Stem cell therapy in coronavirus disease 2019: current evidence and future potential.

The end of 2019 saw the beginning of the coronavirus disease 2019 (COVID-19) pandemic that soared in 2020, affecting 215 countries worldwide, with no signs of abating. In an effort to contain the spread of the disease and treat the infected, researchers are racing against several odds to find an effective solution. The unavailability of timely and affordable or definitive treatment has caused significant morbidity and mortality. Acute respiratory distress syndrome (ARDS) caused by an unregulated host inflammatory response toward the viral infection, followed by multi-organ dysfunction or failure, is one of the primary causes of death in severe cases of COVID-19 infection. Currently, empirical management of respiratory and hematological manifestations along with anti-viral agents is being used to treat the infection. The quest is on for both a vaccine and a more definitive management protocol to curtail the spread. Researchers and clinicians are also exploring the possibility of using cell therapy for severe cases of COVID-19 with ARDS. Mesenchymal stromal cells are known to have immunomodulatory properties and have previously been used to treat viral infections. This review explores the potential of mesenchymal stromal cells as cell therapy for ARDS.

Correlation between tear levels of vascular endothelial growth factor and vitamin D at retinopathy of prematurity stages in preterm infants.

Deregulation of vascular endothelial growth factor (VEGF) levels leads to retinopathy of prematurity (ROP). Vitamin D (VIT-D) is known to regulate VEGF in an oxygen dependent manner. The purpose of this study was to correlate tear levels of VEGF and VIT-D with different ROP stages in preterm infants. In this prospective cross-sectional study, we enrolled 104 pre-term infants. They were grouped into: Group-1 (Classical ROP) and Group-2 (Aggressive ROP), which were further subdivided into Group-1A (progressing), Group-1B (regressing), Group-2A (pre-treatment), and Group-2B (post-treatment). Tear VEGF and VIT-D levels and their association with different ROP stages were assessed. Stage 1 and stage 2 had higher whereas stage 3 had lower VEGF levels in Group-1B compared to Group-1A. Stage 1 and stage 3 showed higher levels of VIT-D with no difference in stage 2 in Group-1B compared to Group-1A., Group-2B showed higher VEGF and lower VIT-D levels compared to Group-2A. Presence of a positive correlation at an early stage (stage 1) of ROP and a negative correlation at a more advanced stage (stage 3) of ROP with VIT-D and VEGF implies stage-specific distinct signaling crosstalk. These findings suggest that VIT-D supplementation may have the potential to modify the course and outcome of ROP.

Safety and efficacy of combination of suberoylamilide hydroxyamic acid and mitomycin C in reducing pro-fibrotic changes in human corneal epithelial cells.

Corneal haze post refractive surgery is prevented by mitomycin c (MMC) treatment though it can lead to corneal endothelial damage, persistent epithelial defects and necrosis of cells. Suberanilohydroxamic acid (SAHA) however has been proposed to prevent corneal haze without any adverse effects. For clinical application we have investigated the short and long term outcome of cells exposed to SAHA. Human donor cornea, cultured limbal epithelial cells, corneal rims and lenticules were incubated with SAHA and MMC. The cells/tissue was then analyzed by RT-qPCR, immunofluorescence and western blot for markers of apoptosis and fibrosis. The results reveal that short term exposure of SAHA and SAHA + MMC reduced apoptosis levels and increased αSMA expression compared to those treated with MMC. Epithelial cells derived from cultured corneal rim that were incubated with the MMC, SAHA or MMC + SAHA revealed enhanced apoptosis, reduced levels of CK3/CK12, ∆NP63 and COL4A compared to other treatments. In SAHA treated lenticules TGFß induced fibrosis was reduced. The results imply that MMC treatment for corneal haze has both short term and long term adverse effects on cells and the cellular properties. However, a combinatorial treatment of SAHA + MMC prevents expression of corneal fibrotic markers without causing any adverse effect on cellular properties.

Resveratrol Rescues Human Corneal Epithelial Cells Cultured in Hyperosmolar Conditions: Potential for Dry Eye Disease Treatment.

PURPOSE: Dry eye disease (DED) is a common ocular surface condition across age groups. Recently, vitamin D deficiency has gained importance as a causative factor, and its supplementation alleviates symptoms of DED. Resveratrol (RES) regulates vitamin D receptors (VDRs) and Notch signaling. We investigated the role of RES on vitamin D levels and Notch signaling under hyperosmolar conditions. METHODS: Human corneal epithelial (HCE-T) cells were treated with RES in hyperosmolar and normal conditions. Quantitative real-time polymerase chain reaction (PCR), immunofluorescence, enzyme-linked immunosorbent assay, and western blot analysis were performed for estimating reactive oxygen species, VDR, secreted 25-hydroxyvitamin D3, and Notch signaling pathway molecules in treated and control cells. RESULTS: HCE-T cells in hyperosmolar conditions had increased reactive oxygen species levels and decreased vitamin D levels that got restored in the presence of RES. Hyperosmolarity also reduced VDR expression and Notch activity that normalized to original levels with RES. In the presence of Notch blocker LY-411575, RES could not restore VDR expression or secreted vitamin D levels in HCE-T cells exposed to hyperosmolar conditions, whereas recombinant Jagged1 restored vitamin D and VDR levels. CONCLUSIONS: RES restores vitamin D levels in hyperosmolar conditions most likely through activation of Notch signaling. Hence, RES can be a potential adjuvant in DED for patients considered for vitamin D treatment.

Vitamin-D3 (α-1, 25(OH) 2D3) Protects Retinal Pigment Epithelium From Hyperoxic Insults.

Purpose: Oxidative stress affects the retinal pigment epithelium (RPE) leading to development of vascular eye diseases. Cholecalciferol (VIT-D) is a known modulator of oxidative stress and angiogenesis. This in vitro study was carried out to evaluate the protective role of VIT-D on RPE cells incubated under hyperoxic conditions. Methods: Cadaver primary RPE (PRPE) cells were cultured in hyperoxia (40% O2) with or without VIT-D (α-1, 25(OH) 2D3). The functional and physiological effects of PRPE cells with VIT-D treatment were analyzed using molecular and biochemical tools. Results: Vascular signaling modulators, such as vascular endothelial growth factor (VEGF) and Notch, were reduced in hyperoxic conditions but significantly upregulated in the presence of VIT-D. Additionally, PRPE conditioned medium with VIT-D induced the tubulogenesis in primary human umbilical vein endothelial cells (HUVEC) cells. VIT-D supplementation restored phagocytosis and transmembrane potential in PRPE cells cultured under hyperoxia. Conclusions: VIT-D protects RPE cells and promotes angiogenesis under hyperoxic insult. These findings may give impetus to the potential of VIT-D as a therapeutic agent in hyperoxia induced retinal vascular diseases.

Matrix metalloproteinases (MMP-8, MMP-9) and the tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2) in patients with fungal keratitis.

PURPOSE: To study the infiltrating cells and quantify the levels of matrix metalloproteinases (MMP-8, MMP-9) and tissue inhibitor of metalloproteinases (TIMP-1, TIMP-2) in the cornea, tear, and serum of patients with fungal keratitis. METHODS: Experimental study. Infected corneal tissue from 4 patients with fungal keratitis (group 1) scheduled to undergo therapeutic keratoplasty accounted for the histopathologic and cytospin smear analysis. Ten patients with fungal keratitis from group 2 served for the quantification of MMPs and TIMPs. Five patients with keratoconus undergoing penetrating keratoplasty and 5 cadaver corneas were chosen as controls for group 2. Corneal buttons obtained during keratoplasty, 15 to 20 microL of tears collected using the capillary flow method, and 3 mL of blood was obtained from patients with fungal keratitis and patients with keratoconus. Corneal button sections from group 1 were stained with hematoxylin and eosin and Grocott methenamine silver nitrate for the histopathologic studies and Giemsa staining for the cytospin smear analysis. Enzyme-linked immunosorbent assay was used for the quantification of total MMP-8, MMP-9, TIMP-1, and TIMP-2 in the corneal homogenates, tear, and serum samples of group 2. RESULTS: Corneal sections from group 1 revealed dense fungal filaments and a large proportion (91.4% +/- 38%) of polymorphonuclear leukocytes (PMNs). Significant elevation in the levels of MMP-8 and MMP-9 (P < 0.05) in the fungal keratitis corneas was observed in group 2 compared with the cadaver and keratoconus corneas. The ratio of MMP/TIMP was also higher in the fungal keratitis corneas. CONCLUSIONS: Infiltrating PMNs in the cornea of patients with fungal keratitis contributed to the increased activities of MMP-8 and MMP-9, thereby enhancing tissue destruction and derangement.

Short Pulse of Clinical Concentration of Bevacizumab Modulates Human Retinal Pigment Epithelial Functionality.

PURPOSE: Cross-talk between Notch signaling and vascular endothelial growth factor (VEGF) is a major driver of angiogenesis. Here we investigated the temporal effect of bevacizumab (BEV) on Notch signaling and the functional features of cultured primary retinal pigment epithelial (PRPE) cells. METHODS: Human (cadaver) PRPE cells were treated with clinical concentrations of BEV (0.25 mg/mL). Notch signaling pathway receptors, ligands, and downstream target genes were analyzed with quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation along with phagocytosis and transmembrane potential was analyzed by fluorescent activated cell sorter (FACS) and immunofluorescence. RESULTS: Bevacizumab-treated PRPE cultures revealed a significant temporal downregulation of notch4 (P < 0.05) and Delta-like-4 (P < 0.005) gene (16% reduced) and protein (29.7% reduced) expression only at the 2-hour exposure, though secreted VEGF levels were significantly blocked (P < 0.005) at all the time points (2, 4, 6 hours). Further, a significant downregulation (P < 0.005) in cell cycle (reduced by 34.1%) and a concurrent (P < 0.005) upregulation of F-actin staining (increased by 2.5-fold) could be detected. Bevacizumab-treated PRPE cells revealed an elevated transmembrane potential (by 63%) and significant decrease (P < 0.01) in phagocytosis (by 19.25%) in comparison to untreated controls. CONCLUSIONS: There is temporal interaction between BEV and the Notch signaling pathway, specifically with Notch4 and Delta-like-ligand-4 in PRPE cultures. This transient decrease in Notch signaling can impact the functionality of RPE cells. These findings can help to provide a better understanding of the effect of long-term usage of anti-VEGF agents in the treatment of retinal degenerative and vitreoretinopathy diseases.

Angiogenic potential of vitreous from Proliferative Diabetic Retinopathy and Eales' Disease patients.

PURPOSE: Proliferative Diabetic Retinopathy (PDR) and Eales' Disease (ED) have different aetiologies although they share certain common clinical symptoms including pre-retinal neovascularization. Since there is a need to understand if the shared end-stage angiogenic pathology of PDR and ED is driven by common stimulating factors, we have studied the cytokines contained in vitreous from both patient groups and analyzed the angiogenic potential of these samples in vitro. MATERIAL AND METHODS: Vitreous samples from patients with PDR (n = 13) and ED (n = 5) were quantified for various cytokines using a cytokine biochip array and sandwich ELISA. An additional group of patients (n = 5) with macular hole (MH) was also studied for comparison. To determine the angiogenic potential of these vitreous samples, they were analyzed for their ability to induce tubulogenesis in human microvascular endothelial cells. Further, the effect of anti-VEGF (Ranibizumab) and anti-IL-6 antibodies were studied on vitreous-mediated vascular tube formation. RESULTS: Elevated levels of IL-6, IL-8, MCP-1 and VEGF were observed in vitreous of both PDR and ED when compared to MH. PDR and ED vitreous induced greater levels of endothelial cell tube formation compared to controls without vitreous (P<0.05). When VEGF in vitreous was neutralized by clinically-relevant concentrations of Ranibizumab, tube length was reduced significantly in 5 of 6 PDR and 3 of 5 ED samples. Moreover, when treated with IL-6 neutralizing antibody, apparent reduction (71.4%) was observed in PDR vitreous samples. CONCLUSIONS: We have demonstrated that vitreous specimens from PDR and ED patients share common elevations of pro-inflammatory and pro-angiogenic cytokines. This suggests that common cytokine profiles link these two conditions.

Proinflammatory cytokines and angiogenic and anti-angiogenic factors in vitreous of patients with proliferative diabetic retinopathy and eales' disease.

PURPOSE: To investigate the mechanism of angiogenesis in proliferative diabetic retinopathy (PDR) and Eales' disease (ED) on the basis of the levels of proinflammatory cytokines, angiogenic growth factor, and antiangiogenic factor in the vitreous humor. METHODS: Twenty-five patients with PDR, 10 patients with ED, and 25 with macular hole (MH) as control subjects were studied. The concentration of the proinflammatory cytokines interleukin-6 (IL-6), IL-8, IL-1 beta; chemokine-monocyte chemoattractant protein-1 (MCP-1); angiogenic factor-vascular endothelial growth factor (VEGF); and antiangiogenic factor-pigment epithelium derived factor (PEDF) in the vitreous fluid obtained from the eyes during vitrectomy were measured by sandwich enzyme linked immunosorbent assay (ELISA). RESULTS: IL-6, IL-8, MCP-1, and VEGF levels in the vitreous were significantly higher in PDR (P < 0.0001) and ED (P < 0.0001) than in MH patients. Conversely, the vitreous level of PEDF was significantly reduced in PDR (P < 0.0001) but not in ED. A significant correlation was observed between VEGF and IL-6 in ED patients. CONCLUSION: The authors demonstrate the importance of VEGF in retinal neovascularization of ED which is an idiopathic inflammatory venous occlusion. Further study is required to understand the interrelationship between VEGF and inflammatory cytokines in PDR and ED.