Lysyl Oxidase Promotes Actin-Dependent Neutrophil Activation and Cytotoxicity Toward Retinal Endothelial Cells in Diabetes.

<p dir="ltr">Activated neutrophils contribute to retinal endothelial cell (EC) death and capillary degeneration associated with early diabetic retinopathy (DR), a major vision-threatening complication of diabetes. However, the factors and mechanisms driving neutrophil activation and cytotoxicity in diabetes remain insufficiently understood. Here we show that lysyl oxidase (LOX), a matrix crosslinking and stiffening enzyme that increases retinal EC susceptibility to activated neutrophils, simultaneously activates neutrophils in its soluble form. Specifically, treatment of diabetic mice with LOX inhibitor β-aminopropionitrile (BAPN) prevented the diabetes-induced increase in neutrophil activation (extracellular release of neutrophil elastase and superoxide) and cytotoxicity towards co-cultured mouse retinal ECs. Mouse neutrophils and differentiated (neutrophil-like) human HL-60 cells treated with recombinant LOX alone exhibited significant activation and cytotoxicity. Mechanistically, this LOX-induced neutrophil activation was associated with biphasic F-actin remodeling, with the initial and rapid (~10 min) F-actin depolymerization followed by a significant increase in F-actin polymerization and polarization. Preventing the initial F-actin depolymerization blocked LOX-induced neutrophil activation and cytotoxicity towards co-cultured retinal ECs. Finally, we show that this biphasic F-actin remodeling is essential for LOX-induced membrane aggregation of azurophilic granule marker CD63 and NADPH organizer p47<sup>phox</sup> that are associated with extracellular release of neutrophil elastase and superoxide, respectively. By revealing a previously unrecognized causal link between LOX and actin-dependent neutrophil activation in diabetes, these findings provide fresh mechanistic insights into the proinflammatory role of LOX in early DR that goes beyond its canonical matrix-stiffening effects.</p>

<p dir="ltr">Activated neutrophils contribute to retinal endothelial cell (EC) death and capillary degeneration associated with early diabetic retinopathy (DR), a major vision-threatening complication of diabetes. However, the factors and mechanisms driving neutrophil activation and cytotoxicity in diabetes remain insufficiently understood. Here we show that lysyl oxidase (LOX), a matrix crosslinking and stiffening enzyme that increases retinal EC susceptibility to activated neutrophils, simultaneously activates neutrophils in its soluble form. Specifically, treatment of diabetic mice with LOX inhibitor β-aminopropionitrile (BAPN) prevented the diabetes-induced increase in neutrophil activation (extracellular release of neutrophil elastase and superoxide) and cytotoxicity towards co-cultured mouse retinal ECs. Mouse neutrophils and differentiated (neutrophil-like) human HL-60 cells treated with recombinant LOX alone exhibited significant activation and cytotoxicity. Mechanistically, this LOX-induced neutrophil activation was associated with biphasic F-actin remodeling, with the initial and rapid (~10 min) F-actin depolymerization followed by a significant increase in F-actin polymerization and polarization. Preventing the initial F-actin depolymerization blocked LOX-induced neutrophil activation and cytotoxicity towards co-cultured retinal ECs. Finally, we show that this biphasic F-actin remodeling is essential for LOX-induced membrane aggregation of azurophilic granule marker CD63 and NADPH organizer p47<sup>phox</sup> that are associated with extracellular release of neutrophil elastase and superoxide, respectively. By revealing a previously unrecognized causal link between LOX and actin-dependent neutrophil activation in diabetes, these findings provide fresh mechanistic insights into the proinflammatory role of LOX in early DR that goes beyond its canonical matrix-stiffening effects.</p>

Activated neutrophils contribute to retinal endothelial cell (EC) death and capillary degeneration associated with early diabetic retinopathy (DR). However, the factors and mechanisms driving neutrophil activation and cytotoxicity in diabetes remain insufficiently understood. Here we show that lysyl oxidase (LOX), a collagen crosslinking and matrix stiffening enzyme that increases retinal EC susceptibility to activated neutrophils, simultaneously activates neutrophils in its alternate soluble form. Specifically, treatment of diabetic mice with LOX inhibitor β-aminopropionitrile (BAPN) prevented the diabetes-induced increase in neutrophil activation (extracellular release of neutrophil elastase and superoxide) and cytotoxicity towards co-cultured mouse retinal ECs. Mouse neutrophils and differentiated (neutrophil-like) human HL-60 cells treated with recombinant LOX alone exhibited similar activation and cytotoxicity. Mechanistically, this LOX-induced neutrophil activation was associated with biphasic F-actin remodeling, with the initial and rapid (<15 min) F-actin depolymerization followed by a significant increase in F-actin polymerization and polarization. Preventing the initial F-actin depolymerization blocked LOX-induced neutrophil activation and cytotoxicity towards co-cultured retinal ECs. Finally, we show that this biphasic F-actin remodeling is essential for LOX-induced membrane clustering of azurophilic granule marker CD63 and NADPH organizer p47 that are associated with extracellular release of neutrophil elastase and superoxide, respectively. By revealing a causal and previously unrecognized link between LOX and actin-dependent neutrophil activation in diabetes, these findings provide fresh mechanistic insights into the proinflammatory role of LOX in early DR that goes beyond its canonical matrix-stiffening effects.

The effect of hyperglycaemia on permeability and the expression of junctional complex molecules in human retinal and choroidal endothelial cells

Diabetic retinopathy (DR) is a severe diabetes-induced eye disease, in which its pathological phenomena basically include abnormal proliferation, migration, and angiogenesis of microvascular endothelial cells in the retina. Long non-coding RNAs (lncRNAs) have been proven to be important regulators in various biological processes, but their participation in DR remains largely undiscovered. In the present study, we aimed to unveil the role of lncRNA small nucleolar RNA host gene 16 (SNHG16) in regulating the functions of human retinal microvascular endothelial cells (hRMECs) under a high-glucose (HG) condition. We found that SNHG16 expression was significantly upregulated in hRMECs treated with HG. Functionally, SNHG16 could facilitate hRMEC proliferation, migration, and angiogenesis. Moreover, SNHG16 was associated with nuclear factor κB (NF-κB) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. Mechanistically, SNHG16 could promote hRMEC dysfunction by sequestering microRNA (miR)-146a-5p and miR-7-5p to act as a competing endogenous RNA (ceRNA) with interleukin-1 receptor-associated kinase 1 (IRAK1) and insulin receptor substrate 1 (IRS1). In conclusion, our results illustrated the potential role of SNHG16 in facilitating hRMEC dysfunction under HG treatment, providing a novel approach for DR therapy.

Objective To investigate whether the advanced glycation end products (AGEs) can induce the apoptosis of rat retinal microvascular endothelial cells and the related mechanisms. Methods Twenty SPF male Wistar rats aged 7 to 8 weeks were used. Microvascular endothelial cells was isolated from rat retinal, and identified by immunofluorescence staining and flow cytometry for vWF, and the tube formation on Matrigel. MTT assay was used to analyze the effect of AGEs on the proliferation of rat retinal microvascular endothelial cells. Flow cytometry was used to detect AGEs-induced apoptosis. Reverse transcription PCR and flow cytometry were used to analyze the expression level of RAGE, PKC, ICAM-1 and iNOS with or without AGEs in culture medium. t-test were used for statistical analysis. Results The isolated and purified rat retinal microvascular endothelial cells express vWF, and can form capillary-like network structure in Matrigel. AGEs can inhibit proliferation of rat retinal endothelial cells in dose- and time-dependent ways: the proliferation of endothelial cells in 200 mg/L AGEs group was lower than control at 5-day(t=8.9, P<0.05), and more significant at 7- and 9-day (t=15.7 or 46.1, both P<0.01). The inhibition to proliferation of endothelial cells by 400 mg/L AGEs was significant at 3-day(t=12.5, P<0.05), and became greatly at 5-, 7- and 9-day(t=22.4, 41.5 or 77.7, all P<0.01). AGEs also induced apoptosis of retinal endothelial cells. Furthermore, AGEs can up-regulate the expression of RAGE, PKC, iNOS and ICAM-1 at mRNA level(t=91.8, 9.22, 16 and 42, both P<0.01) and protein level(t=20.2, 12.3, 7.7 and 13.9, all P<0.01). Conclusion AGEs might induced phenotypic and functional changes of rat retinal microvascular endothelial cells by up-regulating RAGE. Key words: Advanced glycation end products; Retinal endothelial cell; Apoptosis; Phenotype; Function

Angiogenic and Immunologic Proteins Identified by Deep Proteomic Profiling of Human Retinal and Choroidal Vascular Endothelial Cells: Potential Targets for New Biologic Drugs

Increased retinal vascular permeability is one of the earliest manifestations of diabetic retinopathy. The aim of this study was to investigate the role of hyperglycemia-induced platelet endothelial cell adhesion molecule-1 loss on retinal vascular permeability via the β-catenin pathway. Type I diabetes was induced in male Wistar rats using streptozotocin injections, with age-matched non-diabetic rats as controls. Rat retinal microvascular endothelial cells were grown under normal or high glucose conditions for 6days. Small interfering Ribonucleic Acid was used to knock down platelet endothelial cell adhesion molecule-1 in rat retinal microvascular endothelial cells for loss-of-function studies. Retinas and rat retinal microvascular endothelial cells were subjected to Western blot, immunofluorescence labeling, and co-immunoprecipitation analyses to assess protein levels and interactions. A biotinylated gelatin and fluorescein isothiocyanate-avidin assay was used for retinal endothelial cell permeability studies. β-catenin, β-catenin/platelet endothelial cell adhesion molecule-1 interaction, active Src homology 2 domain-containing protein tyrosine phosphatasewere significantly decreased, while β-catenin ubiquitination levels and endothelial permeability were significantly increased, in hyperglycemic retinal endothelial cells. Similar results were observed with platelet endothelial cell adhesion molecule-1 partial knockdown, where β-catenin and active Src homology 2 domain-containing protein tyrosine phosphatase levels were decreased, while phospho-β-catenin and retinal endothelial cell permeability were increased. Platelet endothelial cell adhesion molecule-1 loss may contribute to increased retinal endothelial cell permeability by attenuating β-catenin levels under hyperglycemic conditions.

PurposeTo investigate the effect of reducing high glucose (HG)-induced lysyl oxidase (LOX) overexpression and increased activity on retinal endothelial cell apoptosis.MethodsRat retinal endothelial cells (RRECs) were grown in normal (N) or HG (30 mM glucose) medium for 7 days. In parallel, RRECs were grown in HG medium and transfected with LOX small interfering RNA (siRNA), scrambled siRNA as control, or exposed to β-aminopropionitrile (BAPN), a LOX inhibitor. LOX expression, AKT activation, and caspase-3 activity were determined by Western blot (WB) analysis and apoptosis by differential dye staining assay. Moreover, to determine whether diabetes-induced LOX overexpression alters AKT activation and promotes apoptosis, changes in LOX expression, AKT phosphorylation, caspase-3 activation, and Bax expression were assessed in retinas of streptozotocin (STZ)-induced diabetic mice and LOX heterozygous knockout (LOX+/−) mice.ResultsWB analysis indicated significant LOX overexpression and reduced AKT activation under HG condition in RRECs. Interestingly, when cells grown in HG were transfected with LOX siRNA or exposed to BAPN, the number of apoptotic cells was significantly decreased concomitant with increased AKT phosphorylation. Diabetic mouse retinas exhibited LOX overexpression, decreased AKT phosphorylation, and increased Bax and caspase-3 activation compared to values in nondiabetic mice. In LOX+/− mice, reduced LOX levels were observed with increased AKT activity, and reduced Bax and caspase-3 activity. Furthermore, decreased levels of LOX in the LOX+/− mice was protective against diabetes-induced apoptosis.ConclusionsFindings from this study indicate that preventing LOX overexpression may be protective against HG-induced apoptosis in retinal vascular cells associated with diabetic retinopathy.

Radiation retinopathy (RR) is a common complication following radiation therapy of globe, head, and neck malignancies, and is characterized by microangiopathy, neuroretinopathy, and the irreversible loss of visual function. To date, there is no effective treatment for RR. Stem cells have been clinically used to treat retinal degeneration. CD133+CD34+ cells from human umbilical cord blood (hUCB-CD133+CD34+ cells), a subpopulation of hematopoietic stem cells, were applied to determine their protective efficacy on irradiated rat retinas. After X-ray irradiation on the retinas, rats were intravitreally injected with hUCB-CD133+CD34+ cells. Transplantation of hUCB-CD133+CD34+ cells prevented retinal dysfunction 2 weeks post-operation and lasted at least 8 weeks. CD133+CD34+ cells were distributed along the retinal vessel and migrated to the ganglion cell layer. Moreover, grafted CD133+CD34+ cells reduced the apoptosis of endothelial and ganglion cells in irradiated rats and increased the number of survived CD31+ retinal endothelial cells and Brn3a+ ganglion cells at 2 and 4 weeks, respectively, post-operation. Co-culturing of CD133+CD34+ cells or supernatants with irradiated human retinal microvascular endothelial cells (hRECs) in vitro, confirmed that CD133+CD34+ cells ameliorated hREC apoptosis caused by irradiation. Mechanistically, we found that angioprotective mediators and neurotrophic factors were secreted by CD133+CD34+ cells, which might attenuate irradiation-induced injury of retinal endothelial cells and ganglion cells. hUCB-CD133+CD34+ cell transplantation, as a novel treatment, protects retinal endothelial and ganglion cells of X-irradiated rat retinas, possibly through angioprotective and neurotrophic factors.

Consistent with clinical observations that posterior uveitis frequently involves the retinal vasculature and recent recognition of vascular heterogeneity, the hypothesis for this study was that retinal vascular endothelium was a cell population of unique molecular phenotype. Donor-matched cultures of primary retinal and choroidal endothelial cells from six human cadavers were incubated with either Toxoplasma gondii tachyzoites (10:1, parasites per cell) or Escherichia coli lipopolysaccharide (100 ng/mL); control cultures were simultaneously incubated with medium. Gene expression profiling of endothelial cells was performed using oligonucleotide arrays containing probes designed to detect 8746 human transcripts. After normalization, differential gene expression was assessed by the significance analysis of microarrays, with the false-discovery rate set at 5%. For selected genes, differences in the level of expression between retinal and choroidal cells were evaluated by real-time RT-PCR. Graphic descriptive analysis demonstrated a strong correlation between gene expression of unstimulated retinal and choroidal endothelial cells, but also highlighted distinctly different patterns of expression that were greater than differences noted between donors or between unstimulated and stimulated cells. Overall, 779 (8.9%) of 8746 transcripts were differentially represented. Of note, the 330 transcripts that were present at higher levels in retinal cells included a larger percentage of transcripts encoding molecules involved in the immune response. Differential gene expression was confirmed for 12 transcripts by RT-PCR. Retinal and choroidal vascular endothelial cells display distinctive gene expression profiles. The findings suggest the possibility of treating posterior uveitis by targeting specific interactions between the retinal endothelial cell and an infiltrating leukocyte.

The purpose of this study was to establish and characterize a retinal pericyte cell line from retinal capillaries of transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene (tsA58 Tg rat), and to apply this to the co-culture with a retinal capillary endothelial cell line. The conditionally immortalized rat retinal pericyte cell lines (TR-rPCTs), which express a temperature-sensitive large T-antigen, were obtained from two tsA58 Tg rats. These cell lines had a multicellular nodule morphology and reacted positively with von Kossa staining, a marker of calcification. TR-rPCTs cells expressed mRNA of pericyte markers such as rat intercellular adhesion molecule-1, platelet-derived growth factor-receptor beta, angiopoietin-1, and osteopontin. Western blot analysis indicated that alpha-smooth muscle actin (alpha-SMA) was expressed in TR-rPCT3 and 4 cells. In contrast, alpha-SMA was induced by transforming growth factor-beta1 and its enhancement was reduced by basic fibroblast growth factor in TR-rPCT1 and 2 cells. When TR-rPCT1 cells were cultured with a rat retinal endothelial cell line (TR-iBRB2) in a contact co-culture system, the number of TR-iBRB2 cells were significantly reduced in comparison with that of a single culture of TR-iBRB2 cells, suggesting that physical contact between pericytes and retinal endothelial cells is important for the growth of retinal endothelial cells. In conclusion, conditionally immortalized retinal pericyte cell lines were established from tsA58 Tg rats. These cell lines exhibited the properties of retinal pericytes and can be applied in co-culture systems with a retinal capillary endothelial cell line.

Retinal endothelial cells are more susceptible to oxidative stress and increased permeability than brain-derived endothelial cells

Purpose: Features of cellular senescence have been described in diabetic retinal vasculature. The aim of this study was to investigate how the high glucose microenvironment impacts on the senescence program of retinal endothelial cells. Methods: Human retinal microvascular endothelial cells were cultured under control and high glucose conditions of 5 mM and 25 mM D-glucose, respectively. Isomeric l-glucose was used as the osmotic control. Cells were counted using CASY technology until they reached their Hayflick limit. Senescence-associated β-Galactosidase was used to identify senescent cells. Endothelial cell functionality was evaluated by the clonogenic, 3D tube formation, and barrier formation assays. Cell metabolism was characterized using the Seahorse Bioanalyzer. Gene expression analysis was performed by bulk RNA sequencing. Retinal tissues from db/db and db/+ mice were evaluated for the presence of senescent cells. Publicly available scRNA-sequencing data for retinas from Akimba and control mice was used for gene set enrichment analysis. Results: Long term exposure to 25 mM D-Glucose accelerated the establishment of cellular senescence in human retinal endothelial cells when compared to 5 mM D-glucose and osmotic controls. This was shown from 4 weeks, by a significant slower growth, higher percentages of cells positive for senescence-associated β-galactosidase, an increase in cell size, and lower expression of pRb and HMGB2. These senescence features were associated with decreased clonogenic capacity, diminished tubulogenicity, and impaired barrier function. Long term high glucose-cultured cells exhibited diminished glycolysis, with lower protein expression of GLUT1, GLUT3, and PFKFB3. Transcriptomic analysis, after 4 weeks of culture, identified downregulation of ALDOC, PFKL, and TPI1, in cells cultured with 25 mM D-glucose when compared to controls. The retina from db/db mice showed a significant increase in acellular capillaries associated with a significant decrease in vascular density in the intermediate and deep retinal plexuses, when compared to db/+ mice. Senescent endothelial cells within the db/db retinal vasculature were identified by senescence-associated β-galactosidase staining. Analysis of single cell transcriptomics data for the Akimba mouse retina highlighted an enrichment of senescence and senescence-associated secretory phenotype gene signatures when compared to control mice. Conclusion: A diabetic-like microenvironment of 25 mM D-glucose was sufficient to accelerate the establishment of cellular senescence in human retinal microvascular endothelial cells.

OBJECTIVEIn diabetes, retinal vascular basement membrane (BM) undergoes significant thickening and compromises vessel function including increased vascular permeability, a prominent lesion of early diabetic retinopathy. In this study we determined whether altered expression and activity of lysyl oxidase (LOX), a cross-linking enzyme, may compromise vascular basement membrane functional integrity under high-glucose (HG) conditions.RESEARCH DESIGN AND METHODSRat retinal endothelial cells (RRECs) grown in normal (5 mmol/l) or HG (30 mmol/l glucose) medium for 7 days were assessed for expression of LOX and proLOX by Western blot analysis and LOX enzyme activity. To determine whether HG alters cellular distribution patterns of LOX and proLOX, immunostaining with respective antibodies was performed. Similarly, cells grown in normal or HG medium were subjected to both LOX inhibition with β-aminopropionitrile (BAPN) and by small interfering RNA knockdown, and respectively examined for cell monolayer permeability. Additionally, retinas of streptozotocin (STZ)-induced diabetic rats were analyzed to determine if diabetes altered LOX expression.RESULTSWestern blot analysis revealed significantly increased LOX and proLOX expression in cells grown in HG medium compared with those grown in normal medium. The increased LOX level was strikingly similar to LOX upegulation in the diabetic retinas. In cells grown in HG medium, LOX activity and cell monolayer permeability was significantly increased, as were LOX and proLOX immunostaining. Small interfering RNA- or BAPN–induced-specific blockage of LOX expression or activity, respectively, reduced cell monolayer permeability.CONCLUSIONSHG-induced increased LOX expression and activity compromises barrier functional integrity, a prominent lesion of diabetic retinopathy.