Objective To study the time effect of the gene expression of recombinant adeno-associated virus (rAAV) vector co-expressing human vascular endothel ial growth factor 165 (hVEGF165) and human bone morphogenetic protein 7 (hBMP-7) genes so as to lay a theoretical foundation for gene therapy of osteonecrosis. Methods The best multipl icity of infection (MOI) of BMSCs transfected with rAAV was detected by fluorescent cell counting. The 3rd generation rabbit bone mesenchymal stem cells (BMSCs) were transfected with rAAV-hVEGF165-internal ribosome entry site (IRES)-hBMP-7 (experimental group) and green fluorescent protein (GFP) labeled rAAV-IRES-GFP (control group), respectively. The expression of GFP was observed by inverted fluorescent microscope. The expressions of hVEGF165 and hBMP-7 were assessed by RT-PCR assay and Western blot assay in vitro. The transfected cells in 2 groups were prepared into suspension with 5 × 106 cells/mL, and injected into the rabbit thigh muscles of experimental group 1 (n=9) and control group 1 (n=9), respectively. The muscle injected with rAAV-IRES-GFP was sl iced by frozen section method and the expression of GFP protein was observed by inverted fluorescent microscope. The expressions of hVEGF165 and hBMP-7 were assessed by Western blot assay and ELISA assay in vivo. Results The best MOI of BMSCs transfected with rAAV was 5 × 104 v.g/cell. In vitro, the expressions of GFP, hVEGF165, and hBMP-7 genes started at 1 day after transfection, the expressions obviously increased at 14 days after transfection, and the expression maintained the b level at 28 days after transfection. In vivo, the expressions of GFP, hVEGF165, and hBMP-7 genes could be detected at 2 weeks after injection, and b expressions were shown at 6 to 8 weeks after injection. The values of hVEGF165 and hBMP-7 were (248.67 ± 75.58) pg/mL and (4.80 ± 0.61) ng/mL respectively in experimental group 1, and were (32.28 ± 8.42) pg/mL and (0.64 ± 0.42) ng/mL respectively in control group 1; showing significant differences between 2 groups (P lt; 0.05). Conclusion The rAAV-hVEGF165-IRES-hBMP-7 has efficient gene expression ability.
ObjectiveTo observe changes in peripapillary blood flow before and after combined treatment with anti-vascular endothelial growth factor (VEGF) drugs and Dexamethasone intravitreal implant (DEX) in patients with central retinal vein occlusion (CRVO). MethodsA prospective clinical study. Thirty-three eyes of 33 patients with newly diagnosed non-ischemic CRVO and macular edema (ME) were enrolled from Shanxi Eye Hospital between April 2023 and April 2024. All patients underwent best-corrected visual acuity (BCVA) and swept-source optical coherence tomography angiography (SS-OCTA) examinations. The treatment regimen consisted of three intravitreal injections of ranibizumab and one DEX implant. SS-OCTA was used to scan a 3 mm×3 mm area centered on the optic disc to measure peripapillary retinal nerve fiber layer (RNFL) thickness and blood flow density in the superficial vascular complex (SVC), deep vascular complex (DVC), and radial peripapillary capillaries (RPC). Changes in SVC, DVC, and RPC blood flow density and RNFL thickness were evaluated at 3 and 6 months post-treatment. Shapiro-Wilk test was used to assess normality, and Spearman's rank correlation coefficient was applied for correlation analysis. ResultsCompared with before treatment, the blood flow density changes of SVC and RPC showed a downward trend at 3 and 6 months after treatment. Among them, the difference was statistically significant at 6 months after treatment (Z=?2.592, ?2.070, P=0.012, 0.042), while there was no statistically significant difference at 3 months after treatment (P＞0.05). The blood flow density of DVC showed an upward trend at 3 and 6 months after treatment, but the differences were not statistically significant (P＞0.05). The results of the correlation analysis showed that the thickness of RNFL was negatively correlated with the blood flow density of DVC (r=?0.768, P＜0.001). It was positively correlated with the blood flow densities of SVC and RPC (r=0.288, 0.398; P=0.040, 0.004). ConclusionAnti-VEGF drugs combined with DEX treatment can significantly improve the perioptic disc blood flow distribution in eyes with CRVO, manifested as a decrease in blood flow density of SVC and RPC, while a compensatory increase in blood flow of DVC. The thickness variation of RNFL is closely related to the blood flow density of different vascular layers.
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 explore the effect of vascular endothelial growth factor 165 (VEGF165)-loaded porous poly (ε-caprolactone) (PCL) scaffolds on the osteogenic differentiation of adipose-derived stem cells (ADSCs).MethodsThe VEGF165-loaded porous PCL scaffolds (written, Sf-g/VEGF) were fabricated through a combination of solvent casting/salt leaching and a thermal-induced phase separation technique and then observed under scanning electron microscope (SEM). The release kinetics was determined by ELISA kit. The ADSCs were isolated from inguinal fat pads of 15 Sprague Dawley rats and cultured. The passage 3-4 ADSCs were seeded into the scaffolds, and then cultured in vitro for 7 days. The passage 3-4 ADSCs were seeded into the porous PCL scaffolds (written, Sf-g) as control. The alizarin red S (ARS) staining, ARS activity assay, and real-time quantitative PCR (RT-PCR) were performed to measure the osteogenic differentiation of ADSCs in vitro. Six Sprague Dawley rats were recruited to prepare the bilateral calvarial bone defects models (n=12). The 12 calvarial bone defects were randomly divided into 3 group (n=4). The defects of negative control group were not treated; the defects of Sf-g group and Sf-g/VEGF group were repaired with ADSCs-Sf-g scaffold complex and ADSCs-Sf-g scaffold complex, respectively. At 8 weeks after transplantation, the Micro-CT and HE staining were conducted to evaluate the osteogenic effects in vivo.ResultsThe morphology of the Sf-g/VEGF scaffolds were porous and well-connected, and the cumulative release rate was approximately 80% in 120 hours. The ARS staining showed that the ARS activity of Sf-g/VEGF group were stronger than that of Sf-g group (t=10.761, P=0.000). The mRNA expressions of osteogenic specific markers [special AT-rich sequence protein 2 (Satb2), alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN)] were significantly higher in Sf-g/VEGF group than in Sf-g group (P<0.05). The results of Micro-CT and HE staining also confirmed the promotion effect of Sf-g/VEGF scaffolds. All defects of 2 groups were partially repaired by new bone tissue, especially in Sf-g/VEGF group. The volume and area of new bone tissue were significantly higher in Sf-g/VEGF group than in Sf-g group (P<0.05).ConclusionThe VEGF165-loaded scaffolds can significantly improve the osteogenic differentiation of ADSCs both in vitro and in vivo.
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 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 investigate the effects of cediranib on hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway and proliferation, migration and invasion of liver cancer cells. Methods The hypoxia microenvironment was simulated in vitro, and different doses of cediranib were used to intervene the human hepatoma cell HepG2, MTT assay was used to detect the proliferation of human hepatoma cell HepG2, Transwell chamber assay was used to detect the invasion and migration of human hepatoma cell HepG2, tumor formation in nude mice was used to detect the growth of human hepatoma cell HepG2 in vivo, the angiogenesis of tumor tissue and expression level of HIF-1α/VEGF pathway protein were detected by immunohistochemistry. Results Compared with the control group, the proliferation rate, invasion and migration abilities, and the expression of HIF-1α/VEGF pathway proteins of human hepatoma cell HepG2 were significantly decreased in the different concentration of cediranib treatment group (P<0.05), the tumor volume and microvessel formation of tumor tissues in nude mice were significantly reduced (P<0.05). Conclusion Cediranib may inhibit the proliferation, migration and invasion of liver cancer cells by inhibiting HIF-1α/VEGF signaling pathway.
ObjectiveTo observe the long-term effects of anti-vascular endothelial growth factor (VEGF) drug initiation combined with dexamethasone intravitreal implant (DEX) on the structural integrity of the outer macular region of the eye in patients with macular edema (ME) secondary to central retinal vein occlusion (CRVO). MethodsA retrospective clinical study. From February 2018 to August 2022, 54 patients diagnosed with CRVO combined with ME (CRVO-ME) in Department of Ophthalmology of Central Theater Command General Hospital were included in the study. Among them, there were 30 males and 24 females, all with monocular disease. According to different treatment regiments, patients were divided into anti-VEGF and DEX combination therapy group (initial combination group), anti-VEGF drug monotherapy group (monotherapy group) with 21 eyes and 33 eyes, respectively. Best corrected visual acuity (BCVA), optical coherence tomography (OCT) examination were performed in all eyes. The thickness of foveal retina (CRT) and the deficiency length of outer membrane (ELM), ellipsoid band (EZ) and chimaera band (IZ) in the 1 mm macular area were measured by OCT. The initiating combination group was treated with anti-VEGF agents or DEX as assessed on demand (PRN) after the combination therapy, and the monotherapy group received 3+PRN regimen. Relevant examinations were performed at 1 (V1), 6 (V6), 12 (V12) months and observation cut-off or the last visit (Vf) after treatment using the same equipment before treatment. The deletion length of ELM, EZ and IZ in V1, V6, V12 and Vf after treatment were compared between the two groups. Repeated measurement ANOVA was used to compare BCVA, CRT and deletion length of ELM, EZ and IZ at different follow-up times. Spearman rank correlation test was used to analyze the correlation between the two groups of continuous variables. ResultsThe follow-up time of patients in the initial combination group and monotherapy group was (18.05±5.66) and (21.90±10.80) months, respectively, with no statistical significance (F=13.430, P=0.229). Compared with baseline, the deletion lengths of ELM, EZ and IZ were significantly improved (F=11.848, 10.880, 29.236), BCVA was increased (F=10.541) and CRT was decreased (F=52.278) in the initial combination group and the monotherapy group at different follow-up times after treatment. The differences were statistically significant (P＜0.001). At V1, EZ and IZ deletion lengths were (344.10±413.03), (593.33±372.96) μm and (354.71±321.75), (604.85±385.77) μm in the initial combination group and monotherapy group, respectively. The improvement of EZ and IZ deletion lengths in the initial combination group was better than that in the single drug group, and the difference was statistically significant (F=5.272, 6.106; P=0.026, 0.017). The CRT of the initial combination group and the monotherapy group were (248.86±59.99) and (314.72±214.91) μm, respectively, and the CRT of the initial combination group was significantly lower than that of the monotherapy group, with statistical significance (F=6.102, P=0.017). At V6, V12 and Vf, the deletion length of ELM, EZ and IZ and BCVA and CRT showed no statistical significance (P＞0.05). Correlation analysis showed that ELM, EZ, IZ were positively correlated with BCVA and CRT in the initial combination group and monotherapy group (P＜0.001). In V6, V12 and Vf, the number of anti-VEGF drug injections in the initial combination group and monotherapy group was (2.67±1.32), (4.43±2.27), (6.05±3.51), (4.58±0.90), (7.33±1.93), (11.33±6.10) times, respectively. The number of injections in the initial combination group was significantly lower than that in the monotherapy group, and the difference was statistically significant (F=5.150, 0.646, 3.433; P＜0.001). ConclusionsThe improvement of BCVA and CRT in the initial combination group is similar to that in the monotherapy group. Compared with the monotherapy group, EZ and IZ deletion are improved more significantly in the initial combination group, and CRT decreased more rapidly and significantly. The initial combination group receives fewer anti-VEGF injections than the monocular group.
ObjectiveTo observe the efficacy and safety of vitrectomy combined with subretinal injection of alteplase (tPA) and intravitreal injection of Conbercept in the treatment of large area submacular hemorrhage (SMH) secondary to polypoidal choroidal vasculopathy (PCV). MethodsA retrospective clinical study. From January to September 2021, 32 eyes of 32 patients with massive SMH secondary to PCV diagnosed in the Affiliated Eye Hospital of Nanchang University were included in the study. Large SMH was defined as hemorrhage diameter ≥4 optic disc diameter (DD). There were 32 patients (32 eyes), 20 males and 12 females. The mean age was (72.36±8.62) years. All patients had unilateral disease.The duration from onset of symptoms to treatment was (7.21±3.36) days. All patients underwent best corrected visual acuity (BCVA) and optical coherence tomography (OCT) examination. BCVA examination was performed using the international standard visual acuity chart, which was converted to the logarithm of the minimum angle of resolution (logMAR) visual acuity during statistics. The central macular thickness (CMT) was measured by spectral domain-OCT. The average size of SMH was (6.82±1.53) DD. The logMAR BCVA 1.73±0.44; CMT was (727.96±236.40) μm. All patients were treated with 23G pars plana vitrectomy combined with subretinal injection of tPA and intravitreal injection of Conbercept. At 1, 3, 6 and 12 months after treatment, the same equipment and methods were used for relevant examinations before treatment. The changes of BCVA and CMT, the clearance rate of macular hemorrhage, and the complications during and after surgery were observed. BCVA and CMT before and after treatment were compared by repeated measures analysis of variance. ResultsCompared with before treatment, BCVA gradually increased at 1, 3, 6 and 12 months after treatment, and the differences were statistically significant (F=77.402, P＜0.001). There was no significant difference in BCVA between any two groups at different time points after treatment (P＞0.05). Correlation analysis showed that BCVA at 12 months after treatment was negatively correlated with the course of disease (r=－0.053, P=0.774). One week after treatment, macular hemorrhage was completely cleared in 30 eyes (93.75%, 30/32). The CMT was (458.56±246.21), (356.18±261.46), (345.82±212.38) and (334.64±165.54) μm at 1, 3, 6 and 12 months after treatment, respectively. Compared with before treatment, CMT decreased gradually after treatment, and the difference was statistically significant (F=112.480, P＜0.001). There were statistically significant differences in different follow-up time before and after treatment (P＜0.001). The number of treatments combined with Conbercept during and after surgery was (4.2±1.8) times. At the last follow-up, there was no recurrence of SMH, retinal interlamellar effusion and other complications. Conclusion Subretinal injection of tPA combined with intravitreal injection of Conbercept is safe and effective in the treatment of large SMH secondary to PCV, and it can significantly improve the visual acuity of patients.
