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.

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.

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.

Wide-field corneal subbasal nerve plexus mosaics in age-controlled healthy and type 2 diabetes populations.

A dense nerve plexus in the clear outer window of the eye, the cornea, can be imaged in vivo to enable non-invasive monitoring of peripheral nerve degeneration in diabetes. However, a limited field of view of corneal nerves, operator-dependent image quality, and subjective image sampling methods have led to difficulty in establishing robust diagnostic measures relating to the progression of diabetes and its complications. Here, we use machine-based algorithms to provide wide-area mosaics of the cornea's subbasal nerve plexus (SBP) also accounting for depth (axial) fluctuation of the plexus. Degradation of the SBP with age has been mitigated as a confounding factor by providing a dataset comprising healthy and type 2 diabetes subjects of the same age. To maximize reuse, the dataset includes bilateral eye data, associated clinical parameters, and machine-generated SBP nerve density values obtained through automatic segmentation and nerve tracing algorithms. The dataset can be used to examine nerve degradation patterns to develop tools to non-invasively monitor diabetes progression while avoiding narrow-field imaging and image selection biases.

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.

Reduced Corneal Nerve Fiber Density in Type 2 Diabetes by Wide-Area Mosaic Analysis.

Purpose: To determine if corneal subbasal nerve plexus (SBP) parameters derived from wide-area depth-corrected mosaic images are associated with type 2 diabetes. Methods: One hundred sixty-three mosaics were produced from eyes of 82 subjects by laser-scanning in vivo confocal microscopy (IVCM). Subjects were of the same age, without (43 subjects) or with type 2 diabetes (39 subjects). Mosaic corneal nerve fiber length density (mCNFL) and apical whorl corneal nerve fiber length density (wCNFL) were quantified and related to the presence and duration of diabetes (short duration < 10 years and long duration ≥ 10 years). Results: In mosaics with a mean size of 6 mm2 in subjects aged 69.1 ± 1.2 years, mCNFL in type 2 diabetes was reduced relative to nondiabetic subjects (13.1 ± 4.2 vs. 15.0 ± 3.2 mm/mm2, P = 0.018). Also reduced relative to nondiabetic subjects was mCNFL in both short-duration (14.0 ± 4.0 mm/mm2, 3.2 ± 3.9 years since diagnosis) and long-duration diabetes (12.7 ± 4.2 mm/mm2, 15.4 ± 4.2 years since diagnosis; ANOVA P = 0.023). Lower mCNFL was associated with presence of diabetes (P = 0.032) and increased hemoglobin A1c (HbA1c) levels (P = 0.047). By contrast, wCNFL was unaffected by diabetes or HbA1c (P > 0.05). Global SBP patterns revealed marked degeneration of secondary nerve fiber branches outside the whorl region in long-duration diabetes. Conclusions: Wide-area mosaic images provide reference values for mCNFL and wCNFL and reveal a progressive degeneration of the SBP with increasing duration of type 2 diabetes.

Genome-wide expression datasets of anti-VEGF and dexamethasone treatment of angiogenesis in the rat cornea.

Therapeutics against pathologic new blood vessel growth, particularly those targeting vascular endothelial growth factor (VEGF) are of enormous clinical interest. In the eye, where anti-VEGF agents are in widespread clinical use for treating retinal and corneal blindness, only partial or transient efficacy and resistance to anti-VEGF agents are among the major drawbacks. Conversely, corticosteroids have long been used in ophthalmology for their potency in suppressing inflammation and angiogenesis, but their broad biological activity can give rise to side effects such as glaucoma and cataract. To aid in the search for more targeted and effective anti-angiogenic therapies in the eye, we present here a dataset comparing gene expression changes in dexamethasone versus anti-Vegfa treatment of inflammation leading to angiogenesis in the rat cornea. Global gene expression analysis with GeneChip Rat 230 2.0 microarrays was conducted and the metadata submitted to Expression Omnibus repository. Here, we present a high-quality validated dataset enabling genome-wide comparison of genes differentially targeted by dexamethasone and anti-Vegf treatments, to identify potential alternative therapeutic targets for evaluation.

Genome-wide expression differences in anti-Vegf and dexamethasone treatment of inflammatory angiogenesis in the rat cornea.

Angiogenesis as a pathological process in the eye can lead to blindness. In the cornea, suppression of angiogenesis by anti-VEGF treatment is only partially effective while steroids, although effective in treating inflammation and angiogenesis, have broad activity leading to undesirable side effects. In this study, genome-wide expression was investigated in a suture-induced corneal neovascularization model in rats, to investigate factors differentially targeted by dexamethasone and anti-Vegf. Topical treatment with either rat-specific anti-Vegf, dexamethasone, or normal goat IgG (sham) was given to sutured corneas for 48 hours, after which in vivo imaging, tissue processing for RNA microarray, and immunofluorescence were performed. Dexamethasone suppressed limbal vasodilation (P < 0.01) and genes in PI3K-Akt, focal adhesion, and chemokine signaling pathways more effectively than anti-Vegf. The most differentially expressed genes were confirmed by immunofluorescence, qRTPCR and Western blot. Strong suppression of Reg3g and the inflammatory chemokines Ccl2 and Cxcl5 and activation of classical complement pathway factors C1r, C1s, C2, and C3 occurred with dexamethasone treatment, effects absent with anti-Vegf treatment. The genome-wide results obtained in this study provide numerous potential targets for specific blockade of inflammation and angiogenesis in the cornea not addressed by anti-Vegf treatment, as possible alternatives to broad-acting immunosuppressive therapy.

A microarray whole-genome gene expression dataset in a rat model of inflammatory corneal angiogenesis.

In angiogenesis with concurrent inflammation, many pathways are activated, some linked to VEGF and others largely VEGF-independent. Pathways involving inflammatory mediators, chemokines, and micro-RNAs may play important roles in maintaining a pro-angiogenic environment or mediating angiogenic regression. Here, we describe a gene expression dataset to facilitate exploration of pro-angiogenic, pro-inflammatory, and remodelling/normalization-associated genes during both an active capillary sprouting phase, and in the restoration of an avascular phenotype. The dataset was generated by microarray analysis of the whole transcriptome in a rat model of suture-induced inflammatory corneal neovascularisation. Regions of active capillary sprout growth or regression in the cornea were harvested and total RNA extracted from four biological replicates per group. High quality RNA was obtained for gene expression analysis using microarrays. Fold change of selected genes was validated by qPCR, and protein expression was evaluated by immunohistochemistry. We provide a gene expression dataset that may be re-used to investigate corneal neovascularisation, and may also have implications in other contexts of inflammation-mediated angiogenesis.

Factors regulating capillary remodeling in a reversible model of inflammatory corneal angiogenesis.

Newly formed microcapillary networks arising in adult organisms by angiogenic and inflammatory stimuli contribute to pathologies such as corneal and retinal blindness, tumor growth, and metastasis. Therapeutic inhibition of pathologic angiogenesis has focused on targeting the VEGF pathway, while comparatively little attention has been given to remodeling of the new microcapillaries into a stabilized, functional, and persistent vascular network. Here, we used a novel reversible model of inflammatory angiogenesis in the rat cornea to investigate endogenous factors rapidly invoked to remodel, normalize and regress microcapillaries as part of the natural response to regain corneal avascularity. Rapid reversal of an inflammatory angiogenic stimulus suppressed granulocytic activity, enhanced recruitment of remodelling macrophages, induced capillary intussusception, and enriched pathways and processes involving immune cells, chemokines, morphogenesis, axonal guidance, and cell motility, adhesion, and cytoskeletal functions. Whole transcriptome gene expression analysis revealed suppression of numerous inflammatory and angiogenic factors and enhancement of endogenous inhibitors. Many of the identified genes function independently of VEGF and represent potentially new targets for molecular control of the critical process of microvascular remodeling and regression in the cornea.

RGTA in corneal wound healing after transepithelial laser ablation in a rabbit model: a randomized, blinded, placebo-controlled study.

PURPOSE: To evaluate the efficacy of the agent RGTA for epithelial, stromal and nerve regeneration after laser-induced corneal wounding in rabbits. METHODS: After excimer laser wounding of the anterior cornea in 25 New Zealand white rabbits, topical RGTA or placebo was applied in a randomized, masked manner. Fluorescein epithelial staining was performed on the first 5 postoperative days. In vivo confocal microscopy of corneal subbasal nerves and stroma was performed preoperatively and on week 2, 4, 8 and 16. At 16 weeks, corneas were stained for beta-III tubulin expression. RESULTS: Postoperatively, all epithelia had closed by at least 90% after the third postoperative day. No significant difference in epithelial wound size was found between RGTA and placebo-treated groups, and RGTA did not hinder fluorescein binding. After epithelial wound closure, corneas remained transparent to 16 weeks. By confocal microscopy, subclinical stromal haze was significantly deeper in placebo-treated eyes (mean depth 60 µm) relative to RGTA group (52 µm), p = 0.02. Regenerating beta-III tubulin-positive subbasal nerves were observed in all corneas, but partial masking by haze rendered quantitative analysis unreliable. CONCLUSIONS: RGTA restored stromal microarchitecture and reduced subclinical haze relative to placebo. The mild epithelial wound quickly healed regardless of treatment suggesting an optimal natural healing process in freshly wounded healthy corneas, and indicating that RGTA may be more suitable for healing of chronic or more aggressive wounds. Limitations of the rabbit model for nerve quantification in the presence of haze should also be recognized.

Early effects of dexamethasone and anti-VEGF therapy in an inflammatory corneal neovascularization model.

Inflammatory angiogenesis is the pathogenic mechanism of various sight-threatening eye diseases, among them corneal neovascularization. Current treatment options include steroids which have undesirable side effects, or anti-VEGF which has only limited efficacy. In an inflammatory environment, however, angiogenesis can be stimulated by numerous factors not directly targeted by anti-VEGF therapy. The aim of this study was to induce corneal inflammation leading to angiogenesis, and investigate the early, differential effects of steroid and anti-VEGF therapy at the cellular, tissue, and gene expression levels. Fifty-two Wistar rats received a single intrastromal corneal suture to induce a controlled inflammatory angiogenic response. Rats were subsequently treated with dexamethasone, rat specific anti-VEGF, or goat IgG (control), topically 4 times daily for 7 days. In vivo confocal microscopy of the cornea was performed longitudinally from 5 h up to 7 d to investigate morphology at the cellular and tissue-level. In vivo photographic vessel analysis and immunohistochemistry were also performed. RT-PCR for VEGF-A, FGF-2, IL-6, TNF-α, CXCL2, CCL2, CCL3 and DLL4 was performed at 24 h, and for VEGF-A, IL-6, TNF-α, FGF-2, CXCL2, CCL2, and CCL3 at 7 days. Early infiltration of CD11b + myeloid cells into the cornea at 5 h post-suture was delayed by both treatments relative to controls; however neither treatment was able to suppress accumulation of myeloid cells at day 2 or 7. Limbal vessel dilation was inhibited at 5 h by both treatments, but only dexamethasone showed sustained effect until day 2. Early macrophage recruitment was also suppressed by dexamethasone (but not by anti-VEGF) until day 2. Dexamethasone furthermore suppressed corneal neovascularization at day 7 by over 90%, whereas suppression by anti-VEGF was 14%. Despite differential suppression of vessel dilation, macrophage recruitment, and vascular invasion, anti-VEGF and dexamethasone both down-regulated VEGF-A and IL-6 expression at 24 h with sustained effect to 7 d. They also both down regulated FGF-2 and TNF-α at 24 h and CCL2 at 7 d. In conclusion, anti-angiogenic treatments influence early, pre-angiogenic tissue activity such as limbal vessel dilation, inflammatory cell infiltration of the stroma, and macrophage recruitment. Importantly, the differential effects of steroids and anti-VEGF treatment in suppressing neovascular growth could not be attributed to differential inhibition of several major angiogenic and inflammatory factors in the early pre-sprouting phase, including IL-6, VEGF-A, FGF-2, TNF-α, CCL2, CCL3, CXCL2, or DLL4.

An in vivo method for visualizing flow dynamics of cells within corneal lymphatics.

BACKGROUND: Monitoring the trafficking of specific cell populations within lymphatics could improve our understanding of processes such as transplant rejection and cancer metastasis. Current methods, however, lack appropriate image resolution for single-cell analysis or are incompatible with in vivo and longitudinal monitoring of lymphatics in their native state. We therefore sought to achieve high-resolution live imaging of the dynamic behavior of cells within lymph vessels in the rat cornea. METHODS/RESULTS: Inflammatory angiogenesis was induced by suture placement in corneas of Wistar rats. Pre- and up to 3 weeks post-induction, corneas were noninvasively examined by laser-scanning in vivo corneal confocal microscopy (IVCM) using only endogenous contrast. Lymph vessels and the cells harbored therein were documented by still images, real-time video, and 3D confocal stack reconstruction of live tissue. In vivo, conjunctival and corneal lymphatics were morphologically distinct, those with corneal location being one-quarter the diameter of those in the conjunctiva (p<0.001). Cells were recruited to initially empty pre-existing lymph vessels during the first day of inflammation and maintained a dense occupation of vessels for up to 7 days. A diverse population of cells (diameter range: 1.5-27.5 µm) with varying morphology was observed, and exhibited variable flow patterns and were transported singly and in clusters of at least 2-9 adherent cells. CONCLUSIONS: The in vivo microscopic technique presented enables lymph vessels and cell trafficking to be studied in high resolution in a minimally-perturbed physiologic milieu.

Acute exudative polymorphous vitelliform maculopathy in a young woman: presymptomatic findings and 21-month follow-up.

PURPOSE: To describe ocular findings before and after the diagnosis of acute exudative polymorphous vitelliform maculopathy, in an otherwise healthy 28-year-old woman. METHODS: Case report with 21-months of follow-up. Fundus photography, optical coherence tomography, fluorescein angiography, indocyanine green angiography, and autofluorescence were used for imaging the retina. To examine retinal function, full-field electroretinogram, multifocal electroretinogram, electrooculography, and dark adaptometry were performed. Genetic analysis for mutations associated with Best disease was done. RESULTS: In the asymptomatic patient before diagnosis, white-yellow, drusen-like, subretinal depositions were found in both eyes. A few months later, the patient developed bilateral visual disturbances. Retinal examination at the acute phase revealed a characteristic pattern of multifocal white-yellow subretinal lesions in both posterior poles, imaged by ophthalmoscopy, fluorescein angiography, indocyanine green angiography, and optical coherence tomography. Additionally, electrooculography and dark adaptometry were abnormal. Full-field electroretinogram was normal, but multifocal electroretinogram revealed central depression of peak amplitudes. During the 21-month follow-up without any treatment, visual acuity recovered, electrooculography and dark adaptometry normalized, and the patient experienced one episode of relapse. Genetic studies excluded mutations in the bestrophin gene (BEST1). CONCLUSION: Acute exudative polymorphous vitelliform maculopathy is still a condition of unknown origin, primarily affecting the pigment epithelium. Earlier reports have discussed whether the condition is inherited or acquired. In this report, the presymptomatic retinal findings in acute exudative polymorphous vitelliform maculopathy are described for the first time, indicating that a condition may be associated with primarily affected retinal pigment epithelium.

QALY weights for diabetic retinopathy--a comparison of health state valuations with HUI-3, EQ-5D, EQ-VAS, and TTO.

OBJECTIVE: To estimate quality-adjusted life-year weights for patients with diabetic retinopathy by using various methods and to investigate the empirical validity of the different measures. METHODS: The study population comprised 152 patients with diabetes in Östergötland County, Sweden. Participants were interviewed by telephone by using the time trade-off (TTO) method and a visual analogue scale (EQ-VAS) (direct valuations) as well as the EuroQol five-dimensional questionnaire (EQ-5D) and the health utilities index mark 3 (HUI-3) (indirect valuations). The quality-adjusted life-year weights were adjusted for potential confounders by using analysis of covariance. The empirical validity of the measures was examined by testing their ability to detect hypothetical differences between severity levels of diabetic retinopathy and by investigating the correlation between the measures and the 25-item National Eye Institute Visual Function Questionnaire (NEI VFQ-25). RESULTS: All measures detected significant differences in scores between patient groups classified according to visual impairment in the better eye (analysis of covariance, P < 0.05), but only HUI-3 and EQ-VAS detected significant differences between patient groups classified according to visual impairment or pathological progression in the worse eye. HUI-3 recorded a difference of 0.43 in values between normal vision and blindness in the better eye, which was more than twice the differences captured by the other measures (0.15-0.20). In addition, HUI-3 showed the highest correlation with NEI VFQ-25 (r = 0.54; P < 0.001). CONCLUSIONS: In cost-utility analyses, the choice of quality-adjusted life-year measure may affect whether an intervention is considered cost-effective. Furthermore, if decisions are to be based on values from the general public, HUI-3 can be recommended for cost-utility analyses of interventions directed at diabetic retinopathy.

Cellular level characterization of capillary regression in inflammatory angiogenesis using an in vivo corneal model.

In this study, we introduce a technique for repeated, microscopic observation of single regressing capillaries in vivo in inflamed murine corneas. Natural capillary regression was initiated by removal of inflammatory stimulus during an active pro-angiogenic phase, while the additional impact of anti-angiogenic treatment with triamcinolone or bevazicumab was investigated. Capillaries regressed naturally within 1 week and treatments did not further enhance the natural regression. Morphologically, early-phase regression was characterized by significant lumen narrowing and a significant reduction in CD11b+ myeloid cell infiltration of the extracellular matrix. By 1 week, vascular remodeling occurred concomitant with CD11b+CD68+KiM2R+ mature macrophage localization on capillary walls. Empty conduits without blood flow, positive for collagen IV and devoid of vascular endothelium and pericytes, were apparent in vivo and by 3 weeks were more numerous than perfused capillaries. By 3 weeks, macrophages aggregated around remaining perfused capillaries and were observed in apposition with degrading capillary segments. Abrupt termination of capillary sprouting in our regression model further revealed vascular endothelial abandonment of sprout tips and perfused capillary loop formation within a single angiogenic sprout, possibly as an intussusceptive response to cessation of the stimulus. Finally, we observed lumen constriction and macrophage localization on capillary walls in vivo in a clinical case of corneal capillary regression that paralleled findings in our murine model.