ObjectiveTo investigate the mechanism of G protein coupled receptor kinase interacting protein 1 (GIT1) affecting angiogenesis by comparing the differentiation of bone marrow mesenchymal stem cells (BMSCs) differentiated into endothelial cells between GIT1 wild type mice and GIT1 gene knockout mice.MethodsMale and female GIT1 heterozygous mice were paired breeding, and the genotypic identification of newborn mice were detected by PCR. The 2nd generation BMSCs isolated from GIT1 wild type mice or GIT1 gene knockout mice were divided into 4 groups, including wild type control group (group A), wild type experimental group (group A1), GIT1 knockout control group (group B), and GIT1 knockout experimental group (group B1). The cells of groups A1 and B1 were cultured with the endothelial induction medium and the cells of groups A and B with normal cluture medium. The expressions of vascular endothelial growth factor receptor 2 (VEGFR-2), VEGFR-3, and phospho-VEGFR-2 (pVEGFR-2), and pVEGFR-3 proteins were detected by Western blot. The endothelial cell markers [von Willebrand factor (vWF), platelet-endothelial cell adhesion molecule 1 (PECAM-1), and vascular endothelial cadherin (VE-Cadherin)] were detected by flow cytometry. The 2nd generation BMSCs of GIT1 wild type mice were divided into 4 groups according to the different culture media: group Ⅰ, primary cell culture medium; group Ⅱ, cell culture medium containing SAR131675 (VEGFR-3 blocker); group Ⅲ, endothelial induction medium; group Ⅳ, endothelial induction medium containing SAR131675. The endothelial cell markers (vWF, PECAM-1, and VE-Cadherin) in 4 groups were also detected by flow cytometry.ResultsWestern blot results showed that there was no obviously difference in protein expressions of VEGFR-2 and pVEGFR-2 between groups; and the expressions of VEGFR-3 and pVEGFR-3 proteins in group A1 were obviously higher than those in groups A, B, and B1. The flow cytometry results showed that the expressions of vWF, PECAM-1, and VE-Cadherin were significantly higher in group A1 than in groups A, B, and B1 (P<0.05), and in group B1 than in groups A and B (P<0.05); but no significant difference was found between groups A and B (P>0.05). In the VEGFR-3 blocked experiment, the flow cytometry results showed that the expressions of vWF, PECAM-1, and VE-Cadherin were significantly higher in group Ⅲ than in groupsⅠ, Ⅱ, and Ⅳ, and in group Ⅳ than in groups Ⅰ and Ⅱ (P<0.05); but no significant difference was found between groups Ⅰ and Ⅱ (P>0.05).ConclusionGIT1 mediates BMSCs of mice differentiation into endothelial cells via VEGFR-3, thereby affecting the angiogenesis.
Objective To observe the efficacy and safety of subretinal injection of Aflibercept for the treatment of refractory or recurrent polypoidal choroidal vasculopathy (PCV). MethodsA prospective clinical research. From January to June 2022, 18 patients of 18 eyes with PCV diagnosed in The Affiliated Eye Hospital of Nanchang University were included in the study. All patients underwent best corrected visual acuity (BCVA), indocyanine green angiography and optical coherence tomography (OCT). The BCVA examination was performed using the international standard visual acuity chart, which was converted to logarithm of the minimum angle of resolution (logMAR) visual acuity during statistics. The large choroidal vessel thickness (LVCT), central retinal thickness (CRT), sub-foveal choroidal thickness (SFCT) and retinal pigment epithelium detachment (PED) height were measured by enhanced depth imaging technique of OCT. The choroidal vascular index (CVI) was calculated. There were 18 patients of 18 eyes, 11 males of 11 eyes and 7 females of 7 eyes. The age was (64.22±3.86) years old. The disease duration was (5.22±1.80) years. The patient had received intravitreal injection of anti-vascular endothelial growth factor (VEGF) drugs for (7.72±1.36) times. The logMAR BCVA of the affected eyes was 1.28±0.25. The SFCT, CRT, LVCT, PED height were (436.56±9.80), (432.44±44.29), (283.78±27.10), (342.44±50.18) μm, respectively, and CVI was 0.65±0.01. All eyes were treated with a single subretinal injection of 40 mg/ml Aflibercept 0.05 ml (including Aflibercept 2.0 mg). According to the results of OCT and BCVA after treatment, the lesions were divided into active type and static type. The active lesions were treated with intravitreal injection of Aflibercept at the same dose as before. Quiescent lesions were followed up. Examinations were performed 1-3, 6, 9 and 12 months after treatment using the same equipment and methods before treatment. The BCVA, LVCT, CRT, SFCT, PED height, CVI, interretinal or subretinal fluid, lesion regression rate, injection times, and complications during and after treatment were observed. The BCVA, SFCT, CRT, LVCT, PED height and CVI before and after treatment were compared by repeated measures analysis of variance. ResultsEighteen eyes received subretinal and/or intravitreal injection of Aflibercept (1.61±0.85) times (1-4 times). At the last follow-up, the polypoid lesions regressed in 4 eyes and PED disappeared in 1 eye. Compared with before treatment, BCVA (F=50.298) gradually increased, CRT (F=25.220), PED height (F=144.16), SFCT (F=69.77), LVCT (F=136.69), CVI (F=72.70) gradually decreased after treatment. The differences were statistically significant (P＜0.001). Macular hole occurred in 1 eye after treatment, and the hole closed spontaneously 3 months after treatment. No serious complications such as retinal tear, retinal detachment, endophthalmitis and vitreous hemorrhage occurred during and after treatment. ConclusionSubretinal injection of Aflibercept is safe and effective in the treatment of refractory PCV.
Objective To observe and evaluate the safety and efficacy of anti-vascular endothelial growth factor (VEGF) in the treatment of eyes with macular edema (ME) secondary to branch retinal vein occlusion (BRVO) in Lhasa, Tibet. MethodsA retrospective case series. From September 2018 to January 2022, a total of 41 patients (41 eyes) with BRVO-ME, who were diagnosed in Department of Ophthalmology of Tibet Autonomous Region People’s Hospital, were included in this study. There were 21 eyes in 21 males and 20 eyes in 20 females. The median age was 53 (31,75) years. There were 24 patients with hypertension (58.8%, 24/41). Best corrected visual acuity (BCVA), ocular pressure, fundus color photography and optical coherence tomography (OCT) were performed in all eyes. The BCVA was performed using the international standard logarithmic visual acuity chart, which was converted into logarithm of the minimum angle of resolution (logMAR) BCVA for record. The foveal macular thickness (CMT) was measured by OCT. All eyes were treated with intravitreous injection of anti-VEGF drugs, once a month, among which 23 eyes (56.1%, 23/41) received intravitreous injection of ranibizumab (IVR), and 18 eyes (43.9%, 18/41) received intravitreous injection of conbercept (IVC), and were grouped accordingly. There was no significant difference in age (Z=－0.447), gender composition (Z=－0.485), logMAR BCVA (t=－1.591), intraocular pressure (t=－0.167) and CMT (t=－1.290) between two groups (P＞0.05). During the follow-up, the same devices and methods were used at baseline to perform relevant examinations, and the changes of BCVA, intraocular pressure, CMT and new cardiovascular and cerebrovascular events were compared between baseline and the last follow-up. logMAR BCVA, intraocular pressure and CMT were compared between baseline and last follow-up using Student t test. The comparison of injection times and follow-up time between IVR group and IVC group was conducted by Mann-Whitney U test. ResultsAt baseline, logMAR BCVA, intraocular pressure, and CMT were 0.852±0.431, (12.5±2.5) mm Hg (1 mm Hg= 0.133 kPa), and (578.1±191.1) μm, respectively. At the last follow-up, the number of anti-VEGF drug treatments was (2.7±1.2) times; logMAR BCVA and CMT were 0.488±0.366 and (207.4±108.7) μm, respectively, with CMT > 250 μm in 14 eyes (34.1%, 14/41). Compared with baseline, BCVA (t=4.129) and CMT (t=－0.713) were significantly improved, with statistical significance (P＜0.001). The injection times of IVR group and IVC group were (2.6±0.9) and (3.0±1.5) times, respectively. There were no significant differences in the number of injection times (t=－1.275), logMAR BCVA (t=－0.492), intraocular pressure (t=0.351) and CMT (t=－1.783) between the two groups (P＞0.05). No new hypertension, cardiovascular and cerebrovascular events occurred in all patients during follow-up. At the last follow-up, there were no eye complications related to treatment modalities and drugs. ConclusionShort-term anti-VEGF treatment can improve the visual acuity of BRVO secondary ME patients and alleviate ME in Lhasa, Tibet. The safety and efficacy of ranibizumab and conbercept were similar.
Objective To analyze the diagnostic value of shear wave elastography (SWE) combined with vascular endothelial growth factor B (VEGF-B) and hemoglobin A1c (HbA1c) in early diabetic peripheral neuropathy (DPN). Methods A total of 100 patients with type 2 diabetes mellitus (T2DM) admitted to Mianyang Central Hospital between October 2020 and October 2023 were selected and divided into a T2DM with DPN group (n=31) and a T2DM without DPN group (n=69) based on the presence or absence of DPN. Additionally, 50 healthy individuals from the same hospital’s health examination center were included as a healthy control group. The basic clinical characteristics, mean elasticity (Emean) values of the left and right median and tibial nerves, serum VEGF-B, and HbA1c levels were compared among the three groups. The diagnostic efficacy of SWE, VEGF-B, and HbA1c for DPN was evaluated using receiver operating characteristic (ROC) curves, and Pearson correlation analysis was performed to assess the relationships between median/tibial nerve Emean and VEGF-B/HbA1c. Results The Emean values of the left and right median nerves, Emean values of the left and right tibial nerves, serum VEGF-B, and HbA1c levels in the T2DM with DPN group were significantly higher than those in the T2DM without DPN group and the healthy control group (P<0.05). The Emean values of the left and right median and tibial nerves, Emean values of the left and right tibial nerves, and HbA1c level in the T2DM without DPN group were significantly higher than those in the healthy control group (P<0.05), while no significant difference was observed in serum VEGF-B level between the T2DM without DPN group and the healthy control group (P>0.05). The area under the ROC curve for the combined diagnosis of DPN using SWE, VEGF-B, and HbA1c was 0.859 [95% confidence interval (0.828, 0.955)]. The sensitivity of the combined diagnosis (93.72%) was significantly higher than that of individual diagnoses (78.82%, 75.39%, and 71.05%, respectively; P<0.05), while the specificity (88.64%) showed no significant difference compared to individual diagnoses (80.18%, 78.96%, and 82.88%, respectively; P>0.05). Positive correlations were observed between median/tibial nerve Emean and VEGF-B/HbA1c levels (r=0.428, 0.395, 0.416, and 0.416, respectively; P<0.05). Conclusions Elevated median/tibial nerve Emean, serum VEGF-B, and HbA1c levels are closely associated with DPN. The combination of SWE, VEGF-B, and HbA1c improves diagnostic sensitivity for DPN, demonstrating significant clinical value.
Diabetic macular edema (DME) is the most threatening complication of diabetic retinopathy that affects visual function, which is characterized by intractability and recurrent attacks. Currently, the clinical routine treatments for DME mainly include intravitreal injection, grid laser photocoagulation in the macular area, subthreshold micropulse laser, periocular corticosteroid injection, and vitrectomy. Although conventional treatments are effective for some patients, persistent, refractory, and recurrent DME remains a clinical challenge that needs to be urgently addressed. In recent years, clinical studies have found that certain combination therapies are superior to monotherapy, which can not only restore the anatomical structure of the macular area and effectively reduce macular edema but also improve visual function to some extent while reducing the number of treatments and the overall cost. This makes up for the shortcomings of single treatment modalities and is highly anticipated in the clinical setting. However, the application of combination therapy in clinical practice is relatively short, and its safety and long-term effectiveness need further exploration. Currently, new drugs, new formulations, and new therapeutic targets are still under research and development to address different mechanisms of DME occurrence and development, such as anti-vascular endothelial growth factor agents designed to anchor repetitive sequence proteins with stronger inhibition of vascular leakage, multiple growth factor inhibitors, anti-inflammatory agents, and stem cell therapy. With the continuous improvement of the combination application of existing drugs and treatments and the development of new drugs and treatment technologies, personalized treatment for DME will become possible.
ObjectiveTo observe and analyze the influencing factors for the prognosis of anti-vascular endothelial growth factor (VEGF) drug treatment in patients with macular neovascularization (MNV) under 45 years old. MethodsA retrospective clinical case study. A total of 89 MNV patients with 96 eyes who were diagnosed and treated with anti-VEGF drugs in Department of Ophthalmology of The Second Hospital of Lanzhou University from January 2020 to January 2024 were included in the study. The ages of all patients were ＜45 years old. All patients underwent best corrected visual acuity (BCVA) and optical coherence tomography (OCT) examinations; 49 eyes underwent OCT angiography (OCTA) examination. The BCVA examination was carried out using the international standard visual acuity chart and was converted into the logarithm of the minimum angle of resolution (logMAR) visual acuity for statistics. The macular foveal thickness (CMT) was measured using an OCT instrument. The size of the MNV lesion was measured using the software of the OCTA self-contained device. The affected eyes were given intravitreal injection of anti-VEGF drugs once, and then the drugs were administered as needed after evaluation. The follow-up time after treatment was ≥6 months. During the follow-up, relevant examinations were performed using the same equipment and methods as before treatment. The last follow-up was taken as the time point for efficacy evaluation. According to the OCT image characteristics of the MNV lesions, the affected eyes were divided into the fibrous scar group and the non-fibrous scar group, with 52 (54.16%, 52/96) and 44 (45.83%, 44/96) eyes respectively. Comparing the CMT and BCVA at the last follow-up with those at the baseline, the affected eyes were divided into the CMT reduction group, the CMT increase group, the BCVA improvement group and the BCVA reduction group, with 66 (68.75%, 66/96), 30 (31.25%, 30/96) eyes and 74 (77.08%, 74/96), 22 (22.92%, 22/96) eyes respectively. The Mann-Whitney U test was used for the comparison of non-normally distributed measurement data between groups. Logistic regression analysis was used to analyze the independent factors affecting the prognosis of MNV patients. ResultsThere were no statistically significant differences in the age (Z=−0.928) and gender composition ratio (χ2= 0.123) between the fibrous scar group and the non-fibrous scar group (P＞0.05); there were statistically significant differences in the number of eyes with a follow-up time of ≥36 months and ＜36 months (χ2= 3.906, P=0.048); there were statistically significant differences in the size of the MNV lesions (Z=−2.385, P=0.017); there were statistically significant differences in the number of eyes with different vascular network morphologies (χ2=12.936, P=0.001). Before treatment and at the last follow-up, the CMT of the affected eyes was 267.50 (237.25, 311.75) μm and 242.00 (217.25, 275.75) μm respectively; logMAR BCVA was 0.20 (0.10, 0.50) and 0.35 (0.16, 0.60) logMAR respectively. There were statistically significant differences in the CMT and logMAR BCVA before treatment and at the last follow-up (Z=−3.311,−1.984; P=0.001, 0.047). There were statistically significant differences in different ages (Z=−2.284), myopic diopter (χ2=7.437), etiology (χ2=6.956), and disease course (Z=−1.687) between the CMT reduction group and the CMT increase group (P＜0.05). There were statistically significant differences in the number of eyes with different subjective feelings between the BCVA improvement group and the BCVA reduction group (χ2=10.133, P＜0.05). The results of logistic regression analysis showed that the etiology was an independent risk factor for CMT thickening. ConclusionsAge, etiology, myopic diopter, disease course, follow-up time, lesion size and the morphology of the neovascular network are the influencing factors for the prognosis of anti-VEGF drug treatment in MNV patients under 45 years old. The etiology is an independent risk factor for CMT increase.
ObjectiveTo analyze the status of applying programmed death-1 (PD-1) / programmed death-ligand 1 (PD-L1) inhibitors combined with vascular endothelial growth factor (VEGF) / vascular endothelial growth factor receptor (VEGFR) inhibitors in advanced refractory colorectal cancer. MethodThe relevant literature on domestic and foreign research in recent years was summarized. ResultsThe discovery of immune checkpoint PD-1/PD-L1 and the clinical application of related drugs had changed the treatment pattern of advanced solid tumors, but PD-1/PD-L1 inhibitors had a poor efficacy in the mismatch repair prodicient tumors, and most advanced colorectal cancer belonged to this type. The combination of PD-1/PD-L1 inhibitors and VEGF/VEGFR inhibitors could enhance the therapeutic effect in the advanced refractory colorectal cancer, and their interaction mechanisms and clinical efficacy were continuously being proven. ConclusionsThe combination of PD-1/PD-L1 inhibitors and VEGF/VEGFR inhibitors is a promising treatment strategy for advanced refractory colorectal cancer. More studies need to be further clarified its efficacy.
Diabetic retinopathy (DR) has become an important cause of irreversible vision loss worldwide. Intravitreal injection of anti-vascular endothelial growth factor (VEGF) drugs is an important method to the treatment of DR. However, the current anti-VEGF treatment regimen is not uniform. Anti-VEGF injection was preferred and then delayed combined with laser had better prognostic effect. The best time for operation was 5-7 days after injection of anti-VEGF drugs. Pars plana vitrectomy, intraoperative and postoperative on-demand anti-VEGF drugs injection can significantly improve patient prognosis and reduce complications, but further research is needed to strike a balance between the economic burden and the number of injections. Various anti-VEGF drugs have their own advantages for different diseases and should be selected according to the characteristics of the diseases and drugs. Anti-VEGF drugs combined with antioxidants may further improve DR outcomes. Future studies should pay more attention to the optimization and personalization of anti-VEGF drugs application programs to meet the therapeutic needs of different patients.
Objective To explore the influence on the expressions of vascular endothelial growth factor (VEGF) gene and matrix metalloproteinase-2 (MMP-2) gene in hepatocellular carcinoma of SMMC-7721 cells with RNA interference (RNAi) silencing the expression of hypoxia inducible factor-1α (HIF-1α) gene. Methods Firstly, constructed short hairpin RNA (shRNA) targeting for HIF-1α gene, and then transfected it to SMMC-7721 cells after combining with plasmid. The SMMC-7721 cells were divided into three groups, silencing group, negative control group, and blank control group, which were transfected with HIF-1α-shRNA-pGenesil-1 recombinant vector, shRNA-HK-pGenesil-1 recombinant vector, and pGenesil-1 vector respectively. Transfection cells were screened by the concentration of 500 μg/mL G418, and then positive and negative cell clones with transfection recombination carrier were obtained. Detected the expressions of HIF-1α mRNA, VEGF mRNA, and MMP-2 mRNA in the 3 groups with real time PCR (RT-PCR) technology, under the condition of hypoxic training 6 h, 12 h, and 24 h, as well as conventional oxygen training. Results There was no expression of HIF-1α mRNA at conventional oxygen condition in the 3 groups, and there was no significant difference in expressions of VEGF mRNA and MMP-2 mRNA among the 3 groups (P>0.05) at the condition of conventional oxygen training. The expressions of HIF-1α mRNA, VEGF mRNA, and MMP-2 mRNA in the silencing group, compared with the the negative control group and the blank control group, were obviously decreased (P<0.05) under the condition of hypoxic training (6, 12, and 24 h), while there was no significant difference between the negative control group and the blank control group at each time point (P>0.05), but the expressions of HIF-1α mRNA, VEGF mRNA, and MMP-2 mRNA in the 3 groups under every condition of hypoxic training were all higher than those of conventional oxygen condition (P<0.05). Under the condition of hypoxic training, the expressions of HIF-1α mRNA, VEGF mRNA, and MMP-2 mRNA in the 3 groups decreased over time, and there was significant difference between any 2 time points in each group (P<0.05). Conclusion RNAi technique can effectively silence the expression of HIF-1α mRNA of SMMC-7721 cells, and then silence the expressions of VEGF and MMP-2 mRNA, to inhibit the invasion and metastasis of hepatocellular carcinoma.
Objective To investigate whether human amniotic mesenchymal stem cells (hAMSCs) have the characteristics of mesenchymal stem cells (MSCs) and the differentiation capacity into ligament fibroblastsin vitro. Methods The hAMSCs were separated through trypsin and collagenase digestion from placenta, the phenotypic characteristics of hAMSCs were detected by flow cytometry, the cytokeratin-19 (CK-19) and vimentin expression of hAMSCs were tested through immunofluorescence staining. The hAMSCs at the 3rd passage were cultured with L-DMEM/F12 medium containing transforming growth factor β1 (TGF-β1) and vascular endothelial growth factor (VEGF) as the experimental group and with single L-DMEM/F12 medium as the control group. The morphology of hAMSCs was observed by inverted phase contrast microscope; the cellular activities and ability of proliferation were examined by cell counting kit-8 (CCK-8) method; the ligament fibroblasts related protein expressions including collagen type I, collagen type III, Fibronectin, and Tenascin-C were detected by immunofluorescence staining; specific mRNA expressions of ligament fibroblasts and angiogenesis including collagen type I, collagen type III, Fibronectin, α-smooth muscle actin (α-SMA), and VEGF were measured by real-time fluorescence quantitative PCR. Results The hAMSCs presented monolayer and adherent growth under inverted phase contrast microscope; the flow cytometry results demonstrated that hAMSCs expressed the MSCs phenotypes; the immunofluorescence staining results indicated the hAMSCs had high expression of the vimentin and low expression of CK-19; the hAMSCs possessed the differentiation ability into the osteoblasts, chondroblasts, and lipoblasts. The CCK-8 results displayed that cells reached the peak of growth curve at 7 days in each group, and the proliferation ability in the experimental group was significantly higher than that in the control group at 7 days (P<0.05). The immunofluorescence staining results showed that the expressions of collagen type I, collagen type III, Fibronectin, and Tenascin-C in the experimental group were significantly higher than those in the control group at 5, 10, and15 days after culture (P<0.05). The real-time fluorescence quantitative PCR results revealed that the mRNA relative expressions had an increasing tendency at varying degrees with time in the experimental group (P<0.05). The relative mRNA expressions of collagen type I, collagen type III, Fibronectin, α-SMA, and VEGF in the experimental group were significantly higher than those in the control group at the other time points (P<0.05), but no significant difference was found in the relative mRNA expressions of collagen type I, collagen type III, and VEGF between 2 groups at 5 days (P>0.05). Conclusion The hAMSCs possesses the characteristics of MSCs and good proliferation ability which could be chosen as seed cell source in tissue engineering. The expressions of ligament fibroblasts and angiogenesis related genes could be up-regulated, after inductionin vitro, and the synthesis of ligament fibroblasts related proteins could be strengthened. In addition, the application of TGF-β1 and VEGF could be used as growth factors sources in constructing tissue engineered ligament.