Quantitative proteomic profiling reveals sexual dimorphism in the retina and RPE of C57BL6 mice.

BACKGROUND: Sex as a biological variable is not a common consideration in molecular mechanistic or preclinical studies of retinal diseases. Understanding the sexual dimorphism of adult RPE and retina under physiological conditions is an important first step in improving our understanding of sex-based physio-pathological mechanisms. METHODS: Isobaric tags for relative and absolute quantitation (iTRAQ) were used for quantitative proteomics of male and female mouse retina and RPE (10 mice of each sex for each tissue type). Differentially expressed proteins were subjected to Gene Ontology (GO) analysis and Ingenuity Pathway Analysis (IPA). RESULTS: Differential expression analysis identified 21 differentially expressed proteins in the retina and 58 differentially expressed proteins in the RPE. Ingenuity pathway analysis identified the top canonical pathways differentially activated in the retina to be calcium transport I, nucleotide excision repair, molecular transport and cell death and survival. In the RPE, the top canonical pathways were calcium signaling, dilated cardiomyopathy signaling, actin cytoskeletal signaling and cellular assembly and organization. CONCLUSIONS: These results provide insights into sex differences in the retina and RPE proteome of mice and begin to shed clues into the sexual dimorphism seen in retinal diseases.

Dysregulated platelet function in patients with postacute sequelae of COVID-19.

BACKGROUND: Postacute sequelae of COVID-19 (PASC), also referred to as "Long COVID", sometimes follows COVID-19, a disease caused by SARS-CoV-2. Although SARS-CoV-2 is well known to promote a prothrombotic state, less is known about the thrombosis risk in PASC. Our objective was to evaluate platelet function and thrombotic potential in patients following recovery from SARS-CoV-2, but with clear symptoms of patients with PASC. METHODS: patients with PASC and matched healthy controls were enrolled in the study on average 15 months after documented SARS-CoV-2 infection. Platelet activation was evaluated by light transmission aggregometry (LTA) and flow cytometry in response to platelet surface receptor agonists. Thrombosis in platelet-deplete plasma was evaluated by Factor Xa activity. A microfluidics system assessed thrombosis in whole blood under shear stress conditions. RESULTS: A mild increase in platelet aggregation in patients with PASC through the thromboxane receptor was observed, and platelet activation through the glycoprotein VI (GPVI) receptor was decreased in patients with PASC compared to age- and sex-matched healthy controls. Thrombosis under shear conditions as well as Factor Xa activity were reduced in patients with PASC. Plasma from patients with PASC was an extremely potent activator of washed, healthy platelets - a phenomenon not observed when stimulating healthy platelets after incubation with plasma from healthy individuals. CONCLUSIONS: patients with PASC show dysregulated responses in platelets and coagulation in plasma, likely caused by a circulating molecule that promotes thrombosis. A hitherto undescribed protective response appears to exist in patients with PASC to counterbalance ongoing thrombosis that is common to SARS-CoV-2 infection.

Dysregulated Platelet Function in Patients with Post-Acute Sequelae of COVID-19.

Background: Post-acute sequelae of COVID-19 (PASC), also referred as Long-COVID, sometimes follows COVID-19, a disease caused by SARS-CoV-2. While SARS-CoV-2 is well-known to promote a prothrombotic state, less is known about the thrombosis risk in PASC. Aim: Our objective was to evaluate the platelet function and thrombotic potential in patients following recovery from SARS-CoV-2 with clear symptoms of PASC. Methods: PASC patients and matched healthy controls were enrolled in the study on average 15 months after documented SARS-CoV-2 infection. Platelet activation was evaluated by Light Transmission Aggregometry (LTA) and flow cytometry in response to platelet surface receptor agonists. Thrombosis in platelet-deplete plasma was evaluated by Factor Xa activity. A microfluidics system assessed thrombosis in whole blood under shear stress conditions. Results: A mild increase in platelet aggregation in PASC patients through the thromboxane receptor was observed and platelet activation through the glycoprotein VI (GPVI) receptor was decreased in PASC patients compared to age- and sex-matched healthy controls. Thrombosis under shear conditions as well as Factor Xa activity were reduced in PASC patients. Plasma from PASC patients was an extremely potent activator of washed, healthy platelets - a phenomenon not observed when stimulating healthy platelets after incubation with plasma from healthy individuals. Conclusions: PASC patients show dysregulated responses in platelets and coagulation in plasma, likely caused by a circulating molecule that promotes thrombosis. A hitherto undescribed protective response appears to exists in PASC patients to counterbalance ongoing thrombosis that is common to SARS-CoV-2 infection.

Evaluation of Olfactory Acuity in Patients with Coronavirus Disease 2019 (COVID-19).

Aim and Objectives: To describe the prevalence and characteristics of olfactory dysfunction (OD) in patients with laboratory-confirmed COVID-19 infection. Materials and Methods: This monocentric study was performed at Chest Diseases Hospital during the COVID-19 pandemic and all patients testing positive for COVID-19 over a 5-month period (April to August 2020) were recruited. Detailed history was elicited from subjects and all patients were inquired about olfactory dysfunction (OD). Patients with olfactory dysfunction were asked to complete olfactory questionnaires based on the short version of the Questionnaire of Olfactory Disorders-Negative Statements (sQOD-NS). Results: 655 patients with mild to moderate COVID-19 infection were included in the study. The prevalence rate of olfactory dysfunction was 18.47% (n = 121) with contribution of 11.60% (n = 76) and 6.87% (n = 45) from anosmia and hyposmia respectively, thereby suggesting olfactory dysfunction to be a significant clinical feature in COVID-19 patients. Males were significantly more affected by olfactory dysfunctions than females. Anosmic patients had significantly reduced sQOD-NS results as compared to hyposmic patients (significant at P < 0.05). The mean duration of OD was 7.7 days (± 4.3) and >90% patients in our study showed resolution within 14 days. Conclusion: The early recognition of olfactory dysfunction should help to screen, identify and thereby quickly isolate mildly symptomatic COVID-19 patients from the general population and the existence of these dysfunctions may well be a prognostic factor in the course of the disease.

The retinal pigment epithelium in Sorsby Fundus Dystrophy shows increased sensitivity to oxidative stress-induced degeneration.

Sorsby Fundus Dystrophy (SFD) is a rare inherited autosomal dominant macular degeneration caused by specific mutations in TIMP3. Patients with SFD present with pathophysiology similar to the more common Age-related Macular Degeneration (AMD) and loss of vision due to both choroidal neovascularization and geographic atrophy. Previously, it has been shown that RPE degeneration in AMD is due in part to oxidative stress. We hypothesized that similar mechanisms may be at play in SFD. The objective of this study was to evaluate whether mice carrying the S179C-Timp3 mutation, a variant commonly observed in SFD, showed increased sensitivity to oxidative stress. Antioxidant genes are increased at baseline in the RPE in SFD mouse models, but not in the retina. This suggests the presence of a pro-oxidant environment in the RPE in the presence of Timp3 mutations. To determine if the RPE of Timp3 mutant mice is more susceptible to degeneration when exposed to low levels of oxidative stress, mice were injected with low doses of sodium iodate. The RPE and photoreceptors in Timp3 mutant mice degenerated at low doses of sodium iodate, which had no effect in wildtype control mice. These studies suggest that TIMP3 mutations may result in a dysregulation of pro-oxidant-antioxidant homeostasis in the RPE, leading to RPE degeneration in SFD.

Inhibition of choroidal neovascularization by systemic delivery of gold nanoparticles.

Choroidal neovascularization (CNV) is the abnormal growth of blood vessels that sprout from the choroid vasculature and grow beneath and into the retina. The newly formed blood vessels in CNV often leak blood and fluid which deteriorates vision over time, eventually leading to blindness. In the present study, we examined the efficacy of intravenously injected gold nanoparticles in the laser-induced CNV animal model. Using optical coherence tomography (OCT) and fluorescein angiography, we evaluated CNV lesions longitudinally, over a period of 21 days, with and without nanoparticle treatment. Intravenously injected low concentration of bare gold nanoparticles showed significant anti-angiogenic properties by suppressing CNV development and progression. The treatment group showed significantly decreased fluorescein leakage at the CNV site compared to vehicle injected control mice. OCT assisted CNV volume measurement at all time points showed a significant reduction in lesion size in the treatment group compared with controls.

Role of FGF and Hyaluronan in Choroidal Neovascularization in Sorsby Fundus Dystrophy.

Sorsby's fundus dystrophy (SFD) is an inherited blinding disorder caused by mutations in the tissue inhibitor of metalloproteinase-3 (TIMP3) gene. The SFD pathology of macular degeneration with subretinal deposits and choroidal neovascularization (CNV) closely resembles that of the more common age-related macular degeneration (AMD). The objective of this study was to gain further insight into the molecular mechanism(s) by which mutant TIMP3 induces CNV. In this study we demonstrate that hyaluronan (HA), a large glycosaminoglycan, is elevated in the plasma and retinal pigment epithelium (RPE)/choroid of patients with AMD. Mice carrying the S179C-TIMP3 mutation also showed increased plasma levels of HA as well as accumulation of HA around the RPE in the retina. Human RPE cells expressing the S179C-TIMP3 mutation accumulated HA apically, intracellularly and basally when cultured long-term compared with cells expressing wildtype TIMP3. We recently reported that RPE cells carrying the S179C-TIMP3 mutation have the propensity to induce angiogenesis via basic fibroblast growth factor (FGF-2). We now demonstrate that FGF-2 induces accumulation of HA in RPE cells. These results suggest that the TIMP3-MMP-FGF-2-HA axis may have an important role in the pathogenesis of CNV in SFD and possibly AMD.

Sorsby Fundus Dystrophy Mutation in Tissue Inhibitor of Metalloproteinase 3 (TIMP3) promotes Choroidal Neovascularization via a Fibroblast Growth Factor-dependent Mechanism.

Choroidal neovascularization (CNV) leads to loss of vision in patients with Sorsby Fundus Dystrophy (SFD), an inherited, macular degenerative disorder, caused by mutations in the Tissue Inhibitor of Metalloproteinase-3 (TIMP3) gene. SFD closely resembles age-related macular degeneration (AMD), which is the leading cause of blindness in the elderly population of the Western hemisphere. Variants in TIMP3 gene have recently been identified in patients with AMD. A majority of patients with AMD also lose vision as a consequence of choroidal neovascularization (CNV). Thus, understanding the molecular mechanisms that contribute to CNV as a consequence of TIMP-3 mutations will provide insight into the pathophysiology in SFD and likely the neovascular component of the more commonly seen AMD. While the role of VEGF in CNV has been studied extensively, it is becoming increasingly clear that other factors likely play a significant role. The objective of this study was to test the hypothesis that basic Fibroblast Growth Factor (bFGF) regulates SFD-related CNV. In this study we demonstrate that mice expressing mutant TIMP3 (Timp3S179C/S179C) showed reduced MMP inhibitory activity with an increase in MMP2 activity and bFGF levels, as well as accentuated CNV leakage when subjected to laser injury. S179C mutant-TIMP3 in retinal pigment epithelial (RPE) cells showed increased secretion of bFGF and conditioned medium from these cells induced increased angiogenesis in endothelial cells. These studies suggest that S179C-TIMP3 may promote angiogenesis and CNV via a FGFR-1-dependent pathway by increasing bFGF release and activity.

Tissue inhibitor of metalloproteinases-3 peptides inhibit angiogenesis and choroidal neovascularization in mice.

Tissue inhibitors of metalloproteinases (TIMPs) while originally characterized as inhibitors of matrix metalloproteinases (MMPs) have recently been shown to have a wide range of functions that are independent of their MMP inhibitory properties. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a potent inhibitor of VEGF-mediated angiogenesis and neovascularization through its ability to block the binding of VEGF to its receptor VEGFR-2. To identify and characterize the anti-angiogenic domain of TIMP-3, structure function analyses and synthetic peptide studies were performed using VEGF-mediated receptor binding, signaling, migration and proliferation. In addition, the ability of TIMP-3 peptides to inhibit CNV in a mouse model was evaluated. We demonstrate that the anti-angiogenic property resides in the COOH-terminal domain of TIMP-3 protein which can block the binding of VEGF specifically to its receptor VEGFR-2, but not to VEGFR-1 similar to the full-length wild-type protein. Synthetic peptides corresponding to putative loop 6 and tail region of TIMP-3 have anti-angiogenic properties as determined by inhibition of VEGF binding to VEGFR-2, VEGF-induced phosphorylation of VEGFR-2 and downstream signaling pathways as well as endothelial cell proliferation and migration in response to VEGF. In addition, we show that intravitreal administration of TIMP-3 peptide could inhibit the size of laser-induced choroidal neovascularization lesions in mice. Thus, we have identified TIMP-3 peptides to be efficient inhibitors of angiogenesis and have a potential to be used therapeutically in diseases with increased neovascularization.

Increased neovascularization in mice lacking tissue inhibitor of metalloproteinases-3.

PURPOSE: Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a matrix-bound inhibitor of matrix metalloproteinases (MMPs). The authors have previously determined a novel function of TIMP-3 to inhibit vascular endothelial growth factor (VEGF)-mediated angiogenesis. Here, the authors examined the in vivo angiogenic phenotype of ocular vessels in mice deficient in TIMP-3. METHODS: VEGF-mediated corneal neovascularization and laser-induced choroidal neovascularization (CNV) were examined in TIMP-3-null mice. The effects of the absence of TIMP-3 on the phosphorylation status of the VEGF-receptor-2 (VEGFR-2) and the downstream signaling pathways were evaluated biochemically. In addition, the activation state of MMPs in the retina of TIMP-3-deficient mice was examined by in situ zymography. RESULTS: The results of these studies determine an accentuation of pathologic VEGF-mediated angiogenesis in the cornea and laser-induced CNV in mice lacking TIMP-3. In the absence of the MMP inhibitor, pathophysiological changes were observed in the choroidal vasculature concomitantly with an increase in gelatinolytic activity. These results suggest that an imbalance of extracellular matrix homeostasis, together with a loss of an angiogenesis inhibitor, can prime vascular beds to be more responsive to an angiogenic stimulus. CONCLUSIONS: In light of the recent studies suggesting that genetic variants near TIMP-3 influence susceptibility to age-related macular degeneration, these results imply that TIMP-3 may regulate the development of the choroidal vasculature and is a likely contributor to increased susceptibility to choroidal neovascularization.