Systematic evaluation of combined herbal adjuvant therapy for proliferative diabetic retinopathy

Anti-VEGF Injections vs. Panretinal Photocoagulation Laser Therapy for Proliferative Diabetic Retinopathy: A Systematic Review and Meta-Analysis

Rationale and Application of the Protocol S Anti–Vascular Endothelial Growth Factor Algorithm for Proliferative Diabetic Retinopathy

To analyze the drug combination characteristics of Qingkailing injection for treating abnormal inflammatory factors such as elevated white blood cells and C reactive protein in real world. The patients with Qingkailing injection for abnormal C reactive proteins and abnormal white blood cells were extracted from hospital information system (HIS) of 16 Class 3A hospitals. Then the basic information, traditional Chinese medicine and Western medicine diagnostic information, doctor's advice information, and laboratory information were analyzed; Apriori algorithm was used to construct the models, and Clementine 12.0 was used for correlation analysis to analyze the clinical medication rules and drug combination characteristics in the patients with Qingkailing injection for treatment of elevated C reactive protein and white blood cells in the real world. The results of the study showed that when Qingkailing injection was combined with one kind of western medicine and traditional Chinese medicine in treatment of patients with abnormal C reactive protein, vitamin C (159 cases, 74.30%) and Tanreqing injection (71 cases, 33.18%) were most frequently used; when it was combined with 2 kinds of traditional Chinese medicines, Xueshuantong injection plus Tanreqing injection (support degree 10.75%) were most frequently used. When Qingkailing injection was combined with one kind of western medicine and traditional Chinese medicine in treatment of patients with abnormal white blood cells, vitamin C (596 cases, 56.02%) and Ganmao Qingre granules (247 cases, 23.21%) were most frequently used; when it was combined with 2 kinds of traditional Chinese medicines, Shuanghuanglian+Ganmao Qingre granules (support degree 5.26%) were most frequently used. In the patients with abnormal C-reactive protein and white blood cells, its combinations with antibiotics and nutritional support agents were most common from the pharmacological perspective, indicating that in the treatment of abnormal C-reactive protein, white blood cells and other increased inflammatory indicators, Qingkailing injection was most frequently combined with antibiotic drugs to achieve synergistic effect.

Proliferative diabetic retinopathy (PDR) represents a main cause of acquired blindness. Despite the recognition of the key role exerted by vascular endothelial growth factor (VEGF) in the pathogenesis of PDR, limitations to anti-VEGF therapies do exist. Thus, rapid and cost-effective angiogenesis assays are crucial for the screening of anti-angiogenic drug candidates for PDR therapy. In this context, evaluation of the angiogenic potential of PDR vitreous fluid may represent a valuable tool for preclinical assessment of angiostatic molecules. Here, vitreous fluid obtained from PDR patients after pars plana vitrectomy was used as a pro-angiogenic stimulus in a 3D endothelial cell spheroid/human vitreous assay. The results show that PDR vitreous is able to stimulate the sprouting of fibrin-embedded HUVEC spheroids in a time- and dose-dependent manner. A remarkable variability was observed among 40 individual vitreous fluid samples in terms of sprouting-inducing activity that was related, at least in part, to defined clinical features of the PDR patient. This activity was hampered by various extracellular and intracellular signaling pathway inhibitors, including the VEGF antagonist ranibizumab. When tested on 20 individual vitreous fluid samples, the inhibitory activity of ranibizumab ranged between 0 and 100% of the activity measured in the absence of the drug, reflecting a variable contribution of angiogenic mediators distinct from VEGF. In conclusion, the 3D endothelial cell spheroid/human vitreous assay represents a rapid and cost-effective experimental procedure suitable for the evaluation of the anti-angiogenic activity of novel extracellular and intracellular drug candidates, with possible implications for the therapy of PDR.

We analyzed the expression of ADAMTS proteinases ADAMTS-1, -2, -4, -5 and -13; their activating enzyme MMP-15; and the degradation products of proteoglycan substrates versican and biglycan in an ocular microenvironment of proliferative diabetic retinopathy (PDR) patients. Vitreous samples from PDR and nondiabetic patients, epiretinal fibrovascular membranes from PDR patients, rat retinas, retinal Müller glial cells and human retinal microvascular endothelial cells (HRMECs) were studied. The levels of ADAMTS proteinases and MMP-15 were increased in the vitreous from PDR patients. Both full-length and cleaved activation/degradation fragments of ADAMTS proteinases were identified. The amounts of versican and biglycan cleavage products were increased in vitreous from PDR patients. ADAMTS proteinases and MMP-15 were localized in endothelial cells, monocytes/macrophages and myofibroblasts in PDR membranes, and ADAMTS-4 was expressed in the highest number of stromal cells. The angiogenic activity of PDR membranes correlated significantly with levels of ADAMTS-1 and -4 cellular expression. ADAMTS proteinases and MMP-15 were expressed in rat retinas. ADAMTS-1 and -5 and MMP-15 levels were increased in diabetic rat retinas. HRMECs and Müller cells constitutively expressed ADAMTS proteinases but not MMP-15. The inhibition of NF-κB significantly attenuated the TNF-α-and-VEGF-induced upregulation of ADAMTS-1 and -4 in a culture medium of HRMECs and Müller cells. In conclusion, ADAMTS proteinases, MMP-15 and versican and biglycan cleavage products were increased in the ocular microenvironment of patients with PDR.

The purpose of this study was to investigate whether fibrocytes participate in formation of the fibrovascular membrane (FVM) in patients with proliferative diabetic retinopathy (PDR). Vitreous fluid and FVM samples were obtained during vitrectomy in patients with PDR. Samples from patients with macular hole or epiretinal membrane were used as controls. Vitreous fluid and FVM samples were subjected to immunohistochemical analysis. In addition, cells isolated from the vitreous fluid of PDR and control patients were cultured in serum-free medium. Fibrocytes were identified among these cells by morphological and immunohistochemical analyses. We examined the number of fibrocytes in PDR patients and control patients. Also, the concentrations of monocyte chemoattractant protein-1 (MCP-1), pentraxin3, and serum amyloid P (SAP) in vitreous fluid samples from PDR patients and control patients were determined by enzyme-linked immunosorbent assay. Fibrocytes were observed in the vitreous and FVM samples from PDR patients. Cells cultured from the vitreous samples of PDR patients were spindle shaped and expressed fibrocyte markers. TGF-β1 induced differentiation of these cells into myofibroblasts. The number of fibrocytes was higher in samples from PDR patients than in samples from control patient. The vitreous fluid concentration of MCP-1 was significantly higher in PDR patients than in controls and showed a significant positive correlation with the number of fibrocytes from the vitreous fluid. Vitreous fluid concentrations of pentraxin3 and SAP were also higher in PDR patients than in control patients. These findings indicate that fibrocytes may be involved in development of the FVM in PDR.

Background Compound Xueshuantong capsule (CXC) and Hexuemingmu tablet (HXMMT) are two important Chinese patent medicines (CPMs) frequently used to treat proliferative diabetic retinopathy (PDR), especially when complicated with vitreous hemorrhage (VH). However, a network pharmacology approach to understand the therapeutic mechanisms of these two CPMs in PDR has not been applied. Objective To identify differences in the active ingredients between CXC and HXMMT and to comparatively predict and further analyze the molecular targets shared by these CPMs and PDR. Materials and methods. The differentially expressed messenger RNAs (mRNAs) between normal retinal tissues in healthy individuals and active fibrovascular membranes in PDR patients were retrieved from the Gene Expression Omnibus database. The active ingredients of CXC and HXMMT and the targets of these ingredients were retrieved from the Traditional Chinese Medicine Systems Pharmacology database. The intersections of the CPM (CXC and HXMMT) targets and PDR targets were determined. Then, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed, and the ingredient-target networks, protein-protein interaction networks, and KEGG-target (KEGG-T) networks were constructed. Results CXC contains 4 herbs, and HXMMT contains 19. Radix salviae is the only herb common to both. CXC had 34 potential therapeutic targets in PDR, while HXMMT had these 34 and 10 additional targets. Both CPMs shared the following main processes: response to reactive oxygen species and oxidative stress, regulation of blood vessel diameter and size, vasoconstriction, smooth muscle contraction, hemostasis, and blood coagulation. The shared pathways included the AGE-RAGE signaling pathway in diabetic complications, TNF signaling pathway, relaxin signaling pathway, and IL-17 signaling pathway. Conclusions Both CXC and HXMMT include components effective at treating PDR and affect the following main processes: response to reactive oxygen species and oxidative stress, regulation of blood vessels, and blood coagulation. Radix salviae, the only herb common to both CPMs, contains many useful active ingredients. The PDR-CXC and PDR-HXMMT networks shared 34 common genes (RELA, HSPA8, HSP90AA, HSP90AB1, BRCA, EWSR1, CUL7, HNRNPU, MYC, CTNNB1, MDM2, YWHAZ, CDK2, AR, FN1, HUWE1, TP53, TUBB, EP300, GRB2, VCP, MCM2, EEF1A1, NTRK1, TRAF6, EGFR, PRKDC, SRC, HDAC5, APP, ESR1, AKT1, UBC, and COPS5), and the PDR-HXMMT network has 10 additional genes (RNF2, VNL, RPS27, COPS5, XPO1, PARP1, RACK1, YWHAB, and ITGA4). The top 5 pathways with the highest gene ratio in both networks were the AGE-RAGE signaling pathway in diabetic complications, TNF signaling pathway, relaxin signaling pathway, IL-17 signaling pathway, and focal adhesion. Additional pathways such as neuroactive ligand-receptor interaction, chemokine signaling pathway, and AMPK signaling pathway were enriched with HXMMT targets. Thus, HXMMT has more therapeutic targets shared by different active ingredients and more abundant gene functions than CXC, which may be two major reasons why HXMMT is more strongly recommended than CXC as an auxiliary treatment for new-onset VH secondary to PDR. However, the underlying mechanisms still need to be further explored.

Retinal neovascularization characterizes proliferative diabetic retinopathy (PDR). Pigment epithelium-derived factor (PEDF) has been shown to be a major antiangiogenic growth factor in the mammalian eye. PEDF expression is suppressed by hypoxia, and changes in PEDF have been correlated to the development of retinal neovascularization in animal models of hypoxic eye disease. However, whether this concept of a reduced angiogenesis inhibitor holds true in humans is as yet unclear. In this study, we analyzed the in vivo regulation of PEDF in patients with and without hypoxic eye disease. We used immunoblots to measure PEDF in ocular fluids obtained from 64 nondiabetic and diabetic patients. In addition, immunohistochemistry of PEDF was carried out in specimens of normal human retinas and retinas with various degrees of diabetic retinopathy. The PEDF concentrations in patients with PDR (P < 0.001) or extensive nondiabetic retinal neovascularization caused by retinal-vein occlusion (P < 0.001) were lower than in control patients. Levels of PEDF were replenished in PDR patients with previous retinal scatter photocoagulation compared with PDR patients without previous photocoagulation (P = 0.01). Immunohistochemistry revealed an interstitial staining pattern as expected for a secreted protein, with an intense staining in retinas of patients without proliferative eye disease. However, in patients with PDR, little or no staining was detectable. Our data strongly support the concept that retinal angiogenesis is induced by loss of the major angiogenesis inhibitor in the eye, PEDF, in combination with an increased expression of angiogenic growth factors such as vascular endothelial growth factor. Our findings suggest that substitution of angiogenesis inhibitors may be an effective approach in the treatment of PDR.

In proliferative diabetic retinopathy (PDR), retinal ischemia promotes neovascularization (NV), which can lead to profound vision loss in diabetic patients. Treatment for PDR, panretinal photocoagulation, is inherently destructive and has significant visual consequences. Therapies targeting vascular endothelial growth factor (VEGF) have transformed the treatment of diabetic eye disease but have proven inadequate for treating NV, prompting exploration for additional therapeutic options for PDR patients. In this regard, extracellular proteolysis is an early and sustained activity strictly required for NV. Extracellular proteolysis in NV is facilitated by the dysregulated activity of matrix metalloproteinases (MMPs). Here, we set out to better understand the regulation of MMPs by ischemia in PDR. We demonstrate that accumulation of hypoxia-inducible factor-1α in Müller cells induces the expression of VEGF, which, in turn, promotes increased MMP-2 expression and activity in neighboring endothelial cells (ECs). MMP-2 expression was detected in ECs in retinal NV tissue from PDR patients, whereas MMP-2 protein levels were elevated in the aqueous of PDR patients compared with controls. Our findings demonstrate a complex interplay among hypoxic Müller cells, secreted angiogenic factors, and neighboring ECs in the regulation of MMP-2 in retinal NV and identify MMP-2 as a target for the treatment of PDR.

BackgroundIt’s necessary to analyze the role of VEGF, apelin, and HO-1 in patients with type 2 diabetes (T2DM), and to evaluate its relevance to diabetic retinopathy (DR).MethodsT2DM patients who were treated in our hospital from December 1, 2018 to November 30, 2019 were included. T2DM patients were divided into non-DR (NDR) group, non-proliferative DR (NPDR) group, and proliferative DR (PDR) group. and healthy participants were selected as the control group. The value of VEGF, apelin, and HO1 in predicting PDR were analyzed by receiver operating characteristic (ROC) curve, and the relations of VEGF, apelin, HO-1 and clinical factors in PDR patients were analyzed by Pearson correlation analysis.ResultsA total of 295 participants were included. The level of FPG and HbAlc in PDR group were significantly higher than that of other groups (all p < 0.05); the level of VEGF and apelin in PDR group were significantly higher than that of other groups (all p < 0.05), but the level of HO-1 in PDR group were significantly less than that of other groups(p = 0.017); the AUC of VEGF, apelin, HO-1 and combined use was 0.806(95%CI: 0.779–0.861), 0.819(95%CI: 0.765–0.878), 0.808(95%CI: 0.733–0.869) and 0.902(95%CI: 0.822–0.958) respectively, the AUC, sensitivity, specificity of the three combined use was significantly higher than that of single VEGF, apelin, HO-1 use(all p < 0.05). The cutoff values of serum VEGF, apelin, and HO-1 levels for predicting PDR were 163.85 pg/ml, 8.27 ng/ml, and 26.06 mmol/L respectively. Serum VEGF, apelin, and HO-1 in patients with PDR was related to the time course of DM, FPG and HbAlc (all p < 0.05).ConclusionsVEGF, apelin and HO-1 are related to the progress of DR, and the combined use of VEGF, apelin and HO-1 is beneficial to the diagnosis and treatment of PDR.

Background Vitrectomy is a primary approach to the treatment of proliferative diabetic retinopathy (PDR). However, postoperative rehaemorrhagia often occur.Ranibizumab is an effective drug of anti-vascular endothelial growth factor, but whether the combination of intravitreal injection of ranibizumab with vitrectomy can reduce the incidence of postoperative rehaemorrhagia in PDR patients is still unclear. Objective This study was to investigate the preventive effect of the combination of intravitreal injection of ranibizumab with 25-gauge vitrectomy on postoperative rehaemorrhagia. Methods A retrospective cohort study was perfomed.The clinical data of Ⅴ-Ⅵ stage of PDR patients who received 25-gauge vitrectomy in Peking University International Hospital from January 2014 to July 2015 were collected and analyzed.The PDR patients were divided into only surgery group and drug with surgery group.The patients in the only surgery group (34 eyes of 49 patients ) received 25-gauge vitrectomy, and the patients in the drug with surgery group (32 eyes of 25 patients) received the intravitreal injection of ranibizumab 0.05 ml (0.5 mg) 7 days before 25-gauge vitrectomy.The distribution of eye number in different grades of visual acuities was observed and compared between the two groups in 1 day, 1 week, 1 month and 3 months after surgery, and the incidence of rehaemorrhagia was intergrouply compared in 1 day, 3-7 days and 1 month after surgery. Results The eye number and percentage of the different visual acuities in the drug with surgery group was not significantly different from the only surgery group in 1 day, 1 week, 1 month and 3 months after surgery (1 day: Z=0.673, P=0.412; 1 week: Z=0.113, P=0.737; 1 month: Z=1.755, P=0.185; 3 months: Z=2.474, P=0.116). Rehaemorrhagia occurred in postoperative day 1 and day 3-7 was 1 eye and 1 eye respectively in the drug with surgery group, and that in the only surgery group was 9 eyes and 9 eyes respectively, showing significant difference between the two groups (all at P<0.05). The eye number of rehaemorrhagia in postoperative 1 month was 4 in the only surgery group, and no rehaemorrhagia appeared in the drug with surgery group. Conclusions The combination of intravitreal injection of ranibizumab with 25-gauge vitrectomy can efficiently reduce the incidence of postoperative rehaemorrhagia. Key words: Diabetic retinopathy/urgery; Diabetic retinopathy/drug therapy; Monoclonal antibodies, humanized/ therapeutic use; Ranibizumab; Intravitreal injection; Vitreactomy; Vitreous hemorrhage/prevention & control; Humans

ABSTRACTPurpose: We investigated the expression of the proinflammatory and proangiogenic factor osteoprotegerin (OPG) and its ligands, receptor activator of nuclear factor-κB ligand (RANKL), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and the receptor RANK in proliferative diabetic retinopathy (PDR).Materials and methods: Vitreous samples from PDR and nondiabetic control patients and epiretinal membranes from PDR patients were studied by enzyme-linked immunosorbent assay, immunohistochemistry, and Western blot analysis.Results: Vascular endothelial growth factor, OPG, and soluble RANK levels in vitreous samples from PDR patients were significantly higher than that in nondiabetic controls. Soluble TRAIL levels were significantly lower in PDR patients than that in nondiabetic control, whereas soluble RANKL levels did not differ significantly. RANKL, RANK, and TRAIL were expressed in vascular endothelial cells, myofibroblasts, and CD45-expressing leukocytes in PDR epiretinal membranes.Conclusions: Dysregulated expression of OPG/RANKL/RANK pathway and TRAIL might be related to inflammation and angiogenesis in PDR.

BackgroundDespite great advances in proliferative diabetic retinopathy (PDR) therapy over the last decades, one third of treated patients continue to lose vision. While resident vitreous macrophages called hyalocytes have been implicated in the pathophysiology of vitreoretinal proliferative disease previously, little is known about their exact role in PDR. In this study, we address molecular and cellular alterations in the vitreous of PDR patients as a means towards assessing the potential contribution of hyalocytes to disease pathogenesis.ResultsA total of 55 patients were included in this study encompassing RNA-Sequencing analysis of hyalocytes isolated from the vitreous of PDR and control patients, multiplex immunoassay and ELISA analyses of vitreous samples from PDR and control patients, as well as isolation and immunohistochemical staining of cultured porcine hyalocytes. Transcriptional analysis revealed an enhanced inflammatory response of hyalocytes contributing to the cytokine pool within the vitreous of PDR patients by expressing interleukin-6, among others. Further, increased angiopoietin-2 expression indicated that hyalocytes from PDR patients undergo a proangiogenic shift and may thus mediate the formation of retinal neovascularizations, the hallmark of PDR. Finally, RNA-Sequencing revealed an upregulation of factors known from hemoglobin catabolism in hyalocytes from PDR patients. By immunohistochemistry, cultured porcine hyalocytes exposed to red blood cells were shown to engulf and phagocytose these, which reveals hyalocytes’ potential to dispose of erythrocytes. Thus, our data suggest a potential role for vitreous macrophages in erythrophagocytosis and, thereby, clearance of vitreous hemorrhage, a severe complication of PDR.ConclusionOur results strongly indicate a critical role for vitreous hyalocytes in key pathophysiological processes of proliferative diabetic retinopathy: inflammation, angiomodulation and erythrophagocytosis. Immunomodulation of hyalocytes may thus prove an essential novel therapeutic approach in diabetic vitreoretinal disease.

Objective To compare the outcomes of 23G and 25G plus (25G+) vitrectomy in treatment of proliferative diabetic retinopathy (PDR).Methods This is a prospective randomized study.Fifty-seven PDR patients (75 eyes) with symptoms requiring vitrectomy were randomly divided into 23G vitrectomy group (30 patients,39 eyes) and 25G+ vitrectomy group (27 patients,36 eyes).Visual acuity,intraocular pressures,ophthalmoscopy,B-scan ultrasound was examined before surgery.The follow-up period was 10.0 (23G group) and 8.5 months (25G+ group) respectively.Intraoperative complications,operation time,postoperative visual acuity,intraocular pressure,postoperative complications and postoperative ocular conditions were analyzed.Results The mean surgical times were (53.35± 7.42)minutes and (49.16±5.17) minutes in 23G and 25G+ group respectively,and the difference was significant (t=4.37,P＜0.05).Iatrogenic injuries occurred in 11 eyes (28.21％) and 5 (13.89％) eyes in 23G and 25G + group respectively,and the difference was significant (x2 =4.93,P＜0.05).The postoperative visual acuity of 23G and 25G+ group were improved compared to before surgery (x2=16.81,18.29; P＜0.05).At last follow-up,there was 25 eyes and 24 eyes with visual acuity ≥ 0.05 in 23G and 25G+ groups respectively,and the difference was not significant (x2 =0.13,P＞0.05).Hypotony was detected in 7 and 3 eyes at the third postoperative day in 23G and 25G+ group respectively,and the difference was significant (x2 =5.67,P＜0.05).Conclusion 25G+ vitrectomy is a safe and effective treatment for PDR with shorter surgery time and fewer surgical complications. Key words: Diabetic retinopathy/surgery; Surgical procedures, minimally invasive; Vitrectomy

The activated form of gelatinase B/matrix metalloproteinase-9 is associated with diabetic vitreous hemorrhage