Expression profiles of nestin and synemin in reactive astrocytes and Müller cells following retinal injury: a comparison with glial fibrillar acidic protein and vimentin

Purpose: To examine the expression patterns of the intermediate filament (IF) proteins nestin and synemin following retinal injury. Methods: Wide-scale retinal injuries were created by experimental retinal detachment of 1, 3, 7, or 30 days\u27 duration. Injuries were induced in the right eyes of Long Evans rats, while the left eyes served as internal controls. Vibratome sections of control and injured retinas were labeled with fluorescent probes using a combination of anti-glial fibrillary acidic protein, -vimentin, -nestin, -synemin, -bromodeoxyuridine, and the lectin probe, isolectin B4. Additionally, antibody specificity, as well as protein and mRNA levels of nestin and synemin were determined and quantified using standard western blotting and real time polymerase chain reaction (RT-PCR) techniques. Results: Immunocytochemistry showed increased Müller cell labeling at 1, 3, and 7 days post injury for all four IFs, although the relative levels of nestin expression varied dramatically between individual Müller cells. Nestin was consistently observed in the foremost processes of those Müller cells that grew into the subretinal space, forming glial scars. Elevated levels of nestin expression were also observed in bromodeoxyuridine-labeled Müller cells following retinal insult. Quantitative polymerase chain reaction (qPCR) showed a twofold increase in nestin mRNA 1 day after injury, a level maintained at 3 and 7 days. Western blotting using anti-nestin showed a single band at 220 kDa and the intensity of this band increased following injury. Anti-synemin labeling of control retinas revealed faint labeling of astrocytes; this increased after injury, demonstrating an association with blood vessels. Additionally, there was an upregulation of synemin in Müller cells. qPCR and western blotting with anti-synemin showed a continuous increase in both gene and protein expression over time. Conclusions: Retinal injury induces an upregulation of a complement of four intermediate filament proteins, including synemin and nestin, in Müller cells. The latter provides suggestive support for the concept that these cells may revert to a more developmentally immature state, since these two IF proteins are developmentally regulated and expressed, and thus may serve as cell cycle reentry markers. Nestin and its differential expression patterns with glial fibrillary acidic protein and vimentin networks, as well as its association with proliferating Müller cells and those extending into the subretinal space, suggest a significant role of this protein in glial scar formation and perhaps gliogenesis. Synemin immunopositive astrocytes demonstrate a close relationship to the retinal vasculature, and illustrate a remarkable ability to reorganize their morphology in response to injury. Further examination of the changes in the cytoskeletal signatures of both of these glial cell types may lead to a more comprehensive understanding of mechanisms underway following retinal and other central nervous system injuries. © 2010 Molecular Vision

Expression profiles of nestin and synemin in reactive astrocytes and Müller cells following retinal injury: a comparison with glial fibrillar acidic protein and

Purpose: To examine the expression patterns of the intermediate filament (IF) proteins nestin and synemin following retinal injury. Methods: Wide-scale retinal injuries were created by experimental retinal detachment of 1, 3, 7, or 30 days' duration. Injuries were induced in the right eyes of Long Evans rats, while the left eyes served as internal controls. Vibratome sections of control and injured retinas were labeled with fluorescent probes using a combination of anti-glial fibrillary acidic protein, -vimentin, -nestin, -synemin, -bromodeoxyuridine, and the lectin probe, isolectin B4. Additionally, antibody specificity, as well as protein and mRNA levels of nestin and synemin were determined and quantified using standard western blotting and real time polymerase chain reaction (RT-PCR) techniques. Results: Immunocytochemistry showed increased Müller cell labeling at 1, 3, and 7 days post injury for all four IFs, although the relative levels of nestin expression varied dramatically between individual Müller cells. Nestin was consistently observed in the foremost processes of those Müller cells that grew into the subretinal space, forming glial scars. Elevated levels of nestin expression were also observed in bromodeoxyuridine-labeled Müller cells following retinal insult. Quantitative polymerase chain reaction (qPCR) showed a twofold increase in nestin mRNA 1 day after injury, a level maintained at 3 and 7 days. Western blotting using anti-nestin showed a single band at 220 kDa and the intensity of this band increased following injury. Anti-synemin labeling of control retinas revealed faint labeling of astrocytes; this increased after injury, demonstrating an association with blood vessels. Additionally, there was an upregulation of synemin in Müller cells. qPCR and western blotting with anti-synemin showed a continuous increase in both gene and protein expression over time. Conclusions: Retinal injury induces an upregulation of a complement of four intermediate filament proteins, including synemin and nestin, in Müller cells. The latter provides suggestive support for the concept that these cells may revert to a more developmentally immature state, since these two IF proteins are developmentally regulated and expressed, and thus may serve as cell cycle reentry markers. Nestin and its differential expression patterns with glial fibrillary acidic protein and vimentin networks, as well as its association with proliferating Müller cells and those extending into the subretinal space, suggest a significant role of this protein in glial scar formation and perhaps gliogenesis. Synemin immunopositive astrocytes demonstrate a close relationship to the retinal vasculature, and illustrate a remarkable ability to reorganize their morphology in response to injury. Further examination of the changes in the cytoskeletal signatures of both of these glial cell types may lead to a more comprehensive understanding of mechanisms underway following retinal and other central nervous system injuries

Expression profiles of nestin and synemin in reactive astrocytes and Müller cells following retinal injury: A comparison with glial fibrillar acidic protein and vimentin

Purpose: To examine the expression patterns of the intermediate filament (IF) proteins nestin and synemin following retinal injury. Methods: Wide-scale retinal injuries were created by experimental retinal detachment of 1, 3, 7, or 30 days\u27 duration. Injuries were induced in the right eyes of Long Evans rats, while the left eyes served as internal controls. Vibratome sections of control and injured retinas were labeled with fluorescent probes using a combination of anti-glial fibrillary acidic protein, -vimentin, -nestin, -synemin, -bromodeoxyuridine, and the lectin probe, isolectin B4. Additionally, antibody specificity, as well as protein and mRNA levels of nestin and synemin were determined and quantified using standard western blotting and real time polymerase chain reaction (RT-PCR) techniques. Results: Immunocytochemistry showed increased Müller cell labeling at 1, 3, and 7 days post injury for all four IFs, although the relative levels of nestin expression varied dramatically between individual Müller cells. Nestin was consistently observed in the foremost processes of those Müller cells that grew into the subretinal space, forming glial scars. Elevated levels of nestin expression were also observed in bromodeoxyuridine-labeled Müller cells following retinal insult. Quantitative polymerase chain reaction (qPCR) showed a twofold increase in nestin mRNA 1 day after injury, a level maintained at 3 and 7 days. Western blotting using anti-nestin showed a single band at 220 kDa and the intensity of this band increased following injury. Anti-synemin labeling of control retinas revealed faint labeling of astrocytes; this increased after injury, demonstrating an association with blood vessels. Additionally, there was an upregulation of synemin in Müller cells. qPCR and western blotting with anti-synemin showed a continuous increase in both gene and protein expression over time. Conclusions: Retinal injury induces an upregulation of a complement of four intermediate filament proteins, including synemin and nestin, in Müller cells. The latter provides suggestive support for the concept that these cells may revert to a more developmentally immature state, since these two IF proteins are developmentally regulated and expressed, and thus may serve as cell cycle reentry markers. Nestin and its differential expression patterns with glial fibrillary acidic protein and vimentin networks, as well as its association with proliferating Müller cells and those extending into the subretinal space, suggest a significant role of this protein in glial scar formation and perhaps gliogenesis. Synemin immunopositive astrocytes demonstrate a close relationship to the retinal vasculature, and illustrate a remarkable ability to reorganize their morphology in response to injury. Further examination of the changes in the cytoskeletal signatures of both of these glial cell types may lead to a more comprehensive understanding of mechanisms underway following retinal and other central nervous system injuries. © 2010 Molecular Vision

Novel compstatin family peptides inhibit complement activation by drusen-like deposits in human retinal pigmented epithelial cell cultures

We have used a novel human retinal pigmented epithelial (RPE) cell-based model that mimics drusen biogenesis and the pathobiology of age-related macular degeneration to evaluate the efficacy of newly designed peptide inhibitors of the complement system. The peptides belong to the compstatin family and, compared to existing compstatin analogs, have been optimized to promote binding to their target, complement protein C3, and to enhance solubility by improving their polarity/hydrophobicity ratios. Based on analysis of molecular dynamics simulation data of peptide–C3 complexes, novel binding features were designed by introducing intermolecular salt bridge-forming arginines at the N-terminus and at position −1 of N-terminal dipeptide extensions. Our study demonstrates that the RPE cell assay has discriminatory capability for measuring the efficacy and potency of inhibitory peptides in a macular disease environment

Novel compstatin family peptides inhibit complement activation by drusen-like deposits in human retinal pigmented epithelial cell cultures.

We have used a novel human retinal pigmented epithelial (RPE) cell-based model that mimics drusen biogenesis and the pathobiology of age-related macular degeneration to evaluate the efficacy of newly designed peptide inhibitors of the complement system. The peptides belong to the compstatin family and, compared to existing compstatin analogs, have been optimized to promote binding to their target, complement protein C3, and to enhance solubility by improving their polarity/hydrophobicity ratios. Based on analysis of molecular dynamics simulation data of peptide-C3 complexes, novel binding features were designed by introducing intermolecular salt bridge-forming arginines at the N-terminus and at position -1 of N-terminal dipeptide extensions. Our study demonstrates that the RPE cell assay has discriminatory capability for measuring the efficacy and potency of inhibitory peptides in a macular disease environment

COVID-19 prevalence and mortality in patients with cancer and the effect of primary tumour subtype and patient demographics: a prospective cohort study

Background Patients with cancer are purported to have poor COVID-19 outcomes. However, cancer is a heterogeneous group of diseases, encompassing a spectrum of tumour subtypes. The aim of this study was to investigate COVID-19 risk according to tumour subtype and patient demographics in patients with cancer in the UK. Methods We compared adult patients with cancer enrolled in the UK Coronavirus Cancer Monitoring Project (UKCCMP) cohort between March 18 and May 8, 2020, with a parallel non-COVID-19 UK cancer control population from the UK Office for National Statistics (2017 data). The primary outcome of the study was the effect of primary tumour subtype, age, and sex and on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) prevalence and the case–fatality rate during hospital admission. We analysed the effect of tumour subtype and patient demographics (age and sex) on prevalence and mortality from COVID-19 using univariable and multivariable models. Findings 319 (30·6%) of 1044 patients in the UKCCMP cohort died, 295 (92·5%) of whom had a cause of death recorded as due to COVID-19. The all-cause case–fatality rate in patients with cancer after SARS-CoV-2 infection was significantly associated with increasing age, rising from 0·10 in patients aged 40–49 years to 0·48 in those aged 80 years and older. Patients with haematological malignancies (leukaemia, lymphoma, and myeloma) had a more severe COVID-19 trajectory compared with patients with solid organ tumours (odds ratio [OR] 1·57, 95% CI 1·15–2·15; p<0·0043). Compared with the rest of the UKCCMP cohort, patients with leukaemia showed a significantly increased case–fatality rate (2·25, 1·13–4·57; p=0·023). After correction for age and sex, patients with haematological malignancies who had recent chemotherapy had an increased risk of death during COVID-19-associated hospital admission (OR 2·09, 95% CI 1·09–4·08; p=0·028). Interpretation Patients with cancer with different tumour types have differing susceptibility to SARS-CoV-2 infection and COVID-19 phenotypes. We generated individualised risk tables for patients with cancer, considering age, sex, and tumour subtype. Our results could be useful to assist physicians in informed risk–benefit discussions to explain COVID-19 risk and enable an evidenced-based approach to national social isolation policies