Evidence that the Axis Comprised of TSP1-TGFβ-CTGF Plays a Major Role In the Pathophysiology of Diabetic Retinopathy:Regulatory Role Of IL-4 and IL-13

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4720-4720
Author(s):  
Jaime L Loyola ◽  
Melissa R Lester ◽  
Fabiola E Del Carpio-Cano ◽  
Mario C Rico ◽  
William Foster ◽  
...  
Keyword(s):  
Diabetic Retinopathy ◽  
Proliferative Diabetic Retinopathy ◽  
Transcriptional Activation ◽  
Conflicts Of Interest ◽  
Rolling Circle Amplification ◽  
Protein Profiling ◽  
Control Group ◽  
Rolling Circle ◽  
Mechanistic Pathway

Introduction Diabetic retinopathy is the most severe of several ocular complications of diabetes. Diabetes afflicts approximately 6% of the US population, thus retinopathy remains an important problem (PMID: 14702427). Our laboratory has provided evidence of the significant role of a pro-inflammatory axis during chronic inflammation (PMID: 19485899). The axis is comprised of TSP1-TGFβ-CTGF. We now provide evidence of an additional player within the axis, namely IL-4. A series of biochemical measurements were used to assess the role of the above mentioned axis in the progression of disease, from non-proliferative diabetic retinopathy (NPDR) to proliferative diabetic retinopathy (PDR). Methods Institutionally approved IRB and informed consent allowed this prospective study to recruit a total of 40 individuals from a vulnerable population disproportionately afflicted by this disease and representative of the population that we served in North Philadelphia with type-2 diabetes. Western-blotting technique was utilized to document the presence of fragments from CTGF in plasma, which have been shown in vitreous fluid to be angiogenic (PMID: 14988298). Multiplexed protein profiling on microarrays by rolling-circle amplification was employed to determine IL-4, IL-13, and MIP-1β levels. Commercially available ELISA was the method to determine the plasma concentration of TSP1, TGFβ and CTGF. Results and Discussion TSP1 (p<0.002), TGFβ (p<0.001), CTGF (p<0.05), IL4 (p<0.0002), MIP-1β (p<0.05) but not IL-13 were found significantly elevated in NPDR patients when compared to PDR patients or control group. Fragments of CTGF were more abundant in patients with PDR when compared to NPDR or the control group. Interestingly, the systemic plasma circulation of CTGF during the NPDR phase (elevated) may explain the subsequent vitreous accumulation of CTGF fragments described in the literature during the PDR phase apparently with angiogenic and fibrotic properties. Furthermore, IL-4 regulates CTGF (PMID:11967989) by interfering with TGFβ-induced transcriptional activation of the CTGF gene (blocking 50% of the SMAD pathway). Thus, the elevated levels during the NPDR phase may be a protective response to prevent CTGF synthesis and secretion. The steady-state levels of IL-13 (normal control group) where significantly higher when compared to the NPDR or PDR group with a trend for higher levels of IL-13 in the NPDR group. IL-13 is anti-angiogenic and thus its progressive decrease as a function of length of disease is of interest. Mean H1ac levels were not significantly different in the NPDR patients (8.0 ± 3.5) when compared with PDR patients (8.3 ± 2.2). In summary, our data suggests that the pro-inflammatory axis described above, now including IL-4 and IL-13, overall, plays a major role in the pathophysiology of diabetic retinopathy. The mechanistic pathway is currently under investigation in our laboratory. Disclosures: No relevant conflicts of interest to declare.

Association Between a Pro-Inflammatory Axis Comprised By TSP1-TGFβ-CTGF with Three microRNAs, Mir-19a, miR122 and Mir-133a in the Pathophysiology of Diabetic Retinopathy: A Therapeutic Novel Modality

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2771-2771
Author(s):  
Raul A Dela Cadena ◽  
Melissa Lester ◽  
Fabiola Del Carpio-Cano ◽  
Alina Shevchenko ◽  
John St. Angelo ◽  
...  
Keyword(s):  
Diabetic Retinopathy ◽  
Conflicts Of Interest ◽  
Rolling Circle Amplification ◽  
Active Form ◽  
Protein Profiling ◽  
Differentially Expressed ◽  
Diabetes Clinic ◽  
Alcoholic Fatty Liver ◽  
Rolling Circle ◽  
Potential Biomarker

Abstract Introduction: Diabetic retinopathy (DR) is a severe ocular complication of type 2 diabetes (T2DM). Our laboratory has been studying the role of a pro-inflammatory axis comprised by TSP1-TGFb and CTGF in T2DM. The axis is differentially expressed in patients with T2DM and non-proliferative DR (NPDR) when compared with patients with T2DM and proliferative DR (PDR). The differential expression of such axis is accompanied by interesting cytokine profiles as well. The purpose of this study was to evaluate potential microRNAs associated with the above changes in order to understand better the pathophysiology of T2DM. Recent studies indicate that miRNAs transported to exosomes or HDL can be transferred in an active form to recipients cells potentially involving them in cell-to-cell communication. Methods: Institutional approved IRB and informed consent allowed this prospective study to recruit a total of 40 individuals afflicted by T2DM (n=20 NPDR and n=20 PDR) from the Ophthalmology Department Diabetes Clinic at Temple Hospital, normal controls (n=20) were recruited from the Sol Sherry Thrombosis Research Center at Temple. Western-blotting technique was utilized to document the presence of fragments from CTGF in plasma, which have been documented in vitreous fluid to be angiogenic. Multiplexed protein profiling on microarrays by rolling-circle amplification was employed to determine IL-4 and MIP-1b. Commercially available ELISA was the method to determine the plasma concentration of TSP1, TGFb and CTGF. Total RNA was extracted from human plasma using the miRCURY TM RNA isolation kit by Exiqon at their facility in Denmark. Results and Discussion: TSP1, TGFb, CTGF, IL4, MIP-1b were significantly elevated in NPDR patients when compared to PDR patients. Fragments of CTGF were identified more abundant in patients with PDR when compared to NPDR. Only three microRNAs were differentially expressed in the normal group when compared with either NPDR (decreased), namely miR-133a (p<0.005), miR-19a (p<0.008), miR-122 (p<0.02) or PDR (decreased), miR-19a p<0.000096) and miR-122 (p<0.005). miR19a has been implicated with expression of TSP1 and CTGF, glucose levels, insulin resistance and diabetes-associated pancreatic cancer. miR-133a has been associated with cardiac hyperthrophy, fibrosis and heart failure in diabetes. miR-122 has been identified as a potential biomarker for non-alcoholic fatty liver. In summary our results link for the first time in the literature three circulating microRNAs with a the above axis in the pathophysiology of NPDR and PDR. Disclosures No relevant conflicts of interest to declare.

The Level of Cytokines in the Vitreous Body of Severe Proliferative Diabetic Retinopathy Patients Undergoing Posterior Vitrectomy

2018 ◽  
Vol 24 (27) ◽  
pp. 3276-3281 ◽  
Author(s):  
Dorota Raczyńska ◽  
Katarzyna A. Lisowska ◽  
Krzysztof Pietruczuk ◽  
Joanna Borucka ◽  
Mateusz Ślizień ◽  
...  
Keyword(s):  
Diabetic Retinopathy ◽  
Proliferative Diabetic Retinopathy ◽  
Vitreous Body ◽  
Control Group ◽  
Slit Lamp ◽  
Corrected Visual Acuity ◽  
Cytokine Levels ◽  
Pars Plana ◽  
Eye Examination

Objective: The objective of the study was to compare cytokine levels in the vitreous body of patients with proliferative diabetic retinopathy (PDR) undergoing posterior vitrectomy. Patients and methods: The study included 39 patients (39 eyes) undergoing pars plana vitrectomy (PPV). Patients were divided into three groups: patients with proliferative diabetic retinopathy (PDR) without aflibercept injection prior to the surgery, PDR patients administered aflibercept injection prior to the surgery, and patients without diabetes mellitus (control group). All patients underwent a comprehensive eye examination one day before and 3 weeks after the surgery, including measurements of: best-corrected visual acuity (BVCA) and intraocular pressure (IOP), slit-lamp examination and spectral domain optical coherence tomography (SOCT). Concentrations of cytokines: IL-6, IL-8, IL-12p70, TNF, IL-10, IL-1β were measured in the vitreous body of patients with BD™ Cytometric Bead Array (CBA) Human Inflammatory Cytokines Kit. Results: PDR patients who received pretreatment with aflibercept injection showed significantly lower concentrations of IL-12p70, TNF, IL-10 and IL-1β in the vitreous body compared to the control group. Meanwhile, patients without prior aflibercept injection had a significantly higher concentration of IL-8. There was also a significant positive correlation between IOP before PPV and IL-8 concentration in both PDR patients’ groups. Conclusion: Findings of our study suggest an important role of IL-8 in the development of severe PDR. Aflibercept administration on the day before elective vitrectomy facilitated the surgery.

Proteomic Analysis of the Vitreous Body in Proliferative and Non-Proliferative Diabetic Retinopathy

2020 ◽  
Vol 17 ◽  
Author(s):  
Van-An Duong ◽  
Jeeyun Ahn ◽  
Na-Young Han ◽  
Jong-Moon Park ◽  
Jeong-Hun Mok ◽  
...  
Keyword(s):  
Diabetic Retinopathy ◽  
Proliferative Diabetic Retinopathy ◽  
Microvascular Complications ◽  
Treatment Options ◽  
Vitreous Body ◽  
Control Group ◽  
Diabetic Patients ◽  
Protein Protein Interaction ◽  
Alpha Beta ◽  
New Treatment

Background: Diabetic Retinopathy (DR), one of the major microvascular complications commonly occurring in diabetic patients, can be classified into Proliferative Diabetic Retinopathy (PDR) and Non-Proliferative Diabetic Retinopathy (NPDR). Currently available therapies are only targeted for later stages of the disease in which some pathologic changes may be irreversible. Thus, there is a need to develop new treatment options for earlier stages of DR through revealing pathological mechanisms of PDR and NPDR. Objective: The purpose of this study was to characterize proteomes of diabetic through quantitative analysis of PDR and NPDR. Methods: Vitreous body was collected from three groups: control (non-diabetes mellitus), NPDR, and PDR. Vitreous proteins were digested to peptide mixtures and analyzed using LC-MS/MS. MaxQuant was used to search against the database and statistical analyses were performed using Perseus. Gene ontology analysis, related-disease identification, and protein-protein interaction were performed using the differential expressed proteins. Results: Twenty proteins were identified as critical in PDR and NPDR. The NPDR group showed different expressions of kininogen-1, serotransferrin, ribonuclease pancreatic, osteopontin, keratin type II cytoskeletal 2 epidermal, and transthyretin. Also, prothrombin, signal transducer and activator of transcription 4, hemoglobin subunit alpha, beta, and delta were particularly up-regulated proteins for PDR group. The up-regulated proteins related to complement and coagulation cascades. Statherin was down-regulated in PDR and NPDR compared with the control group. Transthyretin was the unique protein that increased its abundance in NPDR compared with the PDR and control group. Conclusion: This study confirmed the different expressions of some proteins in PDR and NPDR. Additionally, we revealed uniquely expressed proteins of PDR and NPDR, which would be differential biomarkers: prothrombin, alpha-2-HS-glycoprotein, hemoglobin subunit alpha, beta, and transthyretin.

scholarly journals Identification of Diagnostic and Prognostic microRNAs for Recurrent Vitreous Hemorrhage in Patients with Proliferative Diabetic Retinopathy

2019 ◽  
Vol 8 (12) ◽  
pp. 2217 ◽  
Author(s):  
Parviz Mammadzada ◽  
Juliette Bayle ◽  
Johann Gudmundsson ◽  
Anders Kvanta ◽  
Helder André
Keyword(s):  
Diabetic Retinopathy ◽  
Proliferative Diabetic Retinopathy ◽  
Vitreous Hemorrhage ◽  
Control Group ◽  
Ocular Tissues ◽  
Recurrent Hemorrhage ◽  
Pars Plana ◽  
And Control ◽  
Insight Into ◽  
Recurrent Vitreous Hemorrhage

MicroRNAs (miRNAs) can provide insight into the pathophysiological states of ocular tissues such as proliferative diabetic retinopathy (PDR). In this study, differences in miRNA expression in vitreous from PDR patients with and without incidence of recurrent vitreous hemorrhage (RVH) after the initial pars-plana vitrectomy (PPV) were analyzed, with the aim of identifying biomarkers for RVH. Fifty-four consented vitreous samples were analyzed from patients undergoing PPV for PDR, of which eighteen samples underwent a second surgery due to RVH. Ten of the sixty-six expressed miRNAs (miRNAs-19a, -20a, -22, -27a, -29a, -93, -126, -128, -130a, and -150) displayed divergences between the PDR vitreous groups and to the control. A significant increase in the miRNA-19a and -27a expression was determined in PDR patients undergoing PPV as compared to the controls. miRNA-20a and -93 were significantly upregulated in primary PPV vitreous samples of patients afflicted with RVH. Moreover, this observed upregulation was not significant between the non-RVH and control group, thus emphasizing the association with RVH incidence. miRNA-19a and -27a were detected as putative vitreous biomarkers for PDR, and elevated levels of miRNA-20a and -93 in vitreous with RVH suggest their biomarker potential for major PDR complications such as recurrent hemorrhage incidence.

Novel role of erythropoietin in proliferative diabetic retinopathy

2007 ◽  
Vol 77 (3) ◽  
pp. S62-S64 ◽  
Author(s):  
Hitoshi Takagi ◽  
Daisuke Watanabe ◽  
Kiyoshi Suzuma ◽  
Masafumi Kurimoto ◽  
Izumi Suzuma ◽  
...  
Keyword(s):  
Diabetic Retinopathy ◽  
Proliferative Diabetic Retinopathy

scholarly journals Correlations Between Different Angiogenic and Inflammatory Factors in Vitreous Fluid of Eyes With Proliferative Diabetic Retinopathy

2021 ◽  
Vol 8 ◽  
Author(s):  
Guanrong Wu ◽  
Baoyi Liu ◽  
Qiaowei Wu ◽  
Changting Tang ◽  
Zijing Du ◽  
...  
Keyword(s):  
Diabetic Retinopathy ◽  
Growth Factor ◽  
Proliferative Diabetic Retinopathy ◽  
Fasting Blood Glucose ◽  
Intercellular Adhesion Molecule ◽  
Inflammatory Factors ◽  
Control Group ◽  
Intercellular Adhesion Molecule 1 ◽  
Vitreous Fluid ◽  
Inflammation Mediators

Purpose: To investigate the expression of various angiogenesis and inflammation mediators in the vitreous fluid of eyes with proliferative diabetic retinopathy (PDR).Methods: A total of 38 eyes with PDR and 37 control eyes were included. Vitreous fluid was collected during vitrectomy. Vitreous levels of colony stimulating factor-1 receptor (CSF-1R), syndecan-1, placental growth factor (PIGF), and angiopoietin-like protein 4 (ANGPTL-4) were measured by multiplex immunoassay. Vitreous levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), and intercellular adhesion molecule-1 (ICAM-1) were measured by cytometric beads array. Levels of these mediators were compared between the PDR and control eyes. Correlations between levels of different mediators and between these mediators and kidney function metrics in the PDR group were also analyzed.Results: Vitreous levels of syndecan-1, PIGF, ANGPTL-4, VEGF, and IL-8 were significantly higher in the PDR group compared to the control group (all p &lt; 0.05). Levels of VEGF were significantly correlated with levels of syndecan-1, PIGF, and ANGPTL-4 (r = 0.370 to 0.497, all p &lt; 0.05). Significant positive correlations were detected between levels of any two of the following mediators including syndecan-1, PIGF, ANGPTL-4, and IL-8 (r = 0.370 to 0.906, all p &lt; 0.05). Apart from VEGF, levels of these mediators were positively correlated with serum creatinine and blood urea nitrogen (r = 0.328 to 0.638, all p &lt; 0.05), and negatively correlated with fasting blood glucose and estimated glomerular filtration rate (r = −0.325 to −0.603, all p &lt; 0.05).Conclusions: Correlations between different angiogenesis and inflammation mediators were observed in eyes with PDR, suggesting cross-talks of different angiogenesis and inflammation pathways in the pathogenesis of PDR. The levels of angiogenesis and inflammation in PDR are correlated with kidney damage, indicating possible common pathways in diabetic retinopathy and nephropathy.

Mitigating the Non-Specific Amplification in RCA: Assessment of the Role of Ligation and Exonuclease Digestion in Circular DNA Preparation

2021 ◽  
Author(s):  
Vandana Kuttappan Nair ◽  
Chandrika Sharma ◽  
Mrittika Sengupta ◽  
Souradyuti Ghosh
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
False Positive ◽  
Rolling Circle Amplification ◽  
Circular Dna ◽  
Rolling Circle ◽  
Specific Amplification ◽  
Exonuclease Digestion ◽  
Sticky End

<b>Layman Summary: </b>Rolling circle amplification (RCA) is a popular and extensively used bioanalytical tool. Like any nucleic acid amplifications, non-specific amplification may occur in it and risk generating false positive readouts. The work described in the manuscript investigates non-specific amplification in RCA as a function of ligation and exonuclease digestion assays during the synthesis of circular DNA. In particular, it investigates and compares the role of three different ligation techniques, namely splint-padlock ligation, cohesive end (sticky end ligation), and self-annealing ligation. In addition, it also probes the role of single exonuclease vs dual exonuclease digestions. We employed real time fluorescence to quantify the effect of these factors. Finally, our work hypothesizes the possible origins of non-specific amplification in RCA.

scholarly journals A Randomized Controlled Trial of Conbercept Pretreatment before Vitrectomy in Proliferative Diabetic Retinopathy

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Xiaochun Yang ◽  
Jianbiao Xu ◽  
Ruili Wang ◽  
Yan Mei ◽  
Huo Lei ◽  
...  
Keyword(s):  
Visual Acuity ◽  
Diabetic Retinopathy ◽  
Proliferative Diabetic Retinopathy ◽  
Controlled Trial ◽  
Control Group ◽  
Intraoperative Bleeding ◽  
Significant Difference ◽  
Pars Plana ◽  
Effective Adjunct

Purpose.To determine the efficacy and safety of preoperative intravitreal conbercept (IVC) injection before vitrectomy for proliferative diabetic retinopathy (PDR).Methods.107 eyes of 88 patients that underwent pars plana vitrectomy (PPV) for active PDR were enrolled. All patients were assigned randomly to either preoperative IVC group or control group. Follow-up examinations were performed for three months after surgery. The primary bioactivity measures were severity of intraoperative bleeding, incidence of early and late recurrent VH, vitreous clear-up time, and best-corrected visual acuity (BCVA) levels. The secondary safety measures included intraocular pressure, endophthalmitis, rubeosis, tractional retinal detachment, and systemic adverse events.Results.The incidence and severity of intraoperative bleeding were significantly lower in IVC group than in the control group. The average vitreous clear-up time of early recurrent VH was significantly shorter in IVC group compared with that in control group. There was no significant difference in vitreous clear-up time of late recurrent VH between the two groups. Patients that received pretreatment of conbercept had much better BCVA at 3 days, 1 week, and 1 month after surgery than control group. Moreover, both patients with improved BCVA were greater in IVC group than in control group at each follow-up.Conclusions.Conbercept pretreatment could be an effective adjunct to vitrectomy in accelerating postoperative vitreous clear-up and acquiring stable visual acuity restoration for PDR.

Elevated Circulating Levels of Thrombospondin-1 in Plasma Are Associated with Increased Circulating Levels of TGFb and Pro-Inflammatory Cytokines in Patients Afflicted with Rheumatoid Arthritis: Utilization of the Multiplexed Protein Profiling on Microarray by Rolling-Circle Amplification Technique.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1284-1284
Author(s):  
Mario C. Rico ◽  
Joanne M. Manns ◽  
Hien Nguyen ◽  
Nicole Beharry ◽  
Meera Reddy ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Rolling Circle Amplification ◽  
Protein Profiling ◽  
Rolling Circle ◽  
Cytokine Levels ◽  
Thrombospondin 1 ◽  
Circulating Levels ◽  
Pro Inflammatory Cytokines

Abstract Rheumatoid Arthritis (RA) is a chronic, autoimmune disease that affects a vast population worldwide with women being afflicted three times more than men. There is evidence for an increased risk of cardiovascular events in RA patients compared to the general population. These cardiovascular events may be associated with the chronic inflammatory state in which activation of coagulation leads to thrombin generation. Our laboratory has evidence that thrombospondin-1 (TSP1), an adhesive molecule that plays a major role in RA, promotes thrombin generation on the surface of a monocytic cell line (Isordia-Salas et al, Thromb Res.2005;116(6)). We also have documented that disrupting the TSP1 interaction on human neutrophils prevents the development of erosive arthritis in an experimental animal model (Manns et al, Arthritis and Rheumatism54(8), 2006). This observation is mediated by a novel pathway whereby TSP1 induces the up-regulation of Connective Tissue Growth Factor (CTGF). Therefore, to assess whether our in vitro and in vivo observations can be extrapolated to human disease, blood samples were collected from 20 patients afflicted with rheumatoid arthritis and 13 from healthy donors which served as the negative control. Plasma samples were separated and analyzed by Enzyme-Linked Immunosorbent Assay (ELISA) to determine the levels of transforming growth factor beta (TGF-β), protrombin F1+2 fragments (F1+2) and thrombospondin-1, and by multiplexed cytokine protein profiling on microarray by rolling-circle amplification (RCA) to determine cytokine levels. The F1+2 plasma levels showed an elevated trend in the RA group (p=0.06). TSP1 plasma levels were significantly increased in the RA group compared to the normal control (p=0.0004). Pro-inflammatory cytokine levels including IL-1β (p=0.0365), IL-6 (p=0.0029), TNF-α (p=0.0339), interferon-inducible protein 10 (p=0.0003) and macrophage inflammatory protein -1α (p=0.0012) were found elevated in the RA group compared to the normal control group. Some regulatory cytokines such as transforming growth factor-β (0.06), interferon-gamma (p=0.06) and IL-13 (p=0.22) showed no statistically significance between groups, but all of them showed a trend for higher circulation levels in plasma in the RA group. In summary, comparison between normal individuals and RA patients showed an increase in the levels of cytokines in the RA afflicted patients confirming what has been reported in the literature. We were able to correlate an increase in proteolytic factors and TSP1 levels in the RA patients with an increase of pro-inflammatory cytokines. Further studies are needed to elucidate why TSP1 acts as a pro-inflammatory molecule on the neutrophil surface of the RA patients.

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