Abstract: Diabetic retinopathy (DR) is a complex multifactorial disease and one of the leading causes of visual impairment worldwide. DR pathogenesis is still not completely understood and, even if studies performed in the past focused on microvascular dysfunction as the main event, growing body of scientific evidence has demonstrated an important role of inflammation and neurodegeneration in the onset and progression of DR. This review summarizes current literature on the role of inflammation in the pathogenesis and progression of DR. In particular, it focuses on clinical inflammatory biomarkers detectable with non-invasive retinal imaging, suggestive of a local inflammatory condition. Current available treatments are applicable only at advanced stages of disease, therefore, there is the need to detect biomarkers of subclinical or early DR that can help in DR management before irreversible damage occurs. A better understanding of inflammatory pathways involved in DR may permit to implement more specific and personalized therapeutic strategies and clinical biomarkers may be a helpful tool in the everyday clinical practice to direct the patient to the most appropriate treatment option.
Background: Retinal endothelial cells are very active and contribute to the integrity of the neurovascular unit. Vascular dysfunction has been proposed to contribute to the pathogenesis of glaucoma. Here, we evaluated the hypothesis that ocular hypertension triggers mitochondrial alterations in endothelial cells impairing the integrity of the blood retinal barrier (BRB).
Methods: Ocular hypertension was induced by injection of magnetic microbeads into the anterior chamber of EndoMito-EGFP mice, a strain expressing green fluorescent protein selectively in the mitochondria of endothelial cells. Capillary density, mitochondrial volume, and the number of mitochondrial components were quantified in 3D-reconstructed images from whole-mounted retinas using Imaris software. Dynamin-related protein (DRP-1), mitofusin-2 (MFN-2) and optic atrophy-1 (OPA-1) expression were assessed by western blot analysis of enriched endothelial cells. Mitochondrial structure was evaluated by transmission electron microscopy (TEM) and oxygen consumption rate was monitored by Seahorse analysis. The integrity of the BRB was evaluated by quantifying Evans blue leakage.
Results: Our data demonstrate that two and three weeks after ocular hypertension induction, the total mitochondria volume in endothelial cells decreased from 0.140±0.002 μm3 from non-injured retinas to 0.108±0.005 and 0.093±0.007 μm3, respectively in glaucomatous eyes (mean ± S.E.M, ANOVA, P<0.001; N=6/group). Frequency distribution showed a substantial increase of smaller mitochondria complexes (<0.5 μm3) in endothelial cells from glaucomatous retinas. Significant upregulation of DRP-1 was found in vessels isolated from glaucomatous retinas compared to the intact retinas, while MFN-2 and OPA-1 expression was not affected. Structural alteration in endothelial cell mitochondria was confirmed by TEM, which were accompanied by a 1.93-fold reduction in the oxygen consumption rate as well as 2.6-fold increase in vasculature leakage in glaucomatous retinas (n=3–6/group). In addition, this model did not trigger changes in the density of the vascular network, suggesting that mitochondrial fragmentation was not due to endothelial cell loss.
Conclusions: This study shows that ocular hypertension leads to early alterations in the dynamic of endothelial cell mitochondria, contributing to vascular dysfunction in glaucoma.
Background: Sight-threatening diabetic macular edema (DME) is caused by increased microvascular permeability. While few direct vascular targeting strategies are available, VEGF pathway inhibition has shown to be effective in reducing retinal vascular leakage but is associated with non-negligible side effects. Thus, more options are needed. Vascular specific Activin-like kinase receptor type I (ALK1) pathway and its circulating ligand Bone morphogenetic protein-9 (BMP9) is known for its potent quiescent and stabilizing effect on the vasculature. However, little is known about this pathway in the context of microvascular permeability associated with diabetes. We hypothesize that BMP9/ALK1 pathway is inhibited in diabetic (DB) retinas leading to vascular destabilization and leakage and that its activation could re-establish proper vascular endothelial barrier functions (EBF).
Methods: The effect of hyperglycemia (i.e., HG >10 mM of D-glucose) on Alk1 signaling was evaluated in vitro by subjecting endothelial cells (EC) to increasing concentrations of D-glucose (5, 11, 25 mM) and in vivo using DB mice (Streptozotocin-induced diabetes). The contribution of Alk1 signaling on EBF was evaluated using Evans Blue permeation in inducible endothelial specific Alk1 KO mice. To evaluate the potential protective effects of BMP9/Alk1 signaling on EBF, BMP9 overexpression was achieved using adenoviral delivery in DB mice. Statistical-One-Way ANOVA or Student’s t-test was used.
Results: Endothelial tissue from DB mice showed a significant inhibition of BMP9/ALK1-canonical Smad1,5,8 quiescence signaling (DB n=5; CTL n=4; P<0.01), which was associated with reduced expression of target genes (JAG1, Id1,3, Hey1,2 & HES). Moreover, we showed that retinal hyperpermeability associated with diabetes was exacerbated in Alk1 heterozygote mice (n=4–9/group; P<0.0001). Finally, we demonstrated that activation of Alk1 signaling in ECs prevented vascular permeability induced by HG, both in vitro (n=3; P=0.009) and in vivo (n=4–9/group; P<0.0001).
Conclusions: Consistent with our hypothesis, vascular stability and quiescence induced by BMP9-ALK1 signaling is inhibited in the DB/HG endothelium which could be an important factor in vascular leakage leading to DME. Our results show that activation of this pathway could offer a therapeutically interesting future option to slow down the onset of DME.
Background: The goal of this study was to engineer an epithelialized and endothelialized pigmented choroidal substitute using the self-assembly approach of tissue engineering.
Methods: Cells from human choroids were isolated and cultured. Culture purity was assessed using immunostaining (CD31, HMB45, vimentin, keratins 8/18). To engineer the choroid, fibroblasts were cultured in the presence of serum and ascorbic acid to promote extracellular matrix (ECM) assembly. Endothelial cells, melanocytes or RPE cells were separately seeded on the stromal substitutes. Choroidal substitutes were further characterized by histology, mass spectrometry, immunostaining, and compared to native human choroids.
Results: The technique used to isolate choroidal cells yielded pure cultures of fibroblasts, melanocytes and vascular endothelial cells. The stromal substitutes engineered using the self-assembly approach were composed of collagen (types I, VI, XII and XIV), proteoglycans (decorin, lumican) and other ECM proteins. Protein expression was confirmed using immunostaining. Endothelial cells spontaneously assembled into capillary-like structures and vascular networks when cocultured with fibroblast-containing ECM sheets.
Conclusions: This study shows that the self-assembly approach of tissue engineering can be used to reconstruct a choroid using native cells. This model represents a unique tool to better understand the crosstalk between the different choroidal cell types and cell-ECM interactions.
Abstract: Genetic studies have revealed that variants in genes that encode regulators of the complement system are major risk factors for the development of age-related macular degeneration (AMD). The biochemical consequences of the common polymorphism in complement factor H (Tyr402His) include increased formation of the membrane attack complex (MAC), which is deposited at the level of the inner choroid and choriocapillaris. Whereas the MAC is normally protective against foreign pathogens, it can also damage resident bystander cells when it is insufficiently regulated. Indeed, human maculas with early AMD show loss of endothelial cells in the choriocapillaris, the principal site of MAC activation. Modeling of MAC injury of choroidal endothelial cells in vitro reveals that these cells are susceptible to cell lysis by the MAC, and that unlysed cells alter their gene expression profile to undergo a pro-angiogenic phenotype that includes increased expression of matrix metalloproteinase-9. Strategies for protecting choriocapillaris endothelial cells from MAC-mediated lysis and for replacing lysed endothelial cells will be discussed.
Abstract: Corneal blindness represents one of the world’s three major causes of blindness, and the fundamental problem of corneal transplantation is a severe shortage of donor tissues worldwide, resulting in approximately 1.5 million new cases of blindness annually. To address the growing need for corneal transplants two main approaches are being pursued: allogenic and bioengineering cornea. Bioengineering corneas are constructed by naturally generating an extracellular matrix (ECM) component as the scaffold structure with or without corneal cells. It is well established that the scaffold structure directs the fate of cells, therefore, the fabrication of the correct scaffold structure components could produce an ideal corneal substitute, able to mimic the native corneal function. Another key factor in the construction of tissue engineering cornea is seed cells. However, unlike the epithelium and stroma cells, human cornea endothelium cells (HCECs) are notorious for having a limited proliferative capacity in vivo because of the mitotic block at the G1 phase of the cell cycle due to “contact-inhibition”. This review will focus on the main concepts of recent progress towards the scaffold and seed cells, especially endothelial cells for bioengineering cornea, along with future perspectives.
Abstract: Since the 21st century, the development of corneal tissue engineering technology has been developing rapidly. With the progress of biomaterials, cell culture and tissue engineering technology, tissue engineering cornea has gained great development in both basic scientific research and clinical application. In particular, tissue engineered corneal scaffolds are the core components of tissue engineered corneas. It is the focus of current research on tissue engineering cornea to search for scaffolds with good biocompatibility, high safety and good biomechanical properties. In this paper, the recent research progress of tissue engineering corneal materials is reviewed.
Background: To evaluate the association between corneal central endothelial cell count (CECC) with reactivity for hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), human T-lymphotropic virus-1 (HTLV1), and syphilis from an eye bank database.
Methods: Eye bank data included 19,159 donors and 38,318 corneas screened for HBV, HCV, HIV, HTLV1, and syphilis from July 2007–May 2015. Linear and binary mixed effects models were used to determine the adjusted marginal effect a positive viral screening test had on CECC and morphology, respectively. The models were adjusted for age, race, gender, lens status, and death to preservation. Eyes with missing data were excluded from the analysis. Statistical significance was defined as P values <0.05.
Results: A total of 18,097 donors and 35,136 corneas were included in the final analysis. Average CECC for eyes with negative viral screening was 2,597±436 while the average CECC for eyes screening positive for syphilis, HBV, HCV, HIV, and HTLV1 were 2,638±392 (P=0.073), 2,569±419 (P=0.815), 2,603±363 (P=0.207), 2,615±360 (P=0.733), and 2,625±436 (P=0.362) respectively.
Conclusions: The presence of HBV, HCV, HIV, HTLV1, and syphilis display no association with a statistically significant difference in CECC when compared to normal non-diseased donors.
Abstract: The efficacy of selective laser trabeculoplasty (SLT) to treat open-angle glaucoma (OAG) and ocular hypertension (OHT) has been increasingly substantiated lately. Repeated SLT is usually needed to control intraocular pressure (IOP), be it as primary or adjunctive therapy. We review the studies available, in terms of SLT repeatability; and conclude comparable efficacy, with success rate, duration and complications similar to those in initial SLT.
