Nitric oxide, a survival factor for lens epithelial cells

Purpose: Nitric oxide (NO) is capable of promoting either cell death or cell survival depending on cell type and experimental conditions. In this study, the possible effects of NO on the viability of lens epithelial cells were investigated in an explant model used previously to identify cellular changes associated with posterior capsule opacification following cataract surgery. Methods: Rat lens epithelial explants prepared from weanling rats were cultured in a serum-free medium for five days with or without the addition of the nitric oxide synthase inhibitor, L-Nω-nitro-L-arginine methyl ester (L-NAME), using the inactive enantiomer D-NAME as a control. Alternatively, explants were cultured for nine days with or without the NO donor, sodium nitroprusside. Explants were assessed morphologically and immunohistochemically or by determining DNA content. Results: In the presence of L-NAME but not in controls, progressive rounding up and detachment of cells from the lens capsule occurred, leading to extensive cell loss. Affected cells showed apoptosis-like cell-surface blebbing and nuclear fragmentation. Conversely, inclusion of sodium nitroprusside suppressed the morphological changes and spontaneous cell loss that occurred when sparsely covered explants were cultured for nine days, increased cell coverage fourfold during that period, and prevented the expression of the transdifferentiation markers α-smooth muscle actin and fibronectin. In addition, whereas L-NAME exacerbated cell loss induced by culturing with 50 pg/ml transforming growth factor-β2, sodium nitroprusside offered protection. Conclusions: This study points to a previously unidentified role for NO as an endogenously produced survival factor for lens epithelial cells, raising the possibility of using NO deprivation as a means of removing residual lens cells following cataract surgery and thereby preventing posterior capsule opacification

Inhibition of FGF-Induced ␣A-Crystallin Promoter Activity in Lens Epithelial Explants by TGF␤

PURPOSE. Fibroblast growth factor (FGF) plays a key role in normal lens biology, and recent studies suggest that transforming growth factor (TGF)-␤ is involved in the origin of certain forms of cataract. In the current study, the effects of FGF and TGF␤ on ␣A-crystallin promoter activity were investigated. METHODS. Rat lens epithelial explants were cultured with or without growth factors after transfecting with the firefly luciferase reporter gene driven by either the mouse ␣A-crystallin promoter region or a control simian virus (SV)40 promoter. RESULTS. FGF-2, at a concentration that induced lens fiber differentiation, strongly stimulated ␣A-crystallin promoter activity in explants at 3 to 4 days of culture, whereas SV40 promoter control specimens showed no comparable increase. At lower concentrations of FGF, sufficient to induce cell proliferation but not differentiation, there was only a slight increase in ␣A-crystallin promoter activity. Stimulation of ␣A-crystallin promoter activity induced by the fiber-differentiating concentration of FGF was virtually abolished by as little as 25 pg/ml TGF␤2, but the onset of fiber-specific ␤-crystallin accumulation was not prevented at this concentration. Phase-contrast microscopy revealed overt cataractous changes only at concentrations of TGF␤ more than 25 pg/ml. CONCLUSIONS. The stimulation of ␣A-crystallin promoter activity by FGF is consistent with its role in inducing accumulation of crystallins in explants. The blocking effect of TGF␤ on this process, even at a concentration too low to induce obvious pathologic changes, indicates the potential for TGF␤ to disturb ␣A-crystallin gene expression during early fiber differentiation. (Invest Ophthalmol Vis Sci

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Glutathione and catalase suppress TGFß-induced cataract-related changes in cultured rat lenses and lens epithelial explants

Purpose: The damaging effects of oxidative stress and transforming growth factor-β (TGFβ)-induced transdifferentiation of lens epithelial cells have both been implicated independently in the etiology of cataract. The aim of this study was to investigate whether the presence of antioxidant systems in the lens influences the ability of lens epithelial cells to respond to TGFβ. Methods: Whole lenses from young rats were cultured with or without TGFβ in the presence or absence of reduced glutathione (GSH). Lens epithelial explants from weanling rats were used to investigate the effects of GSH and catalase on TGFβ-induced cataract-related changes. Lenses were monitored for opacification for three to four days, photographed, and then processed for routine histology. Explants were assessed by phase contrast microscopy, enzyme-linked immunosorbent assay (ELISA) of α-smooth muscle actin (αSMA), and/or immunolocalization of αSMA and Pax6, markers for transdifferentiation and normal lens epithelial phenotype, respectively. Results: In cultured lenses, GSH strongly suppressed TGFβ-induced opacification and subcapsular plaque formation. In explants, both GSH and catalase suppressed changes typically associated with TGFβ-induced transdifferentiation including wrinkling of the lens capsule, cell-surface blebbing, apoptotic cell loss, induction of αSMA, and loss of Pax6 expression. Conclusions: This study suggests that antioxidant systems present in the normal lens, which protect the epithelium against the damaging effects of reactive oxygen species, may also serve to protect it against the potentially cataractogenic effects of TGFβ. Taken together with other recent studies, it also raises the possibility that TGFβ may induce cataract-related changes in lens epithelial cells via release of hydrogen peroxide

Save Sight Institute

PURPOSE. Cataract is the most common cause of blindness in the world today, and yet there is no generally accepted treatment other than surgical intervention. Studies in rodent models designed to increase understanding of the molecular basis of cataract have shown that transforming growth factor (TGF)-␤ induces morphologic and molecular changes similar to those associated with some forms of human cataract. Because aging is the most widely recognized risk factor for cataract, it is important that any animal model be examined in this context. This was a study of the effects of aging on susceptibility to TGF␤-induced cataract. METHODS. Lenses from weanling, adult, and senile rats were cultured in defined serum-free medium with a range of concentrations of TGF␤2. The lenses were cultured for up to 7 days, photographed daily, fixed, and prepared for histology and immunolocalization. Opacification was quantified by image analysis. RESULTS. Lenses from weanling, adult, and senile rats all underwent similar cataractous changes when exposed to TGF␤. This included opacification, the formation of anterior subcapsular plaques, and accumulation of type I collagen and ␣-smooth muscle actin. Lenses from adult and senile animals, however, were generally more adversely affected by TGF␤ than lenses from weanlings. This study also showed that a low dose of TGF␤ administered over a prolonged period had an effect similar to that of a higher dose administered over a shorter period. CONCLUSIONS. An elevation of TGF␤ activity, either acute or chronic, and/or an age-related increase in lens cell susceptibility to TGF␤ may be triggering factors in the etiology of certain forms of cataract. (Invest Ophthalmol Vis Sci. 2000;41:3544 -3551) C ataract is the leading cause of blindness worldwide. 2 Despite the extent of the problem and considerable research effort over the years, there is no generally accepted pharmacologic agent that can be applied to prevent or slow the onset or progression of this disease. Development of such a therapeutic agent depends on increased understanding of the molecular basis of cataractogenesis. In this laboratory we have been investigating the effects of various growth factors on lens biology and disease. We have shown that in vitro all three mammalian isoforms of transforming growth factor (TGF)-␤ induce responses in lens cells that mimic events in cataractogenesis. Lens epithelial cells in explants 3,4 and cultured whole lenses 5 are induced to undergo molecular and morphologic changes that are typically associated with subcapsular cataracts and with aftercataract, the subcapsular opacification that often arises from lens cells remaining after cataract surgery. 6 TGF␤ induces distinct anterior subcapsular opacities in cultured lenses from weanling rats. 5 These opacities correspond histologically with subcapsular plaques, which contain aberrant cells and are virtually indistinguishable from early-stage anterior subcapsular cataracts in humans. Lenses and lens explants cultured with TGF␤ express molecular markers for subcapsular cataract, type I collagen, and ␣-smooth muscle actin. 7 Taken together, these findings point to a role for TGF␤ in the etiology of major forms of cataract. A variety of risk factors predispose toward cataract, but the most widely recognized of these is aging