Conjunctival signs and symptoms are observed in a subset of patients with COVID-19, and SARS-CoV-2 has been detected in tears, raising concerns regarding the eye both as a portal of entry and carrier of the virus. The purpose of this study was to determine whether ocular surface cells possess the key factors required for cellular susceptibility to SARS-CoV-2 entry/infection. Methods: We analyzed human post-mortem eyes as well as surgical specimens for the expression of ACE2 (the receptor for SARS-CoV-2) and TMPRSS2, a cell surface-associated protease that facilitates viral entry following binding of the viral spike protein to ACE2. Results: Across all eye specimens, immunohistochemical analysis revealed expression of ACE2 in the conjunctiva, limbus, and cornea, with especially prominent staining in the superficial conjunctival and corneal epithelial surface. Surgical conjunctival specimens also showed expression of ACE2 in the conjunctival epithelium, especially prominent in the superficial epithelium, as well as weak or focal expression in the substantia propria. All eye and conjunctival specimens also expressed TMPRSS2. Finally, Western blot analysis of protein lysates from human corneal epithelium obtained during refractive surgery confirmed expression of ACE2 and TMPRSS2.Conclusions: Together, these results suggest that ocular surface cells including conjunctiva are susceptible to infection by SARS-CoV-2, and could therefore serve as a portal of entry as well as a reservoir for person-to-person transmission of this virus. This highlights the importance of safety practices including face masks and ocular contact precautions in preventing the spread of COVID-19 disease.

Microglia activation is recognized as the hallmark of neuroinflammation. However, the activation profile and phenotype changes of microglia during the process of retinal degeneration are poorly understood. This study aimed to elucidate the time-spatial pattern of microglia distribution and characterize the polarized phenotype of activated microglia during retinal neuroinflammation and degeneration in rd1 (Pde6βrd1/rd1) mice, the classic model of inherited retinal degeneration. Retinae of rd1 mice at different postnatal days (P7, P14, P21, P28, P56, and P180) were prepared for further analysis. We found most CD11b+ or IBA1+ microglia expressed Ki-67 and CD68 in rd1 mice and these cells migrated toward the layer of degenerative photoreceptors at the rapid rods degeneration phase from P14 to P28. These microglia exhibited typical ameboid activated shape with round bodies and scarce dendrites, while at late phase at P180, they displayed resting ramified morphology with elongated dendrites. Flow cytometry revealed that the percentage of CD86+CD206- M1 microglia increased markedly in rd1 retinae, however, no significant change was observed in CD206+CD86- M2 microglia. Interestingly, CD86+CD206+ microglia, an intermediate state between the two extremes of M1 and M2, increased markedly at the rapid rods degeneration phase. The immunofluorescence images revealed that microglia in rd1 mice highly expressed M1 markers including CD16/32, CD86, and CD40. In addition, increased expression of pro-inflammatory cytokines (TNF-α, IL-6, and CCL2) was observed in rd1 mice. Our findings unfolded a panorama for the first time that microglia conducted distinctive behaviors with the progression of retinal degeneration in rd1 mice. Microglia is activated and particularly polarized to a pro-inflammatory M1 phenotype at the rapid rods degenerative phase, suggesting that the involvement of M1 microglia in the retinal neuroinflammation and degeneration. Most microglia adopted an intermediate polarization “M1½” state in rd1, revealing that microglia orchestrated a complicated continuous spectrum in degenerative retina.

PurposeConjunctival signs and symptoms are observed in a subset of patients with COVID-19, and SARS-CoV-2 has been detected in tears, raising concerns regarding the eye both as a portal of entry and carrier of the virus. The purpose of this study was to determine whether ocular surface cells possess the key factors required for cellular susceptibility to SARS-CoV-2 entry/infection.MethodsWe analyzed human post-mortem eyes as well as surgical specimens for the expression of ACE2 (the receptor for SARS-CoV-2) and TMPRSS2, a cell surface-associated protease that facilitates viral entry following binding of the viral spike protein to ACE2.ResultsAcross all eye specimens, immunohistochemical analysis revealed expression of ACE2 in the conjunctiva, limbus, and cornea, with especially prominent staining in the superficial conjunctival and corneal epithelial surface. Surgical conjunctival specimens also showed expression of ACE2 in the conjunctival epithelium, especially prominent in the superficial epithelium, as well as the substantia propria. All eye and conjunctival specimens also expressed TMPRSS2. Finally, western blot analysis of protein lysates from human corneal epithelium obtained during refractive surgery confirmed expression of ACE2 and TMPRSS2.ConclusionsTogether, these results indicate that ocular surface cells including conjunctiva are susceptible to infection by SARS-CoV-2, and could therefore serve as a portal of entry as well as a reservoir for person-to-person transmission of this virus. This highlights the importance of safety practices including face masks and ocular contact precautions in preventing the spread of COVID-19 disease.

Birds (Aves) display high metabolic rates and oxygen consumption relative to mammals, increasing reactive oxygen species (ROS) formation. Although excess ROS reduces lifespan by causing extensive cellular dysfunction and damage, birds are remarkably long-lived. We address this paradox by identifying the constitutive activation of the NRF2 master antioxidant response in Neoaves (~95% of bird species), providing an adaptive mechanism capable of counterbalancing high ROS levels. We demonstrate that a KEAP1 mutation in the Neoavian ancestor disrupted the repression of NRF2 by KEAP1, leading to constitutive NRF2 activity and decreased oxidative stress in wild Neoaves tissues and cells. Our evidence suggests this ancient mutation induced a compensatory program in NRF2-target genes with functions beyond redox regulation-including feather development-while enabling significant metabolic rate increases that avoid trade-offs with lifespan. The strategy of NRF2 activation sought by intense clinical investigation therefore appears to have also unlocked a massively successful evolutionary trajectory.

Background/Aims: Platelet-derived growth factors (PDGFs) have emerged as pivotal in pathological angiogenesis, which is a hallmark of various tumors and retinal diseases. Here we evaluated the anti-angiogenic effect of imatinib, an inhibitor of PDGF receptors α and β (PDGFR-α and -β), in retinal neovascularization using an oxygen-induced retinopathy (OIR) model. Methods: The OIR model was established and given imatinib or vehicle treatments daily from P12 to P16. At the peak of angiogenesis at P17, the neovascularization area was quantified on retinal whole-mounts with isolectin B4 staining. Immunofluorescence staining and western blots were used to determine the effect of imatinib on different vascular cells and the pathway molecules involved. Results: Imatinib effectively suppressed pathological angiogenesis in OIR mice and reduced the number of all three types of vascular cells, including endothelial cells, pericytes, and smooth muscle cells. Moreover, the expression and activation of PDGFR-α and -β were inhibited by imatinib. The imatinib-treated OIR mice presented with reduced expression of other potent pro-angiogenic factors such as VEGF and FGF2. No obvious retinal or systemic side effects were observed in the imatinib treatment group. Conclusions: Imatinib appears to be safe and effective in suppressing retinal neovascularization. Targeting PDGFs/PDGFRs may also be important for anti-angiogenic treatment and offer a viable alternative treatment for retinal angiogenic diseases.

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The Nrf2-Keap1 pathway regulates transcription of a wide array of antioxidant and cytoprotective genes and offers critical protection against oxidative stress. This pathway has demonstrated benefit for a variety of retinal conditions. Retinal ischemia plays a pivotal role in many vision threatening diseases. Retinal vascular endothelial cells are an important participant in ischemic injury. In this setting, Nrf2 provides a protective pathway via amelioration of oxidative stress and inflammation. In this study, we investigated a potent small molecule inhibitor of the Nrf2-Keap1 protein-protein interaction (PPI), CPUY192018, for its therapeutic potential in retinal cells and retinal ischemiareperfusion injury. In human retinal endothelial cells (HREC), treatment with CPUY192018 increased Nrf2 protein levels and nuclear translocation, stimulated Nrf2-ARE-induced transcriptional capacity, and induced Nrf2 target gene expression. Furthermore, CPUY192018 protected HREC against oxidative stress and inflammatory activation. CPUY192018 also activated Nrf2 and suppressed inflammatory response in macrophages. In the retinal ischemia-reperfusion (I/R) model, administration of CPUY192018 induced Nrf2 target gene activation in the retina. Both systemic and topical treatment with CPUY192018 rescued visual function after ischemiareperfusion injury. Taken together, these findings indicate that small molecule Keap1-Nrf2 PPI inhibitors can activate the Nrf2 pathway in the retina and provide protection against retinal ischemic and inflammatory injury, suggesting Keap1-Nrf2 PPI inhibition in the treatment of retinal conditions.

PURPOSE.To investigate the expression of angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2 in human retina. METHODS.Human post-mortem eyes from 13 non-diabetic control cases and 11 diabetic retinopathy cases were analyzed for the expression of ACE2. To compare the vascular ACE2 expression between different organs that involve in diabetes, the expression of ACE2 was investigated in renal specimens from nondiabetic and diabetic nephropathy patients. Expression of TMPRSS2, a cell-surface protease that facilitates SARS-CoV-2 entry, was also investigated in human nondiabetic retinas. Primary human retinal endothelial cells (HRECs) and primary human retinal pericytes (HRPCs) were further used to confirm the vascular ACE2 expression in human retina. RESULTS.We found that ACE2 was expressed in multiple nonvascular neuroretinal cells, including the retinal ganglion cell layer, inner plexiform layer, inner nuclear layer, and photoreceptor outer segments in both nondiabetic and diabetic retinopathy specimens. Strikingly, we observed significantly more ACE2 positive vessels in the diabetic retinopathy specimens. By contrast, in another end-stage organ affected by diabetes, the kidney, ACE2 in nondiabetic and diabetic nephropathy showed apical expression of ACE2 tubular epithelial cells, but no endothelial expression in glomerular or peritubular capillaries. Western blot analysis of protein lysates from HRECs and HRPCs confirmed expression of ACE2. TMPRSS2 expression was present in multiple retinal neuronal cells, vascular and perivascular cells, and Müller glia. CONCLUSIONS.Together, these results indicate that retina expresses ACE2 and TMPRSS2. Moreover, there are increased vascular ACE2 expression in diabetic retinopathy retinas.