A double-crosslinked nanocellulose-reinforced dexamethasone-loaded collagen hydrogel for corneal application and sustained anti-inflammatory activity.

In cases of blinding disease or trauma, hydrogels have been proposed as scaffolds for corneal regeneration and vehicles for ocular drug delivery. Restoration of corneal transparency, augmenting a thin cornea and postoperative drug delivery are particularly challenging in resource-limited regions where drug availability and patient compliance may be suboptimal. Here, we report a bioengineered hydrogel based on porcine skin collagen as an alternative to human donor corneal tissue for applications where long-term stability of the hydrogel is required. The hydrogel is reinforced with cellulose nanofibers extracted from the Ciona intestinalis sea invertebrate followed by double chemical and photochemical crosslinking. The hydrogel is additionally loaded with dexamethasone to provide sustained anti-inflammatory activity. The reinforced double-crosslinked hydrogel after drug loading maintained high optical transparency with significantly improved mechanical characteristics compared to non-reinforced hydrogels, while supporting a gradual sustained drug release for 60 days in vitro. Dexamethasone, after exposure to crosslinking and sterilization procedures used in hydrogel production, inhibited tube formation and cell migration of TNFα-stimulated vascular endothelial cells. The drug-loaded hydrogels suppressed key pro-inflammatory cytokines CCL2 and CXCL5 in TNFα-stimulated human corneal epithelial cells. Eight weeks after intra-stromal implantation in the cornea of 12 New-Zealand white rabbits subjected to an inflammatory suture stimulus, the dexamethasone-releasing hydrogels suppressed TNFα, MMP-9, and leukocyte and fibroblast cell invasion, resulting in reduced corneal haze, sustained corneal thickness and stromal morphology, and reduced overall vessel invasion. This collagen-nanocellulose double-crosslinked hydrogel can be implanted to treat corneal stromal disease while suppressing inflammation and maintaining transparency after corneal transplantation. STATEMENT OF SIGNIFICANCE: To treat blinding diseases, hydrogel scaffolds have been proposed to facilitate corneal restoration and ocular drug delivery. Here, we improve on a clinically tested collagen-based scaffold to improve mechanical robustness and enzymatic resistance by incorporating sustainably sourced nanocellulose and dual chemical-photochemical crosslinking to reinforce the scaffold, while simultaneously achieving sustained release of an incorporated anti-inflammatory drug, dexamethasone. Evaluated in the context of a corneal disease model with inflammation, the drug-releasing nanocellulose-reinforced collagen scaffold maintained the cornea's transparency and resisted degradation while suppressing inflammation postoperatively. This biomaterial could therefore potentially be applied in a wider range of sight-threatening diseases, overcoming suboptimal administration of postoperative medications to maintain hydrogel integrity and good vision.

Association of Placental Growth Factor and Angiopoietin in Human Retinal Endothelial Cell-Pericyte co-Cultures and iPSC-Derived Vascular Organoids.

PURPOSE: Placental growth factor (PlGF) and Angiopoietin (Ang)-1 are two proteins that are involved in the regulation of endothelial cell (EC) growth and vasculature formation. In the retina and endothelial cells, pericytes are the major source of both molecules. The purpose of this study is to examine the association of PlGF and Ang-1 with human EC/pericyte co-cultures and iPSC-derived vascular organoids. METHODS: In this study, we used co-cultures of human primary retinal endothelial cells (HREC) and primary human retinal pericytes (HRP), western blotting, immunofluorescent analysis, TUNEL staining, LDH-assays, and RNA seq analysis, as well as human-induced pluripotent stem cells (iPSC), derived organoids (VO) to study the association between PlGF and Ang-1. RESULTS: Inhibition of PlGF by PlGF neutralizing antibody in HREC-HRP co-cultures resulted in the increased expression of Ang-1 and Tie-2 in a dose-dependent manner. This upregulation was not observed in HREC and HRP monocultures but only in co-cultures suggesting the association of pericytes and endothelial cells. Furthermore, Vascular endothelial growth factor receptor 1 (VEGFR1) inhibition abolished the Ang-1 and Tie-2 upregulation by PlGF inhibition. The pericyte viability in high-glucose conditions was also reduced by VEGFR1 neutralization. Immunofluorescent analysis showed that Ang-1 and Ang-2 were expressed mainly by perivascular cells in the VO. RNA seq analysis of the RNA isolated from VO in high glucose conditions indicated increased PlGF and Ang-2 expressions in the VO. PlGF inhibition increased the expression of Ang-1 and Tie-2 in VO, increasing the pericyte coverage of the VO microvascular network. CONCLUSION: Combined, these results suggest PlGF's role in the regulation of Ang-1 and Tie-2 expression through VEGFR1. These findings provide new insights into the neovascularization process in diabetic retinopathy and new targets for potential therapeutic intervention.

Bioengineered corneal tissue for minimally invasive vision restoration in advanced keratoconus in two clinical cohorts.

Visual impairment from corneal stromal disease affects millions worldwide. We describe a cell-free engineered corneal tissue, bioengineered porcine construct, double crosslinked (BPCDX) and a minimally invasive surgical method for its implantation. In a pilot feasibility study in India and Iran (clinicaltrials.gov no. NCT04653922 ), we implanted BPCDX in 20 advanced keratoconus subjects to reshape the native corneal stroma without removing existing tissue or using sutures. During 24 months of follow-up, no adverse event was observed. We document improvements in corneal thickness (mean increase of 209 ± 18 µm in India, 285 ± 99 µm in Iran), maximum keratometry (mean decrease of 13.9 ± 7.9 D in India and 11.2 ± 8.9 D in Iran) and visual acuity (to a mean contact-lens-corrected acuity of 20/26 in India and spectacle-corrected acuity of 20/58 in Iran). Fourteen of 14 initially blind subjects had a final mean best-corrected vision (spectacle or contact lens) of 20/36 and restored tolerance to contact lens wear. This work demonstrates restoration of vision using an approach that is potentially equally effective, safer, simpler and more broadly available than donor cornea transplantation.

Data mining and network analysis reveals C-X-C chemokine receptor type 5 is involved in the pathophysiology of age-related macular degeneration.

Our previous studies found that the C-X-C motif chemokine receptor 5 (CXCR5) loss leads to retinal pigment epithelium (RPE) dysfunction and AMD pathogenesis. The current study aimed to characterize the G protein-coupled receptor (GPCR) structure of CXCR5 and analyze its interactions with AMD-related risk genes. The sequence alignments, homology model of CXCR5 and structural assessment analysis were performed. Data and text mining were then performed to identify AMD-related risk genes and their interaction with CXCR5 using statistical and mathematical algorithms. Sequence alignment and phylogenetic tree analysis revealed that human CXCR5 was highly similar (85.4839%) to the rabbit. The least similarity (33.871%) was found to be in zebrafish compared to the other species. The CXCR5 model structural assessment and secondary structure analysis exhibited an excellent model. Network analysis revealed that IL10, TNF, ICAM1, CXCL1, CXCL8, APP, TLR4, SELL, C3, IL17A and CCR2 were the most connected genes CXCR5. These findings suggest that CXCR5 signaling may regulate the biological function of RPE and modulate AMD pathophysiology via GPCR signaling and interacting with identified AMD risk genes. In summary, the data presented here provide novel and crucial insights into the molecular mechanisms of CXCR5 involvement in AMD.Communicated by Ramaswamy H. Sarma.

NF-κB activation in retinal microglia is involved in the inflammatory and neovascularization signaling in laser-induced choroidal neovascularization in mice.

PURPOSE: To evaluate Nuclear Factor NF-κB (NF-κB) signaling on microglia activation, migration, and angiogenesis in laser-induced choroidal neovascularization (CNV). METHODS: Nine-week-old C57BL/6 male mice were randomly assigned to IMD-0354 treated or untreated groups (5 mice, 10 eyes per group). CNV was induced with a 532-nm laser. Laser spots (power 250 mW, spot size 100 µm, time of exposure 50 ms) were created in each eye using a slit-lamp delivery system. Selective inhibitor of nuclear factor kappa-B kinase subunit beta (IKK2) inhibitor IMD-0354 (10 µg) was delivered subconjunctivally; vehicle-treated mice were the control. The treatment effect on CNV development was assessed at five days post-CNV induction in vivo in C57BL/6 and Cx3cr1gfp/wt mice by fluorescent angiography, fundus imaging, and ex vivo by retinal flatmounts immunostaining and Western blot analysis of RPE/Choroidal/Scleral complexes (RCSC) lysates. In vitro evaluations of IMD-0354 effects were performed in the BV-2 microglial cell line using lipopolysaccharide (LPS) stimulation. RESULTS: IMD-0354 caused a significant reduction in the fluorescein leakage and size of the laser spot, as well as a reduction in microglial cell migration and suppression of phospho-IκBα, Vascular endothelial growth factor (VEGF-A), and Prostaglandin-endoperoxide synthase 2 (COX-2). In vivo and ex vivo observations demonstrated reduced lesion size in mice, CD68, and Allograft inflammatory factor 1 (IBA-1) positive microglia cells migration to the laser injury site in IMD-0354 treated eyes. The data further corroborate with GFP-positive cells infiltration of the CNV site in Cx3cr1wt/gfp mice. In vitro IMD-0354 (10-25 ng/ml) treatment reduced NF-κB activation, expression of COX-2, caused decreased Actin-F presence and organization, resulting in reduced BV-2 cells migration capacity. CONCLUSION: The present data indicate that NF-κB activation in microglia and it's migration capacity is involved in the development of laser CNV in mice. Its suppression by NF-κB inhibition might be a promising therapeutic strategy for wet AMD.

Relapse of pathological angiogenesis: functional role of the basement membrane and potential treatment strategies.

Blinding eye diseases such as corneal neovascularization, proliferative diabetic retinopathy, and age-related macular degeneration are driven by pathological angiogenesis. In cancer, angiogenesis is key for tumor growth and metastasis. Current antiangiogenic treatments applied clinically interfere with the VEGF signaling pathway-the main angiogenic pathway-to inhibit angiogenesis. These treatments are, however, only partially effective in regressing new pathologic vessels, and the disease relapses following cessation of treatment. Moreover, the relapse of pathological angiogenesis can be rapid, aggressive and more difficult to treat than angiogenesis in the initial phase. The manner in which relapse occurs is poorly understood; however, recent studies have begun to shed light on the mechanisms underlying the revascularization process. Hypotheses have been generated to explain the rapid angiogenic relapse and increased resistance of relapsed disease to treatment. In this context, the present review summarizes knowledge of the various mechanisms of disease relapse gained from different experimental models of pathological angiogenesis. In addition, the basement membrane-a remnant of regressed vessels-is examined in detail to discuss its potential role in disease relapse. Finally, approaches for gaining a better understanding of the relapse process are discussed, including prospects for the management of relapse in the context of disease.

Deficiency of C-X-C chemokine receptor type 5 (CXCR5) gene causes dysfunction of retinal pigment epithelium cells.

Homeostasis of the retinal pigment epithelium (RPE) is essential for the health and proper function of the retina. Regulation of RPE homeostasis is, however, largely unexplored, yet dysfunction of this process may lead to retinal degenerative diseases, including age-related macular degeneration (AMD). Here, we report that chemokine receptor CXCR5 regulates RPE homeostasis through PI3K/AKT signaling and by suppression of FOXO1 activation. We used primary RPE cells isolated from CXCR5-deficient mice and wild type controls, as well as ex vivo RPE-choroidal-scleral complexes (RCSC) to investigate the regulation of homeostasis. CXCR5 expression in mouse RPE cells was diminished by treatment with hydrogen peroxide. Lack of CXCR5 expression leads to an abnormal cellular shape, pigmentation, decreased expression of the RPE differentiation marker RPE65, an increase in the undifferentiated progenitor marker MITF, and compromised RPE barrier function, as well as compromised cell-to-cell interaction. An increase in epithelial-mesenchymal transition (EMT) markers (αSMA, N-cadherin, and vimentin) was noted in CXCR5-deficient RPE cells both in vitro and in age-progression specimens of CXCR5-/- mice (6, 12, 24-months old). Deregulated autophagy in CXCR5-deficient RPE cells was observed by decreased LC3B-II, increased p62, abnormal autophagosomes, and impaired lysosome enzymatic activity as shown by GFP-LC3-RFP reporter plasmid. Mechanistically, deficiency in CXCR5 resulted in the downregulation of PI3K and AKT signaling, but upregulation and nuclear localization of FOXO1. Additionally, inhibition of PI3K in RPE cells resulted in an increased expression of FOXO1. Inhibition of FOXO1, however, reverts the degradation of ZO-1 caused by CXCR5 deficiency. Collectively, these findings suggest that CXCR5 maintains PI3K/AKT signaling, which controls FOXO1 activation, thereby regulating the expression of genes involved in RPE EMT and autophagy deregulation.

Synergistic interactions of PlGF and VEGF contribute to blood-retinal barrier breakdown through canonical NFκB activation.

To investigate the role of placental growth factor/vascular endothelial growth factor (PlGF-VEGF) heterodimers are involved in the blood-retinal barrier (BRB) breakdown and the associated mechanism, human retinal endothelial cells (HRECs) were treated with recombinant human (rh)PlGF-VEGF heterodimers and rhPlGF and studied in normal and high-glucose conditions. HREC barrier function was evaluated by the measurement of trans-endothelial electrical resistance (TEER). Adeno-Associated Virus Type 5 (AAV5) vectors overexpressed PlGF in the retina by intravitreal injection into the C57BL6 mouse eye. AAV5-GFP vector and naïve animals were used as controls. Immunofluorescence (IF) and western blots examined the protein expression of PlGF-VEGF heterodimers, VEGF, PlGF, NFκB, p-IκBα, ZO-1, and VE-cadherin in HREC and mouse retina. PlGF-VEGF heterodimers were detected predominantly in the HREC cell nuclei based on IF and cytoplasmic and nuclear fractionation experiments. High glucose treatment increased PlGF-VEGF nuclear abundance. Dot immunoblotting demonstrated a strong affinity of the 5D11D4 antibody to PlGF-VEGF heterodimers. rhPlGF-VEGF disrupted the barrier function of HREC, which was prevented by the neutralization of PlGF-VEGF by the 5D11D4 antibody. Stimulation of HRECs with rhPlGF also led to an increase in the nuclear signals for PlGF-VEGF, p-IκBα, and colocalization of NFκB p65 and PlGF-VEGF in the nuclei. The selective IKK2 inhibitor IMD0354 disrupted the nuclear colocalization. Treatment with IMD0354 restored the barrier function of HREC, as indicated by the ZO-1 and VE-cadherin expression. In the mouse retinas, PlGF overexpression by AAV5 vector reduced ZO-1 expression and increased abundance of pIκBα. PIGF/VEGF heterodimers mediate BRB breakdown potentially through the canonical NFκB activation.

RNA-Seq reveals placental growth factor regulates the human retinal endothelial cell barrier integrity by transforming growth factor (TGF-ß) signaling.

Placental growth factor (PlGF or PGF) is a member of the VEGF (vascular endothelial growth factor) family. It plays a pathological role in inflammation, vascular permeability, and pathological angiogenesis. The molecular signaling by which PlGF mediates its effects in non-proliferative diabetic retinopathy (DR) remains elusive. This study aims to characterize the transcriptome changes of human retinal endothelial cells (HRECs) with the presence and the absence of PlGF signaling. Primary HRECs were treated with the PlGF antibody (ab) to block its activity. The total RNA was isolated and subjected to deep sequencing to quantify the transcripts and their changes in both groups. We performed transcriptome-wide analysis, gene ontology, pathway enrichment, and gene-gene network analyses. The results showed that a total of 3760 genes were significantly differentially expressed and were categorized into cell adhesion molecules, cell junction proteins, chaperone, calcium-binding proteins, and membrane traffic proteins. Functional pathway analyses revealed that the TGF-ß pathway, pentose phosphate pathway, and cell adhesion pathway play pivotal roles in the blood-retina barrier and antioxidant defense system. Collectively, the data provide new insights into the molecular mechanisms of PlGF's biological functions in HRECs relevant to DR and diabetic macular edema (DME). The newly identified genes and pathways may act as disease markers and target molecules for therapeutic interventions for the patients with DR and DME refractory to the current anti-VEGF therapy.

Transcriptome-Wide Analysis of CXCR5 Deficient Retinal Pigment Epithelial (RPE) Cells Reveals Molecular Signatures of RPE Homeostasis.

Age-related macular degeneration (AMD) is the most common cause of irreversible blindness in the elderly population. In our previous studies, we found that deficiency of CXCR5 causes AMD-like pathological phenotypes in mice, characterized by abnormalities and dysfunction of the retinal pigment epithelium (RPE) cells. The abnormalities included abnormal cellular shape and impaired barrier function. In the present study, primary RPE cells were derived separately from CXCR5 knockout (KO) mice and from C57BL6 wild type (WT). The isolated primary cells were cultured for several days, and then total RNA was isolated and used for library preparation, sequencing, and the resultant raw data analyzed. Relative to the WT, a total of 1392 differentially expressed genes (DEG) were identified. Gene ontology analysis showed various biological processes, cellular components, and molecular functions were enriched. Pathway enrichment analysis revealed several pathways, including the PI3K-Akt signaling, mTOR signaling, FoxO, focal adhesion, endocytosis, ubiquitin-mediated proteolysis, TNFα-NF-kB Signaling, adipogenesis genes, p53 signaling, Ras, autophagy, epithelial-mesenchymal transition (EMT), and mitochondrial pathway. This study explores molecular signatures associated with deficiency of CXCR5 in RPE cells. Many of these signatures are important for homeostasis of this tissue. The identified pathways and genes require further evaluation to better understand the pathophysiology of AMD.

Discovery of novel L-type voltage-gated calcium channel blockers and application for the prevention of inflammation and angiogenesis.

BACKGROUND: The ways in which microglia activate and promote neovascularization (NV) are not fully understood. Recent in vivo evidence supports the theory that calcium is required for the transition of microglia from a surveillance state to an active one. The objectives of this study were to discover novel L-type voltage-gated channel (L-VGCC) blockers and investigate their application for the prevention of inflammation and angiogenesis. METHODS: Pharmacophore-based computational modeling methods were used to screen for novel calcium channel blockers (CCBs) from the ZINC compound library. The effects of CCBs on calcium blockade, microglial pro-inflammatory activation, and cell toxicity were validated in BV-2 microglial cell and freshly isolated smooth muscle cell (SMC) cultures. Laser-induced choroidal neovascularization (NV) and the suture-induced inflammatory corneal NV models of angiogenesis were used for in vivo validation of the novel CCBs. CX3CR1gfp/+ mice were used to examine the infiltration of GFP-labeled microglial cells. RESULTS: We identified three compounds from the ZINC database (Zinc20267861, Zinc18204217, and Zinc33254827) as new blockers of L-type voltage-gated calcium channels (L-VGCC) using a structure-based pharmacophore approach. The effects of the three CCBs on Ca2+ influx into cells were verified in BV-2 microglial cells using Fura-2 fluorescent dye and in freshly isolated SMCs using the voltage-patch clamp. All three CCBs reduced microglial cell migration, activation stimulated by lipopolysaccharide (LPS), and reduced the expression of the inflammatory markers NF-κB (phospho-IκBα) and cyclooxygenase-2 (COX-2) as well as reactive oxygen species. Of the three compounds, we further examined the in vivo activity of Zinc20267861. Topical treatment with Zinc20267861 in a rat model of suture-induced inflammatory cornea neovascularization demonstrated efficacy of the compound in reducing monocyte infiltration and overall corneal NV response. Subconjunctival administration of the compound in the choroidal NV mouse model effectively prevented CNV and microglial infiltration. CONCLUSIONS: Our findings suggest that the novel CCBs identified here are effective anti-inflammatory agents that can be further evaluated for treating NV disorders and can be potentially applied in the treatment of ocular inflammatory and pathological angiogenetic disorders.

Photoreceptor Degeneration Accompanies Vascular Changes in a Zebrafish Model of Diabetic Retinopathy.

Purpose: Diabetic retinopathy (DR) is a leading cause of vision impairment and blindness worldwide in the working-age population, and the incidence is rising. Until now it has been difficult to define initiating events and disease progression at the molecular level, as available diabetic rodent models do not present the full spectrum of neural and vascular pathologies. Zebrafish harboring a homozygous mutation in the pancreatic transcription factor pdx1 were previously shown to display a diabetic phenotype from larval stages through adulthood. In this study, pdx1 mutants were examined for retinal vascular and neuronal pathology to demonstrate suitability of these fish for modeling DR. Methods: Vessel morphology was examined in pdx1 mutant and control fish expressing the fli1a:EGFP transgene. We further characterized vascular and retinal phenotypes in mutants and controls using immunohistochemistry, histology, and electron microscopy. Retinal function was assessed using electroretinography. Results: Pdx1 mutants exhibit clear vascular phenotypes at 2 months of age, and disease progression, including arterial vasculopenia, capillary tortuosity, and hypersprouting, could be detected at stages extending over more than 1 year. Neural-retinal pathologies are consistent with photoreceptor dysfunction and loss, but do not progress to blindness. Conclusions: This study highlights pdx1 mutant zebrafish as a valuable complement to rodent and other mammalian models of DR, in particular for research into the mechanistic interplay of diabetes with vascular and neuroretinal disease. They are furthermore suited for molecular studies to identify new targets for treatment of early as well as late DR.

Revascularization after angiogenesis inhibition favors new sprouting over abandoned vessel reuse.

Inhibiting pathologic angiogenesis can halt disease progression, but such inhibition may offer only a temporary benefit, followed by tissue revascularization after treatment stoppage. This revascularization, however, occurs by largely unknown phenotypic changes in pathologic vessels. To investigate the dynamics of vessel reconfiguration during revascularization, we developed a model of reversible murine corneal angiogenesis permitting longitudinal examination of the same vasculature. Following 30 days of angiogenesis inhibition, two types of vascular structure were evident: partially regressed persistent vessels that were degenerate and barely functional, and fully regressed, non-functional empty basement membrane sleeves (ebms). While persistent vessels maintained a limited flow and retained collagen IV+ basement membrane, CD31+ endothelial cells (EC), and α-SMA+ pericytes, ebms were acellular and expressed only collagen IV. Upon terminating angiogenesis inhibition, transmission electron microscopy and live imaging revealed that revascularization ensued by a rapid reversal of EC degeneracy in persistent vessels, facilitating their phenotypic normalization, vasodilation, increased flow, and subsequent new angiogenic sprouting. Conversely, ebms were irreversibly sealed from the circulation by excess collagen IV deposition that inhibited EC migration and prevented their reuse. Fully and partially regressed vessels therefore have opposing roles during revascularization, where fully regressed vessels inhibit new sprouting while partially regressed persistent vessels rapidly reactivate and serve as the source of continued pathologic angiogenesis.

Autoimmune-Mediated Retinopathy in CXCR5-Deficient Mice as the Result of Age-Related Macular Degeneration Associated Proteins Accumulation.

Previous research has shown that CXCR5-/- mice develop retinal degeneration (RD) with age, a characteristic related to age macular degeneration (AMD). RD in these mice is not well-understood, and in this study, we sought to characterize further the RD phenotype and to gain mechanistic insights into the function of CXCR5 in the retina. CXCR5-/- and WT control mice were used. Fundus images demonstrated a significant (p < 0.001) increase of hypo-pigmented spots in the retina of aged CXCR5-/- mice compared with WT control mice. PAS staining indicated localization of deposits in the sub-retinal pigment epithelia (RPE) layer. AMD-associated proteins Cryab, amyloid beta, and C3d were detected within the RPE/sub-RPE tissues by immunofluorescence (IF). In addition, western blot analysis of COX-2, Arg1, and VEGF-a revealed an increase in the signaling of these molecules within the RPE/choroid complex. Transmission electron microscopy (TEM) indicated a drusen-like structure of sub-RPE deposits with an accumulation of vacuolated cellular debris. Loss of photoreceptors was detected by peanut lectin staining and was corroborated by a reduction in MAP2 signaling. Loss of blood-retinal barrier integrity was demonstrated by a reduction of ZO-1 expression. Inflammatory cells were detected in the sub-RPE space, with an increase in IBA-1 positive microglia cells on the surface of the RPE. Mass spectrometry analysis of CXCR5-/- mouse RPE/choroid proteins extracts, separated by SDS-page and incubated with autologous serum, identified autoantibodies against AMD-associated proteins: Cryaa, Cryab, and Anxa2. In vitro evaluations in BV-2 cell culture indicated a significant increase in production of Arg-1 (p < 0.001) and COX-2 (p < 0.01) in the presence of anti-CXCR5 antibody when compared with Igg-treated control BV-2 cells stimulated with IL-4 and TNFα/IFNγ, respectively. Anti-CXCR5 antibody treatment without stimulating agents did not affect Arg-1 and COX-2 expression; this suggests that CXCR5 may have a regulatory role in microglia cells activation. These results indicate that with age, CXCR5-/- mice develop RD characterized by microglia dysfunction, increased production of CXCL13 in the RPE progressive photoreceptor, neuronal loss, and sub-RPE deposition of cellular debris, resulting in the production of immunogenic proteins and autoimmune-mediated RD.

Repeat Corneal Neovascularization is Characterized by More Aggressive Inflammation and Vessel Invasion Than in the Initial Phase.

Purpose: Treatment of corneal neovascularization can lead to vessel regression and recovery of corneal transparency. Here, we examined the response of the cornea to a repeated stimulus after initial vessel regression comparing the second wave of neovascularization with the first. Methods: Corneal neovascularization was induced by surgical suture placement in the rat cornea for 7 days, followed by suture removal and a 30-day regression period. Corneas were then re-sutured and examined for an additional 4 days. Longitudinal slit-lamp imaging, in vivo confocal microscopy, and microarray analysis of global gene expression was conducted to assess the inflammatory and neovascularization response. Inhibitory effect of topical dexamethasone for repeat neovascularization was assessed. Results: After initial robust neovascularization, 30 days of regression resulted in the recovery of corneal transparency; however, a population of barely functional persistent vessels remained at the microscopic level. Upon re-stimulation, inflammatory cell invasion, persistent vessel dilation, vascular invasion, and gene expression of Vegfa, Il1ß, Il6, Ccl2, Ccl3, and Cxcl2 all doubled relative to initial neovascularization. Repeat neovascularization occurred twice as rapidly as initially, with activation of nitric oxide and reactive oxygen species, matrix metalloproteinase, and leukocyte extravasation signaling pathways, and suppression of anti-inflammatory LXR/RXR signaling. While inhibiting initial neovascularization, a similar treatment course of dexamethasone did not suppress repeat neovascularization. Conclusions: Persistent vessels remaining after the initial resolution of neovascularization can rapidly reactivate to facilitate more aggressive inflammation and repeat neovascularization, highlighting the importance of achieving and confirming complete vessel regression after an initial episode of corneal neovascularization.

Intussusceptive Vascular Remodeling Precedes Pathological Neovascularization.

Objective- Pathological neovascularization is crucial for progression and morbidity of serious diseases such as cancer, diabetic retinopathy, and age-related macular degeneration. While mechanisms of ongoing pathological neovascularization have been extensively studied, the initiating pathological vascular remodeling (PVR) events, which precede neovascularization remains poorly understood. Here, we identify novel molecular and cellular mechanisms of preneovascular PVR, by using the adult choriocapillaris as a model. Approach and Results- Using hypoxia or forced overexpression of VEGF (vascular endothelial growth factor) in the subretinal space to induce PVR in zebrafish and rats respectively, and by analyzing choriocapillaris membranes adjacent to choroidal neovascular lesions from age-related macular degeneration patients, we show that the choriocapillaris undergo robust induction of vascular intussusception and permeability at preneovascular stages of PVR. This PVR response included endothelial cell proliferation, formation of endothelial luminal processes, extensive vesiculation and thickening of the endothelium, degradation of collagen fibers, and splitting of existing extravascular columns. RNA-sequencing established a role for endothelial tight junction disruption, cytoskeletal remodeling, vesicle- and cilium biogenesis in this process. Mechanistically, using genetic gain- and loss-of-function zebrafish models and analysis of primary human choriocapillaris endothelial cells, we determined that HIF (hypoxia-induced factor)-1α-VEGF-A-VEGFR2 signaling was important for hypoxia-induced PVR. Conclusions- Our findings reveal that PVR involving intussusception and splitting of extravascular columns, endothelial proliferation, vesiculation, fenestration, and thickening is induced before neovascularization, suggesting that identifying and targeting these processes may prevent development of advanced neovascular disease in the future. Visual Overview- An online visual overview is available for this article.

MicroRNAs in the cornea: Role and implications for treatment of corneal neovascularization.

With no safe and efficient approved therapy available for treating corneal neovascularization, the search for alternative and effective treatments is of great importance. Since the discovery of miRNAs as key regulators of gene expression, knowledge of their function in the eye has expanded continuously, facilitated by high throughput genomic tools such as microarrays and RNA sequencing. Recently, reports have emerged implicating miRNAs in pathological and developmental angiogenesis. This has led to the idea of targeting these regulatory molecules as a therapeutic approach for treating corneal neovascularization. With the growing volume of data generated from high throughput tools applied to study corneal neovascularization, we provide here a focused review of the known miRNAs related to corneal neovascularization, while presenting new experimental data and insights for future research and therapy development.

Transcriptome-wide analysis of differentially expressed chemokine receptors, SNPs, and SSRs in the age-related macular degeneration.

BACKGROUND: Age-related macular degeneration (AMD) is the most common, progressive, and polygenic cause of irreversible visual impairment in the world. The molecular pathogenesis of the primary events of AMD is poorly understood. We have investigated a transcriptome-wide analysis of differential gene expression, single-nucleotide polymorphisms (SNPs), indels, and simple sequence repeats (SSRs) in datasets of the human peripheral retina and RPE-choroid-sclera control and AMD. METHODS AND RESULTS: Adaptors and unbiased components were removed and checked to ensure the quality of the data sets. Molecular function, biological process, cellular component, and pathway analyses were performed on differentially expressed genes. Analysis of the gene expression datasets identified 5011 upregulated genes, 11,800 downregulated genes, 42,016 SNPs, 1141 indels, and 6668 SRRs between healthy controls and AMD donor material. Enrichment categories for gene ontology included chemokine activity, cytokine activity, cytokine receptor binding, immune system process, and signal transduction respectively. A functional pathways analysis identified that chemokine receptors bind chemokines, complement cascade genes, and create cytokine signaling in immune system pathway genes (p value < 0.001). Finally, allele-specific expression was found to be significant for Chemokine (C-C motif) ligand (CCL) 2, 3, 4, 13, 19, 21; C-C chemokine receptor (CCR) 1, 5; chemokine (C-X-C motif) ligand (CXCL) 9, 10, 16; C-X-C chemokine receptor type (CXCR) 6; as well as atypical chemokine receptor (ACKR) 3,4 and pro-platelet basic protein (PPBP). CONCLUSIONS: Our results improve our overall understanding of the chemokine receptors' signaling pathway in AMD conditions, which may lead to potential new diagnostic and therapeutic targets.

Time-dependent LXR/RXR pathway modulation characterizes capillary remodeling in inflammatory corneal neovascularization.

Inflammation in the normally immune-privileged cornea can initiate a pathologic angiogenic response causing vision-threatening corneal neovascularization. Inflammatory pathways, however, are numerous, complex and are activated in a time-dependent manner. Effective resolution of inflammation and associated angiogenesis in the cornea requires knowledge of these pathways and their time dependence, which has, to date, remained largely unexplored. Here, using a model of endogenous resolution of inflammation-induced corneal angiogenesis, we investigate the time dependence of inflammatory genes in effecting capillary regression and the return of corneal transparency. Endogenous capillary regression was characterized by a progressive thinning and remodeling of angiogenic capillaries and inflammatory cell retreat in vivo in the rat cornea. By whole-genome longitudinal microarray analysis, early suppression of VEGF ligand-receptor signaling and inflammatory pathways preceded an unexpected later-phase preferential activation of LXR/RXR, PPARα/RXRα and STAT3 canonical pathways, with a concurrent attenuation of LPS/IL-1 inhibition of RXR function and Wnt/ß-catenin signaling pathways. Potent downstream inflammatory cytokines such as Cxcl5, IL-1ß, IL-6 and Ccl2 were concomitantly downregulated during the remodeling phase. Upstream regulators of the inflammatory pathways included Socs3, Sparc and ApoE. A complex and coordinated time-dependent interplay between pro- and anti-inflammatory signaling pathways highlights a potential anti-inflammatory role of LXR/RXR, PPARα/RXRα and STAT3 signaling pathways in resolving inflammatory corneal angiogenesis.

Selective IKK2 inhibitor IMD0354 disrupts NF-κB signaling to suppress corneal inflammation and angiogenesis.

Corneal neovascularization is a sight-threatening condition caused by angiogenesis in the normally avascular cornea. Neovascularization of the cornea is often associated with an inflammatory response, thus targeting VEGF-A alone yields only a limited efficacy. The NF-κB signaling pathway plays important roles in inflammation and angiogenesis. Here, we study consequences of the inhibition of NF-κB activation through selective blockade of the IKK complex IκB kinase ß (IKK2) using the compound IMD0354, focusing on the effects of inflammation and pathological angiogenesis in the cornea. In vitro, IMD0354 treatment diminished HUVEC migration and tube formation without an increase in cell death and arrested rat aortic ring sprouting. In HUVEC, the IMD0354 treatment caused a dose-dependent reduction in VEGF-A expression, suppressed TNFα-stimulated expression of chemokines CCL2 and CXCL5, and diminished actin filament fibers and cell filopodia formation. In developing zebrafish embryos, IMD0354 treatment reduced expression of Vegf-a and disrupted retinal angiogenesis. In inflammation-induced angiogenesis in the rat cornea, systemic selective IKK2 inhibition decreased inflammatory cell invasion, suppressed CCL2, CXCL5, Cxcr2, and TNF-α expression and exhibited anti-angiogenic effects such as reduced limbal vessel dilation, reduced VEGF-A expression and reduced angiogenic sprouting, without noticeable toxic effect. In summary, targeting NF-κB by selective IKK2 inhibition dampened the inflammatory and angiogenic responses in vivo by modulating the endothelial cell expression profile and motility, thus indicating an important role of NF-κB signaling in the development of pathologic corneal neovascularization.