves negative pressure wound therapy in pigs.

Negative pressure wound therapy (NPWT) is considered to be an effective technique to promote the healing of various wounds. The aim of this study was to evaluate different wound dressings combined with NPWT in treating wounds in Wuzhishan pigs. Excisions were made in the backs of the pigs and were covered with polyvinyl alcohol (PVA) dressing, polyurethane (PU) dressing or PU dressing with non-adherent membrane (PU-non-ad). NPWT was applied to the wound site. In the control group, basic occlusive dressing (gauze) without NPWT was applied. On days 0, 3, 7, 14, 21 and 28 post-surgery, the wound size was measured during dressing change, and wound healing rate (WHR) was calculated. In addition, blood perfusion within 2cm of the surrounding wound was measured by laser doppler flowmetry. Dressing specimen was collected and microbiology was analysed. Granulation tissues from the central part of the wounds were analysed for histology, vascular endothelial growth factor (VEGF) and cluster of differentiation 31 (CD31) mRNA expression. The PU-non-ad-NPWT significantly (p<0.01) accelerated wound healing in the pigs. Further pathological analysis revealed that the non-adherent membrane effectively protected granulation tissue formation in PU-NPWT treated wounds. The blood perfusion analysis suggested that the non-adherent membrane improved the blood supply to the wound area. Microbiological analysis showed that non-adherent membrane decreased the bacterial load in the PU-NPWT dressing. VEGF and CD31 mRNA expression was upregulated in the wound tissue from the PU-non-ad-NPWT treated groups. In this study, the PU dressing with non-adherent membrane was an ideal dressing in NPWT-assisted wound healing.

Co-treatment with bone marrow-derived mesenchymal stem cells and curcumin improved angiogenesis in myocardium in a rat model of MI.

The cardioprotective properties of mesenchymal stem cells and the therapeutic potential of curcumin (CUR) have been explored. Combining these approaches may enhance stem cell effectiveness and expedite healing. This study aimed to investigate the synergistic effects of co-treating bone marrow mesenchymal stem cells (BMSCs) with curcumin on vascular endothelial growth factor (VEGF) levels, in a rat model of myocardial ischemia (MI). Sixty-five male rats were divided into four groups: G1 (healthy control), G2 (MI induced by isoproterenol hydrochloride), G3 (treated with BMSCs), and G4 (co-treated with curcumin and BMSCs). Blood and tissue samples were collected at specific time points (day 1, 7, 15 and 21) after MI induction. Serum levels of lactate dehydrogenase (LDH), creatine kinase (CK), cardiac troponin I (cTnI), aspartate aminotransferase (AST), CK-MB and VEGF were measured. VEGF mRNA and protein expression were evaluated using RT-qPCR and Western blot techniques. Histopathological assessments were performed using H&E staining and CD31 immunofluorescence staining. VEGF expression significantly increased on days 7 and 15 in the CUR-BMSCs group, peaking on day 7. Western blot analysis confirmed elevated VEGF protein expression on days 7 and 15 post-MI. ELISA results demonstrated increased serum VEGF levels on days 7 and 15, reaching the highest level on day 7 in CUR-BMSCs-treated animals. Treated groups showed lower levels of LDH, AST, CK, CK-MB and cTnI compared to the untreated MI group. H&E staining revealed improved myocardial structure, increased formation of new capillaries, in both treatment groups compared to the MI group. Combining curcumin with BMSCs promotes angiogenesis in the infarcted myocardium after 15days of MI induction. These findings suggest the potential of this combined therapy approach for enhancing cardiac healing and recovery.

CRISPR/Cas genome editing revealed non-angiogenic role of VEGFA gene in porcine luteal cells: a preliminary report.

The angiogenic cytokine vascular endothelial growth factor A (VEGFA) also exerts non-angiogenic effects on endocrine functionality of porcine luteal cells critical for progesterone (P4) production. The expression dynamics of VEGFA-FLT/KDR system were investigated using RT-qPCR during luteal stages and VEGFA gene knock out (KO) porcine luteal cells were generated using CRISPR/Cas9 technology. The downstream effects of VEGFA ablation were studied using RT-qPCR, Annexin V, MTT, ELISA for P4 estimation and scratch wound assay. Bioinformatics analysis of RNA-Seq data of porcine mid-luteal stage was conducted for exploring protein-protein interaction network, KEGG pathways, transcription factors and kinase mapping for VEGFA-FLT/KDR interactomes. The VEGFA-FLT/KDR system expressed throughout the luteal stages with highest expression during mid- luteal stage. Cellular morphology, structure and oil-red-o staining for lipid droplets did not differ significantly between VEGFA KO and wild type cells, however, VEGFA KO significantly decreased (p < 0.05) viability and proliferation efficiency of edited cells on subsequent passages. Expression of apoptotic gene, CASP3 and hypoxia related gene, HIF1A were significantly (p < 0.05) upregulated in KO cells. The relative mRNA expression of VEGFA and steroidogenic genes STAR, CYP11A1 and HSD3B1 decreased significantly (p < 0.05) upon KO, which was further validated by the significant (p < 0.05) decrease in P4 output from KO cells. Bioinformatics analysis mapped VEGFA-FLT/KDR system to signalling pathways associated with steroidogenic cell functionality and survival, which complemented the findings of the study. The ablation of VEGFA gene resulted in decreased steroidogenic capability of luteal cells, which suggests that VEGFA exerts additional non-angiogenic regulatory effects in luteal cell functionality.

Exploring the anti-angiogenic properties of diosgenin saponin from Trigonella graecum (fenugreek): A study on Zebrafish embryos

Background: Angiogenesis plays a role in both physiological and pathological processes such as wound healing, embryonic development, and cancer metastasis, and it is the process by which new blood vessels are formed from existing ones. Diosgenin is a naturally occurring steroidal sapogenin found in plants such as yam and fenugreek, which is a compound of interest because of its pharmacological properties, including anti-carcinogenic effects. Zebrafish embryos are used due to their translucent nature, which facilitates the direct observation of vascular development. Aims and Objectives: The purpose of this study is to utilize zebrafish embryos to examine the impact of diosgenin on angiogenesis. It specifically aims to determine whether diosgenin could inhibit the development of inter-segmental vessels (ISV) in zebrafish embryos and also to investigate its effect on vascular endothelial growth factor (VEGF-A), a key regulator of angiogenesis. Materials and Methods: To explore the anti-angiogenic properties of diosgenin, we performed in vivo experiments using the zebrafish angiogenesis model. This involved staining red blood cells with O-dianisidine and conducting semiquantitative real-time polymerase chain reaction analysis to assess VEGF-A mRNA expression levels. Results: Diosgenin was shown to hinder the growth and formation of ISV in dosage-dependent development using a red blood cell staining assay when compared to the positive control, SU5416. In addition, there was a significant decrease in VEGF-A expression. Conclusion: In the presence of diosgenin, there is an evidence of inhibition of ISV development and suppression of VEGF mRNA expression. These findings underscore the therapeutic potential of diosgenin for targeting angiogenesis in cancer. However, further research is needed to understand the mechanism underlying diosgenin’s effect and its clinical relevance.

MicroRNA-299-3p inhibits cell proliferation, motility, invasion and angiogenesis via VEGFA in upper tract urothelial carcinoma.

Upper tract urothelial carcinoma (UTUC) is a rare tumor with extraordinarily different features between Eastern and Western countries. Vascular endothelial growth factor-A (VEGFA) was originally identified as a secreted signaling protein and regulator of vascular development and cancer progression. In this study, we aimed to elucidate the molecular mechanisms underlying the regulation of VEGFA by microRNA in UTUC. VEGFA expression was evaluated by immunohistochemistry in 140 human UTUC tissue samples. Next, we assessed the regulatory relationship between VEGFA and miR-299-3p by real-time PCR, western blotting, ELISA and dual-luciferase reporter assays using two UTUC cell lines. The role of miR-299-3p/VEGFA in cell proliferation, motility, invasion, and tube formation was analyzed in vitro. High VEGFA expression was significantly associated with tumor stage, grade, distant metastasis and cancer-related death and correlated with poor progression-free and cancer-specific survival. VEGFA knockdown repressed proliferation, migration, invasion and angiogenesis in UTUC cell lines. miR-299-3p significantly reduced VEGFA protein expression and miR-299-3p overexpression inhibited VEGFA mRNA and protein expression by directly targeting its 3'-UTR. Functional studies indicated that VEGFA overexpression reversed the miR-299-3p-mediated suppression of tumor cell proliferation, migration, invasion and angiogenesis. In addition, miR-299-3p/VEGFA suppressed cellular functions in UTUC by modulating the expression of P18 and cyclin E2. Our findings suggest that miR-299-3p possibly suppresses UTUC cell proliferation, motility, invasion and angiogenesis via VEGFA. VEGFA may act as a prognostic predictor, and both VEGFA and miR-299-3p could be potential therapeutic targets for UTUC.

Inhibiting MiR-320a promotes myocardial apoptosis in myocardial infarction rats through activating VEGF pathway.

To detect the expressions of vascular endothelial growth factor (VEGF) and micro ribonucleic acid (miR)-320a in myocardial cells of rats with myocardial infarction (MI), and to study the detailed mechanism of the role of miR-320a in myocardial apoptosis in MI rats. The Sprague-Dawley rat model of MI was established, and the rats were randomly divided into a control group (n=8), recombinant adeno-associated virus (rAAV)-miR-320a group (n=8) and rAAV-miR-320a TuDs group (n=8). The corresponding rAAV (1×1011 virion-like particles) was intravenously injected. At 2 weeks after the injection of rAAV, all rats were euthanatized, and the organs were collected, frozen in liquid nitrogen and stored at -80°C for further experiments. The total RNA and total protein were extracted from heart tissues, and the expression levels of rAAV-miR-320a and rAAV-miR-320aTuDs in heart tissues were determined via reverse transcription-polymerase chain reaction (RT-PCR). Moreover, RT-PCR and Western blotting were performed to detect the mRNA and protein expressions in heart tissues, respectively. At the same time, myocardial apoptosis was evaluated through flow cytometry. After treatment with miR-320a TuDs, the mRNA and protein expressions of VEGF in heart tissues in MI were significantly increased (P<0.05). The results of flow cytometry showed that miR-320a TuDs intervention could promote myocardial apoptosis in MI (P<0.05). In addition, the results of Western blotting revealed that miR-320a TuDs could facilitate the activation of the VEGF signaling pathway in heart tissues in MI (P<0.05). Inhibiting miR-320a can promote myocardial apoptosis through activating the VEGF signaling pathway in myocardial cells in MI.

Tetramethylpyrazine inhibits keratitis and neovascularization induced by corneal alkali burn by suppressing the TLR4/NF-κB pathway activation and NLRP1/NLRP3 inflammasomes in rats

The role and related mechanisms of tetramethylpyrazine (TMP) in corneal alkali burn in rats were expected to be explored in this article. After construction of corneal alkali burn rat models, TMP eye drops were given four times daily for consecutive 7 days. H&E staining was utilized for observing the histopathological changes of corneas on the 3rd and 7th days of treatment; immunohistochemistry for detecting the Nestin protein expression changes; qRT-PCR for determining the expression changes of genes correlated with neovascularization [C-X-C Motif Chemokine Ligand 1 (CXCL-1), vascular endothelial growth factor A (VEGFA) and CD31] and inflammation-related factors [monocyte chemoattractant protein-1 (MCP-1), interleukin-1β (IL-1β), tumour necrosis factor α (TNF-α), and IL-6]; Western blot for testing NLR Family Pyrin Domain Containing 1 (NLRP1)/NLRP3 inflammasomes and toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) pathway-related protein expression changes. All above trials were completed based on rat corneal tissue. TMP ameliorated the pathological damage in alkali-burned rat corneal tissue. Specifically, TMP treatment decreased Nestin-positive cell expression and the CXCL-1, VEGFA and CD31 mRNA expression in alkali-burned rat corneal tissue dose-dependently. TMP also down-regulated the IL-1β, TNF-α, MCP-1 and IL-6 mRNA expression and inhibited the NLRP1, caspase-1, NLRP3, pro-IL-1β and mature IL-1β protein expression in the alkali-burned rat corneal tissue. In addition, TMP treatment down-regulated the myeloid differentiation factor 88 (MyD88) and TLR4 protein expression and decreased the p–NF–κB p65/NF-κB p65 ratio in the alkali-burned rat corneal tissue. The mechanism of TMP relieving the inflammatory response and inhibiting neovascularization caused by corneal alkali burn in rats might have a correlation with the suppression of acitivation of TLR4/NF-κB pathway and NLRP1/NLRP3 inflammasomes in rats.

Effect of a 630 nm light on vasculogenic mimicry in A549 lung adenocarcinoma cells in vitro

ObjectiveThe objective of this study was to investigate the effect of photodynamic therapy (PDT) on the formation of vasculogenic mimicry (VM) in the human lung adenocarcinoma A549 cell line in vitro. MethodsThe participants were divided into a blank control group, a photosensitizer group, a light group, and a PDT group. Cells from each group were cultured in three dimensions using Matrigel, and vasculogenic mimicry generation was observed microscopically. Periodic Acid-Schiff (PAS) staining was used to verify the vasculogenic mimicry structure. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was used to detect the expression levels of cellular osteopontin (OPN) and vascular endothelial growth factor (VEGF) mRNA. Western blotting was used to detect the expression levels of cellular OPN and VEGF protein. ResultsA549 cells cultured on Matrigel for about six hours revealed VM on PAS staining, and the number of formations was significantly reduced in the PDT group compared with other groups (P < 0.05). The RT-PCR results showed that the PDT group downregulated OPN and VEGF mRNA expression compared with each control group (P < 0.05). Western blot results showed that OPN and VEGF protein expression was downregulated in the PDT group compared with each control group (P < 0.05). The results of RT-PCR showed that the expression of OPN and VEGF mRNA was downregulated in the PDT group compared with each control group (P < 0.05). The results of Western blotting showed that the expression of OPN and VEGF was downregulated in the protein PDT group compared with each control group (P < 0.001). ConclusionPhotodynamic therapy significantly inhibited the formation of vasculogenic mimicry in human lung adenocarcinoma A549 cells in vitro and downregulated the expression of OPN, VEGF mRNA, and protein levels.

Effect of comprehensive nursing based on evidence-based nursing on reducing the incidence of pressure ulcers in patients undergoing posterior orthopedic surgery.

To analyze the effect of comprehensive nursing based on evidence-based nursing during the perioperative period on reducing the incidence of pressure ulcers in patients undergoing posterior orthopedic surgery. Data on 120 patients who underwent orthopedic posterior surgery in our hospital from February 2021 to December 2022 were retrospectively analyzed. The patients were divided into an observation group (n = 60) and a control group (n = 60) based on different nursing methods. Patients in the control group received routine nursing, whereas those in the observation group received comprehensive nursing under the guidance of the concept of evidence-based nursing. The incidence of postoperative pressure ulcer was also recorded. Fasting venous blood (5 mL) was collected from patients before and after surgery and used to measure levels of myeloperoxidase (MPO) and superoxide dismutase (SOD) using enzyme-linked immunosorbent assay. Ulcer tissue samples of patients with pressure ulcers were collected and used to detect the expression of caspase-3 protein, vascular endothelial growth factor (VEGF) mRNA, tumor necrosis factor-α (TNF-α) mRNA, and interleukin-1β (IL-1β) mRNA. The incidence of postoperative pressure ulcers was 8% in the observation group and 23% in the control group (P = .024). The scores of sensory perceptions of the patients in the observation group were significantly lower than those in the control group (P < .001), as were the scores for moisture (P < .001), activity (P = .008), mobility (P < .001), nutrition (P = .003), friction, and shear (P < .001). After surgery, the serum MPO level in the observation group was significantly lower than that in the control group (P < .001), whereas the SOD level in the observation group was significantly higher than that in the control group (P < .001). The expression of TNF-α, IL-1β, VEGF mRNA, and caspase-3 protein in pressure ulcer tissues in the observation group was significantly lower than that in the control group. Comprehensive nursing based on the concept of evidence-based nursing can significantly reduce the incidence of postoperative pressure ulcers following posterior orthopedic surgery.

17β-estradiol inhibits TGF-β-induced collagen gel contraction mediated by human Tenon fibroblasts via Smads and MAPK signaling pathways.

To investigate the impact of 17β-estradiol on the collagen gels contraction (CGC) and inflammation induced by transforming growth factor (TGF)-β in human Tenon fibroblasts (HTFs). HTFs were three-dimensionally cultivated in type I collagen-generated gels with or without TGF-β (5 ng/mL), 17β-estradiol (12.5 to 100 µmol/L), or progesterone (12.5 to 100 µmol/L). Then, the collagen gel diameter was determined to assess the contraction, and the development of stress fibers was analyzed using immunofluorescence staining. Immunoblot and gelatin zymography assays were used to analyze matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) being released into culture supernatants. Enzyme-linked immunosorbent assay (ELISA) and reverse transcription-quantitative polymerase chain reaction (RT-PCR) were used to detect interleukin (IL)-6, monocyte chemoattractant proteins (MCP)-1, and vascular endothelial growth factor (VEGF) in HTFs at the translational and transcriptional levels. The phosphorylation levels of Sma- and Mad-related proteins (Smads), mitogen-activated protein kinases (MAPKs), and protein kinase B (AKT) were measured by immunoblotting. Statistical analysis was performed using either the Tukey-Kramer test or Student's unpaired t-test to compare the various treatments. The CGC caused by TGF-β in HTFs was significantly inhibited by 17β-estradiol (25 to 100 µmol/L), and a statistically significant difference was observed when comparing the normal control group with 17β-estradiol concentrations exceeding 25 µmol/L (P<0.05). The suppressive impact of 17β-estradiol became evident 24h after administration and peaked at 72h (P<0.05), whereas progesterone had no impact. Moreover, 17β-estradiol attenuated the formation of stress fibers, and the production of MMP-3 and MMP-1 in HTFs stimulated by TGF-β. The expression of MCP-1, IL-6, and VEGF mRNA and protein in HTFs were suppressed by 100 µmol/L 17β-estradiol (P<0.01). Additionally, the phosphorylation of Smad2 Smad3, p38, and extracellular signal-regulated kinase (ERK) were downregulated (P <0.01). 17β-estradiol significantly inhibits the CGC and inflammation caused by TGF-β in HTFs. This inhibition is likely related to the suppression of stress fibers, inhibition of MMPs, and attenuation of Smads and MAPK (ERK and p38) signaling. 17β-estradiol may have potential clinical benefits in preventing scar development and inflammation in the conjunctiva.

Correlation Analysis of Vascular Endothelial Growth Factor Level with Clinicopathological Features and Prognosis in Patients with Diabetic Nephropathy: A Biopsy-based Study.

Globally, Type 2 diabetes mellitus (T2DM) is one of the most prevalent chronic diseases, which poses a great potential threat to the human body. Diabetic nephropathy (DN), a very common complication in T2DM, is also the main trigger for end-stage renal disease. A thorough understanding of the pathogenesis is the key as well as the breakthrough for future diagnosis and treatment of DN. This investigation aims to provide more in-depth and accurate guidance for future follow-up research by analyzing the role of vascular endothelial growth factor (VEGF) in the kidney tissue of DN patients. Seventy-nine patients with suspected DN who underwent renal needle biopsy in our hospital from January 2015 to June 2019 were selected as the research participants. After the biopsy, 36 cases were confirmed as DN, and the other 43 were T2DM with primary glomerulonephritis. Determination of VEGF mRNA and protein expression in renal tissue employed PCR and Western blot, and the connection between VEGF mRNA level and clinical pathology (such as renal function, inflammatory factors and pathological manifestations) was discussed. The disease recurrence in DN patients was recorded through the 3-year prognostic followed up, and the related influencing factors were analyzed. In kidney tissue, VEGF mRNA level and protein expression were notably higher in DN patients than in diabetic patients (P<0.05). Pearson correlation coefficient analysis identified that VEGF mRNA and protein had a positive connection with blood urea nitrogen (BUN), serum creatinine (Scr), 24-hoururinetotalprotein(24hUTP) and C-reactive protein (CRP) (P<0.05). Among the various clinicopathological features of DN patients, age, BMI, sex, family history, smoking, drinking, exercise habits, clinical presentations and pathological changes had no significant relationship with VEGF level (P>0.05), but the course of the disease, fasting blood glucose (FBG), glycosylated hemoglobin (HBALC) and pathological stages of nephropathy had a close connection with VEGF level (P<0.05). Prognostic follow-up revealed that VEGF mRNA was noticeably higher in patients with recurrence than in those without (P<0.05). When VEGF mRNA >5.20 in kidney tissue, the sensitivity and specificity for predicting the 3-year recurrence were 85.71% and 84.00% respectively (P<0.05). Finally, Logistic regression analysis identified the independence of FBG, HBALC and VEGF levels as the influencing factors for the prognostic recurrence of DN (P<0.05).VEGF expression in kidney tissue of DN patients is closely linked to renal function and increases as the disease progresses, which is an independent risk factor associated with the prognostic recurrence of DN, with great potential significance for future DN diagnosis and treatment.

Effect and mechanism of Xihuang Pills on rats with precancerous lesions of breast

The purpose of this study was to explore the effect and mechanism of Xihuang Pills on rats with precancerous lesions of the breast. Of 48 healthy female rats, 8 were randomly selected as blank group, and the other 40 were treated with 7,12-dimethylbenzanthracene(DMBA) combined with estrogen and progestin to establish a model of precancerous lesions of the breast. The successfully modeled rats were randomly divided into a model group, a tamoxifen group(1.8 mg·kg~(-1)·d~(-1)), a Xihuang Pills low-dose group(0.3 g·kg~(-1)·d~(-1)), a medium-dose group(0.6 g·kg~(-1)·d~(-1)) and a high-dose group(1.2 g·kg~(-1)·d~(-1)). After 30 days of admi-nistration, the histopathological changes of viscera and breast were observed by haematoxylin and eosin(HE) staining, and the visceral index was calculated. Enzyme linked immunosorbent assay(ELISA) was used to detect the contents of estradiol(E_2) and progesterone(P) in serum. The protein expressions of vascular endothelial growth factor(VEGF) and fibroblast growth factor 2(FGF2) were detected by immunohistochemistry. The protein expressions of VEGF, vascular endothelial growth factor receptor 2(VEGFR2), phosphorylated-vascular endothelial growth factor receptor 2(p-VEGFR2), B-cell lymphoma-2(Bcl-2), and Bcl-2 associated X protein(Bax) were detected by Western blot and the mRNA expressions of VEGF, FGF2, CXC-chemokine receptor 4(CXCR4), cysteine aspartic acid-specific protease(caspase-3), and stromal cell-derived factor 1(SDF-1) were detected by real-time polymerase chain reaction(RT-PCR). HE staining revealed that the model group had some liver and kidney damages and severe hyperplastic mammary tissue, while the Xihuang Pills high-dose group had mild hyperplasia. Compared with the model group, the Xihuang Pills groups had lo-wer ovarian coefficient(P&lt;0.05 or P&lt;0.01) and Xihuang Pills high-dose group had lower uterine coefficient(P&lt;0.01). ELISA results showed that compared with the model group, expressions of E_2 and P in Xihuang Pills high-dose group were significantly decreased(P&lt;0.05 or P&lt;0.01). Immunohistochemistry, Western blot and RT-PCR indicated that compared with the conditions in the model group, the protein and mRNA expressions of VEGF and FGF2 in the Xihuang Pills groups were down-regulated(P&lt;0.05 or P&lt;0.01), and the protein expression of Bcl-2 was lowered(P&lt;0.01); there was a decrease in the protein expressions of VEGFR2 and p-VEGFR2(P&lt;0.01), a down-regulation in the mRNA expressions of CXCR4 and SDF-1(P&lt;0.01), while an increase in the mRNA expression of caspase-3(P&lt;0.01) in both Xihuang Pills medium-dose and high-dose groups; the protein expression of Bax in Xihuang Pills high-dose group was increased(P&lt;0.01). The above results indicated that Xihuang Pills can effectively intervene in precance-rous lesions of the breast, and the mechanism may be related to the regulation of E_2 and P secretion as well as the inhibition of angiogenesis and chemokine receptor expression, thus controlling the occurrence of precancerous lesions of the breast in rats.

Expression of neurotransmitters, vasculogenesis markers and myosin heavy chain isoforms in the masseter muscle of senescence-accelerated mouse prone 8 mice.

Learning and memory deficits and pathologic changes in the hippocampus caused by toothlessness and soft diet feeding are related to reduced masseter muscle (MM) function. Myosin heavy chain (MyHC) isoform expression in the MM also changes under different chewing conditions. The neurotransmitter calcitonin gene-related peptide (CGRP) and vascular endothelial growth factor A (VEGF-A) are involved in MM formation. However, the relationship between CGRP, VEGF-A and MyHC isoforms in the MM in the senescence-accelerated mouse prone 8 (SAMP8) strain, a model of learning and memory deficits, remains unclear. Changes in CGRP, VEGF-A, vasculogenesis marker, and MyHC isoform mRNA expression in the MMs of aging SAMP8 and senescence-accelerated mouse resistant 1 (SAMR1) mice was investigated through quantitative real-time polymerase chain reaction (qRT-PCR) and in situ hybridization. qRT-PCR revealed obviously high CGRP levels in the SAMP8 mouse MM (p<0.001). MyHC-IId/x mRNA expression in the MM was higher in 24-week-old SAMP8 mice than 24-week-old SAMR1 mice (p<0.001) but lower in slow-MyHC SAMP8 mice than SAMR1 mice (p<0.001). CGRP mRNA was observed on the muscle fibers of the SAMP8 mouse MM but not the SAMR1 mouse MM through in situ hybridization. Principal component analysis (PCA) revealed strong positive contributions of SAMP8-MyHC-IId/x, SAMP8-CGRP, SAMR1-MyHC-emb, SAMR1-CGRP, SAMR1-VEGF-A, SAMR1-CD31, SAMP8-VEGF-A, and SAMP8-CD31 in the MM at 12 and 24 weeks. CGRP is also key for the MyHC-IId/x and slow-MyHC patterns in the MMs of SAMP8 mice.

Inhibition of Xanthine Oxidase Protects against Diabetic Kidney Disease through the Amelioration of Oxidative Stress via VEGF/VEGFR Axis and NOX-FoxO3a-eNOS Signaling Pathway.

Xanthine oxidase (XO) is an important source of reactive oxygen species. This study investigated whether XO inhibition exerts renoprotective effects by inhibiting vascular endothelial growth factor (VEGF) and NADPH oxidase (NOX) in diabetic kidney disease (DKD). Febuxostat (5 mg/kg) was administered to streptozotocin (STZ)-treated 8-week-old male C57BL/6 mice via intraperitoneal injection for 8 weeks. The cytoprotective effects, its mechanism of XO inhibition, and usage of high-glucose (HG)-treated cultured human glomerular endothelial cells (GECs) were also investigated. Serum cystatin C, urine albumin/creatinine ratio, and mesangial area expansion were significantly improved in febuxostat-treated DKD mice. Febuxostat reduced serum uric acid, kidney XO levels, and xanthine dehydrogenase levels. Febuxostat suppressed the expression of VEGF mRNA, VEGF receptor (VEGFR)1 and VEGFR3, NOX1, NOX2, and NOX4, and mRNA levels of their catalytic subunits. Febuxostat caused downregulation of Akt phosphorylation, followed by the enhancement of dephosphorylation of transcription factor forkhead box O3a (FoxO3a) and the activation of endothelial nitric oxide synthase (eNOS). In an in vitro study, the antioxidant effects of febuxostat were abolished by a blockade of VEGFR1 or VEGFR3 via NOX-FoxO3a-eNOS signaling in HG-treated cultured human GECs. XO inhibition attenuated DKD by ameliorating oxidative stress through the inhibition of the VEGF/VEGFR axis. This was associated with NOX-FoxO3a-eNOS signaling.

Expression of Acetabular Labral Vascular Endothelial Growth Factor and Nerve Growth Factor Is Directly Associated with Hip Osteoarthritis Pain: Investigation by Immunohistochemical Staining.

The acetabular labrum enhances hip joint stability and plays a key role in osteoarthritis (OA) progression. Labral nerve endings contribute to hip OA pain. Moreover, vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) are associated with pain. Consequently, we analysed VEGF and NGF expression levels in the labrum and their roles in OA. Labra obtained from OA patients were stained immunohistochemically, and labral cells were cultured and subjected to a reverse transcription (RT)-polymerase chain reaction (PCR) to analyse VEGF and NGF mRNA expression. VEGF and NGF expression were compared in each region of the labrum. Correlations between VEGF and NGF expression and age, body mass index, Kellgren-Lawrence grade, Harris Hip Score, the visual analogue scale (VAS), and Krenn score were analysed, and the RT-PCR confirmed the findings. VEGF and NGF expression were high on the labral articular side, negatively correlated with the Krenn score, and positively correlated with the VAS in early OA. VEGF and NGF mRNA expression increased significantly in patients with severe pain and decreased significantly in severely degenerated labra. In early OA, VEGF and NGF expression in the acetabular labrum was associated with the occurrence of hip pain; therefore, these factors could be effective targets for pain management.

Protective Effect of NO2-OA on Oxidative Stress, Gliosis, and Pro-Angiogenic Response in Müller Glial Cells.

Inflammation and oxidative and nitrosative stress are involved in the pathogenesis of proliferative retinopathies (PR). In PR, a loss of balance between pro-angiogenic and anti-angiogenic factors favors the secretion of vascular endothelial growth factor (VEGF). This vascular change results in alterations in the blood-retinal barrier, with extravasation of plasma proteins such as α2-macroglobulin (α2M) and gliosis in Müller glial cells (MGCs, such as MIO-M1). It is well known that MGCs play important roles in healthy and sick retinas, including in PR. Nitro-fatty acids are electrophilic lipid mediators with anti-inflammatory and cytoprotective properties. Our aim was to investigate whether nitro-oleic acid (NO2-OA) is beneficial against oxidative stress, gliosis, and the pro-angiogenic response in MGCs. Pure synthetic NO2-OA increased HO-1 expression in a time- and concentration-dependent manner, which was abrogated by the Nrf2 inhibitor trigonelline. In response to phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS), NO2-OA prevented the ROS increase and reduced the gliosis induced by α2M. Finally, when hypoxic MGCs were incubated with NO2-OA, the increase in VEGF mRNA expression was not affected, but under hypoxia and inflammation (IL-1β), NO2-OA significantly reduced VEGF mRNA levels. Furthermore, NO2-OA inhibited endothelial cell (BAEC) tubulogenesis. Our results highlight NO2-OA's protective effect on oxidative damage, gliosis; and the exacerbated pro-angiogenic response in MGCs.

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.

A Novel "Inside-Out" Intraocular Nanomedicine Delivery Mode for Nanomaterials' Biological Effect Enhanced Choroidal Neovascularization Occlusion and Microenvironment Regulation.

Photodynamic therapy (PDT) is commonly used in choroidal neovascularization (CNV) treatment due to the superior light transmittance of the eye. However, PDT often leads to surrounding tissue damage and further microenvironmental deterioration, including exacerbated hypoxia, inflammation, and secondary neovascularization. In this work, Pt nanoparticles (NPs) and Au NPs decorated zeolitic imidazolate framework-8 nanoplatform is developed to load indocyanine green for precise PDT and microenvironment amelioration, which can penetrate the internal limiting membrane through Müller cells endocytosis and target to CNV by surface-grafted cyclo(Arg-Gly-Asp-d-Phe-Lys) after intravitreal injection. The excessive H2 O2 in the CNV microenvironment is catalyzed by catalase-like Pt NPs for hypoxia relief and enhanced PDT occlusion of neovascular. Meanwhile, Au NPs show significant anti-inflammatory and anti-angiogenesis properties in regulating macrophages and blocking vascular endothelial growth factor (VEGF). Compared with verteporfin treatment, the mRNA expressions of hypoxia-inducible factor-1α and VEGF in the nanoplatform group are downregulated by 90.2% and 81.7%, respectively. Therefore, the nanoplatform realizes a comprehensive CNV treatment effect based on the high drug loading capacity and biosafety. The CNV treatment mode developed in this work provides a valuable reference for treating other diseases with similar physiological barriers that limit drug delivery and similar microenvironment.