Mechanisms regulating vascular and lymphatic regeneration in the heart after myocardial infarction.

Myocardial infarction, caused by a thrombus or coronary vascular occlusion, leads to irreversible ischaemic injury. Advances in early reperfusion strategies have significantly reduced short-term mortality after myocardial infarction. However, survivors have an increased risk of developing heart failure, which confers a high risk of death at 1 year. The capacity of the injured neonatal mammalian heart to regenerate has stimulated extensive research into whether recapitulation of developmental regeneration programmes may be beneficial in adult cardiovascular disease. Restoration of functional blood and lymphatic vascular networks in the infarct and border regions via neovascularisation and lymphangiogenesis, respectively, is a key requirement to facilitate myocardial regeneration. An improved understanding of the endogenous mechanisms regulating coronary vascular and lymphatic expansion and function in development and in adult patients after myocardial infarction may inform future therapeutic strategies and improve translation from pre-clinical studies. In this review, we explore the underpinning research and key findings in the field of cardiovascular regeneration, with a focus on neovascularisation and lymphangiogenesis, and discuss the outcomes of therapeutic strategies employed to date. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Recent Advances in Pathology: the 2023 Annual Review Issue of The Journal of Pathology.

The 2023 Annual Review Issue of The Journal of Pathology, Recent Advances in Pathology, contains 12 invited reviews on topics of current interest in pathology. This year, our subjects include immuno-oncology and computational pathology approaches for diagnostic and research applications in human disease. Reviews on the tissue microenvironment include the effects of apoptotic cell-derived exosomes, how understanding the tumour microenvironment predicts prognosis, and the growing appreciation of the diverse functions of fibroblast subtypes in health and disease. We also include up-to-date reviews of modern aspects of the molecular basis of malignancies, and our final review covers new knowledge of vascular and lymphatic regeneration in cardiac disease. All of the reviews contained in this issue are written by expert groups of authors selected to discuss the recent progress in their particular fields and all articles are freely available online (https://pathsocjournals.onlinelibrary.wiley.com/journal/10969896). © 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Efficacy, durability, and safety of faricimab up to every 16 weeks in patients with neovascular age-related macular degeneration: 2-year results from the Japan subgroup of the phase III TENAYA trial.

PURPOSE: To evaluate 2-year efficacy, durability, and safety of faricimab in the TENAYA Japan subgroup and pooled global TENAYA/LUCERNE cohort of patients with neovascular age-related macular degeneration (nAMD). METHODS: Subgroup analysis of TENAYA/LUCERNE (NCT03823287/NCT03823300): phase III, multicentre, randomised, active comparator-controlled, double-masked, non-inferiority trials. Treatment-naïve patients aged ≥ 50 years with nAMD were randomised (1:1) to intravitreal faricimab (6.0 mg up to every 16 weeks [Q16W] after 4 initial Q4W doses) or aflibercept (2.0 mg Q8W after 3 initial Q4W doses). Outcomes were assessed through year 2 for the TENAYA Japan subgroup (N = 133) and global pooled TENAYA/LUCERNE cohort (N = 1329). RESULTS: Vision and anatomic improvements achieved with faricimab at year 1 were maintained over 2 years and were generally comparable between the TENAYA Japan subgroup and pooled TENAYA/LUCERNE cohort. Adjusted mean best-corrected visual acuity (BCVA) change from baseline at year 2 for the TENAYA Japan subgroup and global pooled TENAYA/LUCERNE cohort was +7.1 (3.7-10.5) and +4.4 (3.2-5.5) letters in the faricimab arm, respectively, and +5.2 (1.9-8.6) and +4.3 (3.1-5.4) letters in the aflibercept arm, respectively. At week 112, the proportion of faricimab-treated patients on Q16W dosing was 61.0% and 63.1% in the TENAYA Japan subgroup and pooled TENAYA/LUCERNE cohort. Faricimab was well tolerated through year 2. CONCLUSION: Year 2 TENAYA Japan subgroup findings for faricimab were generally consistent with the pooled global TENAYA/LUCERNE results in patients with nAMD. Vision and anatomical benefits with faricimab were similar to those with aflibercept but with fewer injections.

Progression of macular retinoschisis following intravitreal aflibercept injection for myopic macular neovascularization-a case report and review of literature.

BACKGROUND: Macular retinoschisis (MRS) and myopic macular neovascularization (mMNV) are both potentially blinding complications of high myopia. In this case report, we highlight the progression of MRS after intravitreal anti-vascular endothelial growth factor (anti-VEGF) treatment for mMNV, as well as an extensive review of the literature on this topic. CASE DESCRIPTION: A 49-year-old woman presented with two weeks of recent onset blurring and metamorphopsia in her right eye. She had high myopia in both eyes (right eye - 20/60 with - 16D, left eye - 20/20 with - 13D). Slit-lamp ophthalmoscopy found a normal anterior segment in both eyes. On fundus examination, features of pathological myopia with posterior staphyloma and peripapillary atrophy were observed in both eyes. An active mMNV, as well as intraretinal fluid, minimal perifoveal inner and outer MRS, and focal posterior vitreous traction along the inferotemporal retinal arcade, were detected on optical coherence tomography (OCT) of the right eye. The patient received an intravitreal injection of Aflibercept (2 mg/0.05 ml). RESULTS: OCT scans at two- and four-month follow-up visits revealed regressed mMNV with a taut epiretinal membrane, progressive worsening of outer MRS, and the development of multiple perifoveal retinal detachment inferior to the fovea. Pars plana vitrectomy surgery was performed for the progressive MRS with good anatomical (resolved MRS) and functional outcome (maintained visual acuity at 20/60) at the last one-month post-surgery visit. CONCLUSION: Intravitreal anti-VEGF injections for mMNV can cause vitreoretinal interface changes, exacerbating MRS and causing visual deterioration. Vitrectomy for MRS could be one of several treatment options.

An endogenous inhibitor of angiogenesis downregulated by hypoxia in human aortic valve stenosis promotes disease pathogenesis.

Aortic valve stenosis is the most common valve disease in the western world. Central to the pathogenesis of this disease is the growth of new blood vessels (angiogenesis) within the aortic valve allowing infiltration of immune cells and development of intra-valve inflammation. Identifying the cellular mediators involved in this angiogenesis is important as this may reveal new therapeutic targets which could ultimately prevent the progression of aortic valve stenosis. Aortic valves from patients undergoing surgery for aortic valve replacement or dilation of the aortic arch were examined both ex vivo and in vitro. We now demonstrate that the anti-angiogenic protein, soluble fms-like tyrosine kinase 1 (sFlt1), a non-signalling soluble receptor for vascular endothelial growth factor, is constitutively expressed in non-diseased valves. sFlt-1 expression was, however, significantly reduced in aortic valve tissue from patients with aortic valve stenosis while protein markers of hypoxia were simultaneously increased. Exposure of primary-cultured valve interstitial cells to hypoxia resulted in a decrease in the expression of sFlt-1. We further reveal using a bioassay that siRNA knock-down of sFlt1 in valve interstitial cells directly results in a pro-angiogenic environment. Finally, incubation of aortic valves with sphingosine 1-phosphate, a bioactive lipid-mediator, increased sFlt-1 expression and inhibited angiogenesis within valve tissue. In conclusion, this study demonstrates that sFlt1 expression is directly correlated with angiogenesis in aortic valves and the observed decrease in sFlt-1 expression in aortic valve stenosis could increase valve inflammation, promoting disease progression. This could be a viable therapeutic target in treating this disease.

Microcirculation surrounding end-stage human chronic skin wounds is associated with endoglin/CD146/ALK-1 expression, endothelial cell proliferation and an absence of p16Ink4a.

Angiogenesis is an essential part of normal skin healing, re-establishing blood flow in developing granulation tissue. Non-healing skin wounds are associated with impaired angiogenesis and although the role of re-establishing macroscopic blood flow to limbs to prevent wound chronicity is well investigated, less is known about vascular alterations at the microcirculatory level. We hypothesised that significant phenotypic changes would be evident in blood vessels surrounding chronic skin wounds. Wound edge tissue, proximal to wound (2 cm from wound edge) and non-involved skin (>10 cm from wound edge) was harvested under informed consent from 20 patients undergoing elective amputation due to critical limb ischemia. To assess blood vessel structure and viability, tissue was prepared for histological analysis and labelled with antibodies specific for PECAM-1 (CD31), CD146, endoglin, ALK-1, ALK-5, and p16Ink4a as a marker of cellular senescence. Density of microvasculature was significantly increased in wound edge dermis, which was concomitant with increased labelling for endoglin and CD146. The number of CD31 positive vessel density was unchanged in wound edge tissue relative to non-involved tissue. Co-labelling of endoglin with the transforming growth factor receptor ALK-1, and to a lesser extent ALK-5, demonstrated activation of endothelial cells which correlated with PCNA labelling indicative of proliferation. Analysis of p16Ink4a staining showed a complete lack of immunoreactivity in the vasculature and dermis, although staining was evident in sub-populations of keratinocytes. We conclude that the endoglin-ALK-1-endothelial proliferation axis is active in the vasculature at the edge of chronic skin wounds and is not associated with p16Ink4a mediated senescence. This information could be further used to guide treatment of chronic skin wounds and optimise debridement protocols.

Adipose Derived Stromal Vascular Fraction and Mesenchymal Stem Cells Improve Angiogenesis in a Rat Hindlimb Ischaemia Model.

OBJECTIVE: This study aimed to investigate the effect of human adipose tissue derived stromal vascular fraction (AD-SVF) and mesenchymal stem cells (AD-MSCs) on blood flow recovery and neovascularisation in a rat hindlimb ischaemia model. METHODS: SVF was isolated using an automated centrifugal system, and AD-MSCs were obtained from adherent cultures of SVF cells. Rats were divided into four groups of six rats each: non-ischaemia (Group 1); saline treated ischaemia (Group 2); SVF treated ischaemia (Group 3); and AD-MSC treated ischaemia (Group 4). Unilateral hindlimb ischaemia was induced in Sprague-Dawley rats via femoral artery ligation. Saline, SVF, or AD-MSCs were injected intramuscularly into the adductor muscle intra-operatively. Cell viability was calculated as the percentage of live cells relative to total cell number. Blood flow improvement, muscle fibre injury, and angiogenic properties were validated using thermal imaging and histological assessment. RESULTS: The viabilities of SVF and AD-MSCs were 83.3% and 96.7%, respectively. Group 1 exhibited no significant temperature difference between hindlimbs, indicating a lack of blood flow changes. The temperature gradient gradually decreased in SVF and AD-MSC treated rats compared with saline treated rats. In addition, only normal muscle fibres with peripherally located nuclei were observed in Group 1. Groups 3 and 4 exhibited significantly fewer centrally located nuclei, indicating less muscle damage compared with Group 2. Regarding angiogenic properties, CD31 staining of endothelial cells showed similar patterns among all groups, whereas expression of vascular endothelial growth factor, as a crucial angiogenesis factor, was enhanced in the SVF and AD-MSC treated groups. CONCLUSION: SVF and AD-MSCs improved blood flow and neovascularisation in a rat hindlimb ischaemia model, suggesting their potential ability to promote angiogenesis. Further extensive research is warranted to explore their potential applications in the treatment of severe lower extremity arterial disease.

Resident vascular endothelial progenitor definition and function: the age of reckoning.

The cardiovascular system is composed around the central function of the endothelium that lines the inner surfaces of its vessels. In recent years, the existence of a progenitor population within the endothelium has been validated through the study of endothelial colony-forming cells (ECFCs) in human peripheral blood and certain vascular beds. However, our knowledge on endothelial populations in vivo that can give rise to ECFCs in culture has been limited. In this review we report and analyse recent attempts at describing progenitor populations in vivo from murine studies that reflect the self-renewal and stemness capacity observed in ECFCs. We pinpoint seminal discoveries within the field, which have phenotypically defined, and functionally scrutinised these endothelial progenitors. Furthermore, we review recent publications utilising single-cell sequencing technologies to better understand the endothelium in homeostasis and pathology.

The effects of oestrogen on vaginal wound healing: A systematic review and meta-analysis.

AIMS: To determine the effects of oestrogen or oestrogen deprivation on vaginal wound healing. Impaired wound healing following prolapse surgery may increase the risk of recurrent prolapse in the future. Vaginal oestrogen therapy may improve wound healing, hereby possibly improving surgical outcomes. METHODS: A systematic search of OVID MEDLINE, OVID Embase, and Web of Science was conducted up to January 28, 2020. We included original studies comparing wound healing-related outcomes of oestrogen exposed subjects (female animals and women) to hypo-oestrogenic subjects after vaginal surgery. Data on wound healing-related outcome measures were extracted. For each individual comparison, the standardised mean difference (Hedges' g; SMD) and 95% confidence interval (CI) were calculated. RESULTS: Of the 1474 studies reviewed, 14 studies were included for review, and 11 provided data for meta-analysis. Oestrogen improves neovascularisation (SMD: 1.13, 95% CI: 0.67-1.60), microscopic wound closure (SMD: 0.98, 95% CI: 0.66-1.29), collagen synthesis (SMD: 1.08, 95% CI: 0.42-1.74), and tissue strength (SMD: 1.26, 95% CI: 0.53-1.99) in animals. Oestrogen increases granulation (SMD: 1.67, 95% CI: 0.54-2.79) and accelerates macroscopic wound closure (SMD: 1.82, 95% CI: 1.22-2.42) in women and animals. Oestrogen decreases the inflammatory response (SMD: -0.58, 95% CI: -1.14 to -0.02) in women and animals and reduces levels of transforming growth factor (TGF)-ß1 (SMD: -1.68, 95% CI: -2.52 to -0.83) in animals. All results were statistically significant. CONCLUSIONS: Oestrogen therapy has a positive effect on vaginal wound healing. Future studies should determine whether oestrogen therapy has the potential to improve surgical outcomes.

Using methanol to preserve retinas for immunostaining.

BACKGROUND: Experimental studies on retinal vasculature and retinal ganglion cells (RGCs) investigating the developmental and pathological conditions of the retina mainly rely on whole-mount retinal immunostaining. Methanol, an auxiliary fixed medium for retinal whole-mount preparations, has been used in some studies; however, its application in short- and long-term storage of retinas for further study has not been well described. We aimed to evaluate methanol use as a preservation treatment for further immunostaining of the retina. METHODS: We generated oxygen-induced retinopathy (OIR) and optic nerve crush (ONC) mouse models and used their retinas for analysis. We pipetted cold methanol (-20°C) on the surface of the retina to help fix the tissues while promoting permeability, after which the retinas were stored in cold methanol (-20°C) for 1, 6, or 12 months before being evaluated using various optical techniques. Thereafter, retinal whole-mount immunostaining was performed to analyse retinal neovascularisation and retinal hypoxia in OIR model, and retinal ganglion cell survival rate in ONC model. RESULTS: Quantitative analysis revealed no significant differences in the fixed retinas after long-term storage in terms of retinal vasculature or retinal hypoxia in the OIR model. Similarly, no significant difference was found in RGC survival rate after long-term storage in methanol. These results suggest that methanol can be used as a storage medium when preserving retinal whole-mount samples. CONCLUSIONS: Cold (-20°C) methanol can serve as an effective medium for long-term storage of fixed retinas, which is useful for further research.

Excising or preserving perforation margins in endoscopic transtympanic cartilage myringoplasty does not affect surgical success.

OBJECTIVE: To compare the outcome of endoscopic transtympanic cartilage myringoplasty with and without removal of perforation edges for repairing chronic perforations with mucosal chronic otitis media (COM). STUDY DESIGN: Quasi-randomised clinical trial. SETTING: Tertiary referral centre. MATERIALS AND METHODS: Patients with chronic perforations and mucosal COM undergoing endoscopic transtympanic cartilage myringoplasty were allocated to a control group for whom the perforation edges were preserved (n = 40) and an intervention group for whom the edges were removed (n = 39). Mean operation time, graft success rate, mean scores of graft neovascularisation and epithelialisation, and hearing were compared between the groups at 4 weeks and/or 6 months postoperatively. RESULTS: Graft success rate was 95% (38/40) in the control group and 97% (38/39) in the intervention group at 6 months postoperatively; the difference was not significant. Mean graft neovascularisation scores 4 weeks postoperatively were 2.52 ± 0.59 in the control group and 2.58 ± 0.55 in the intervention group; the difference was not significant. Mean graft epithelisation scores 4 weeks postoperatively were 1.48 ± 0.57 in the control group and 1.68 ± 0.51 in the intervention group; the difference was not significant and remained nonsignificant 6 months postoperatively (2.5 ± 0.55 vs. 2.76 ± 0.36). Audiological outcomes at 6 months did not differ between the groups. CONCLUSION: Endoscopic, transtympanic cartilage underlay myringoplasty with preservation of the perforation margins did not affect graft neovascularisation, epithelialisation or success. Longer-term outcomes and risk of cholesteatoma require further study.

Vitreous metabolomics profiling of proliferative diabetic retinopathy.

AIMS/HYPOTHESIS: Proliferative diabetic retinopathy (PDR) with retinal neovascularisation (NV) is a leading cause of vision loss. This study identified a set of metabolites that were altered in the vitreous humour of PDR patients compared with non-diabetic control participants. We corroborated changes in vitreous metabolites identified in prior studies and identified novel dysregulated metabolites that may lead to treatment strategies for PDR. METHODS: We analysed metabolites in vitreous samples from 43 PDR patients and 21 non-diabetic epiretinal membrane control patients from Japan (age 27-80 years) via ultra-high-performance liquid chromatography-mass spectrometry. We then investigated the association of a novel metabolite (creatine) with retinal NV in mouse oxygen-induced retinopathy (OIR). Creatine or vehicle was administered from postnatal day (P)12 to P16 (during induced NV) via oral gavage. P17 retinas were quantified for NV and vaso-obliteration. RESULTS: We identified 158 metabolites in vitreous samples that were altered in PDR patients vs control participants. We corroborated increases in pyruvate, lactate, proline and allantoin in PDR, which were identified in prior studies. We also found changes in metabolites not previously identified, including creatine. In human vitreous humour, creatine levels were decreased in PDR patients compared with epiretinal membrane control participants (false-discovery rate <0.001). We validated that lower creatine levels were associated with vascular proliferation in mouse retina in the OIR model (p = 0.027) using retinal metabolomics. Oral creatine supplementation reduced NV compared with vehicle (P12 to P16) in OIR (p = 0.0024). CONCLUSIONS/INTERPRETATION: These results suggest that metabolites from vitreous humour may reflect changes in metabolism that can be used to find pathways influencing retinopathy. Creatine supplementation could be useful to suppress NV in PDR. Graphical abstract.

Pharmacological restoration of visual function in a zebrafish model of von-Hippel Lindau disease.

Von Hippel-Lindau (VHL) syndrome is a rare, autosomal dominant disorder, characterised by hypervascularised tumour formation in multiple organ systems. Vision loss associated with retinal capillary hemangioblastomas remains one of the earliest complications of VHL disease. The mortality of Vhl-/- mice in utero restricted modelling of VHL disease in this mammalian model. Zebrafish harbouring a recessive germline mutation in the vhl gene represent a viable, alternative vertebrate model to investigate associated ocular loss-of-function phenotypes. Previous studies reported neovascularisation of the brain, eye and trunk together with oedema in the vhl-/- zebrafish eye. In this study, we demonstrate vhl-/- zebrafish almost entirely lack visual function. Furthermore, hyaloid vasculature networks in the vhl-/- eye are improperly formed and this phenotype is concomitant with development of an ectopic intraretinal vasculature. Sunitinib malate, a multi tyrosine kinase inhibitor, market authorised for cancer, reversed the ocular behavioural and morphological phenotypes observed in vhl-/- zebrafish. We conclude that the zebrafish vhl gene contributes to an endogenous molecular barrier that prevents development of intraretinal vasculature, and that pharmacological intervention with sunitinib can improve visual function and hyaloid vessel patterning while reducing abnormally formed ectopic intraretinal vessels in vhl-/- zebrafish.

The role of interferon regulatory factor 8 for retinal tissue homeostasis and development of choroidal neovascularisation.

BACKGROUND: Microglia cells represent the resident innate immune cells of the retina and are important for retinal development and tissue homeostasis. However, dysfunctional microglia can have a negative impact on the structural and functional integrity of the retina under native and pathological conditions. METHODS: In this study, we examined interferon-regulatory factor 8 (Irf8)-deficient mice to determine the transcriptional profile, morphology, and temporospatial distribution of microglia lacking Irf8 and to explore the effects on retinal development, tissue homeostasis, and formation of choroidal neovascularisation (CNV). RESULTS: Our study shows that Irf8-deficient MG exhibit a considerable loss of microglial signature genes accompanied by a severely altered MG morphology. An in-depth characterisation by fundus photography, fluorescein angiography, optical coherence tomography and electroretinography revealed no major retinal abnormalities during steady state. However, in the laser-induced CNV model, Irf8-deficient microglia showed an increased activity of biological processes critical for inflammation and cell adhesion and a reduced MG cell density near the lesions, which was associated with significantly increased CNV lesion size. CONCLUSIONS: Our results suggest that loss of Irf8 in microglia has negligible effects on retinal homeostasis in the steady state. However, under pathological conditions, Irf8 is crucial for the transformation of resident microglia into a reactive phenotype and thus for the suppression of retinal inflammation and CNV formation.

A novel phenotype in a family with autosomal dominant retinal dystrophy due to c.1430A > G in retinoid isomerohydrolase (RPE65) and c.37C > T in bestrophin 1 (BEST1).

PURPOSE: The c.1430A > G (Asp477Gly) variant in RPE65 has been reported in Irish and Scottish families with either an autosomal dominant retinal dystrophy (adRD) that resembles choroideremia, a vitelliform macular dystrophy or an isolated macular atrophy. We report novel features on multimodal imaging and the natural history of a family harbouring this variant in combination with the BEST1 c.37C > T (Arg13Cys) variant. METHODS: Members of a family with an adRD were examined clinically to ascertain phenotype and underwent genetic testing. Multimodal imaging included widefield colour fundus photography, quantitative autofluorescence (qAF) and spectral domain optical coherence tomography. Electrophysiology and microperimetry were also performed. RESULTS: Vision loss was attributed to foveal atrophy in the proband and choroidal neovascularisation and a vitello-eruptive lesion in one affected son. Peripheral retinal white dots corresponding to subretinal deposits were seen in three patients. The median qAF8 values in the proband (I:1) were low (40 and 101 in OD and OS) at age 79. Similarly, the qAF8 values for the middle son (II:2) were also low (100 and 87 in ODS and OS) at age 60. Electrophysiology showed disproportionate reduction in Arden ratio prior to the gradual loss of full-field responses. Microperimetry demonstrated an enlarging scotoma in the proband. CONCLUSIONS: The coexistence of the pathogenic BEST1 c.37C > T variant may modify clinical features observed in RPE65 adRD. This study expands our understanding of RPE65 adRD as a retinoid cycle disorder supported by the reduced qAF, fine white retinal dots and corresponding subretinal deposits on OCT in affected members.

2-deoxy-d-ribose (2dDR) upregulates vascular endothelial growth factor (VEGF) and stimulates angiogenesis.

BACKGROUND: Delayed neovascularisation of tissue-engineered (TE) complex constructs is a major challenge that causes their failure post-implantation. Although significant progress has been made in the field of angiogenesis, ensuring rapid neovascularisation still remains a challenge. The use of pro-angiogenic agents is an effective approach to promote angiogenesis, and vascular endothelial growth factor (VEGF) has been widely studied both at the biological and molecular levels and is recognised as a key stimulator of angiogenesis. However, the exogenous use of VEGF in an uncontrolled manner has been shown to result in leaky, permeable and haemorrhagic vessels. Thus, researchers have been actively seeking alternative agents to upregulate VEGF production rather than exogenous use of VEGF in TE systems. We have previously revealed the potential of 2-deoxy-d-ribose (2dDR) as an alternative pro-angiogenic agent to induce angiogenesis and accelerates wound healing. However, to date, there is not any clear evidence on whether 2dDR influences the angiogenic cascade that involves VEGF. METHODS: In this study, we explored the angiogenic properties of 2dDR either by its direct application to human aortic endothelial cells (HAECs) or when released from commercially available alginate dressings and demonstrated that when 2dDR promotes angiogenesis, it also increases the VEGF production of HAECs. RESULTS: The VEGF quantification results suggested that VEGF production by HAECs was increased with 2dDR treatment but not with other sugars, including 2-deoxy-l-ribose (2dLR) and d-glucose (DG). The stability studies demonstrated that approximately 40-50% of the 2dDR had disappeared in the media over 14 days, either in the presence or absence of HAECs, and the reduction was higher when cells were present. The concentration of VEGF in the media also fell after day 4 associated with the reduction in 2dDR. CONCLUSION: This study suggests that 2dDR (but not other sugars tested in this study) stimulates angiogenesis by increasing the production of VEGF. We conclude 2dDR appears to be a practical and effective indirect route to upregulating VEGF for several days, leading to increased angiogenesis.

Lutein and zeaxanthin attenuates VEGF-induced neovascularisation in human retinal microvascular endothelial cells through a Nox4-dependent pathway.

Age-related macular degeneration (AMD) and proliferative diabetic retinopathy (DR) are two of the most common and severe causes of vision loss in the population. Both conditions are associated with excessive levels of vascular endothelial growth factor (VEGF) in the eye which results in an increase in the formation of new blood vessels through a process called neovascularisation. As such, anti-VEGF therapies are currently utilised as a treatment for patients with AMD however they are associated with painful administration of injections and potential degeneration of healthy endothelium. There is therefore growing interest in alternate treatment options to reduce neovascularisation in the eye. The use of carotenoids, lutein (L) and zeaxanthin (Z), has been shown to improve vision loss parameters in patients with AMD, however the underlying mechanisms are not well-understood. We studied the impact of these compounds on neovascularisation processes using an in vitro cell model of the retinal microvascular endothelium. Our findings show that L and Z reduced VEGF-induced tube formation whilst, in combination (5:1 ratio), the compounds significantly blocked VEGF-induced neovascularisation. The carotenoids, individually and in combination, reduced VEGF-induced oxidative stress concomitant with increased activity of the NADPH oxidase, Nox4. We further demonstrated that the Nox4 inhibitor, GLX7013114, attenuated the protective effect of L and Z. Taken together, these findings indicate the protective effect of the carotenoids, L and Z, in reducing VEGF-mediated neovascularisation via a Nox4-dependent pathway. These studies implicate the potential for these compounds to be used as a therapeutic approach for patients suffering from AMD and proliferative DR.

Association of Longitudinal Changes in Drusen Characteristics and Retinal Layer Volumes with Subsequent Subtype of Choroidal Neovascularisation.

AIM: To investigate the longitudinal correlation between drusen characteristics and retinal layer volumes pre conversion and subsequent type of choroidal neovascularisation (CNV). METHODS: This was a single-centre retrospective study. The study participants were patients with wet age-related macular degeneration (AMD) in one eye who developed wet AMD in the contralateral eye, with at least 2 years of follow-up prior to conversion. The Moorfields Eye Hospital database was searched for eligible patients and their data were recorded. Eyes were classified as occult or classic based on fundus fluorescein angiography. Optical coherence tomography (OCT) images were analysed for drusen characteristics and retinal layer volumes were analysed over time using automated software (Topcon 3D OCT-2000 and Orion, Voxeleron LLC, respectively). All values were obtained at baseline as well as year 1 and year 2 before conversion to wet AMD. RESULTS: Fifty-one eyes with bilateral CNV showed high correlation of type of CNV between eyes (kappa statistic 0.89). A total of 49 wet AMD eyes (29 occult, 20 classic) were analysed for drusen parameters. Two patients with retinal angiomatous proliferation were excluded. Drusen count, area, and volume did not differ by CNV type, but the rates of change of drusen area (p = 0.046) and drusen volume (0.022) were higher in the occult group in the year preceding CNV development. Of the 49 eyes, 17 (10 occult, 7 classic) with available good quality OCT were analysed for retinal layer volumes. There was a progressive reduction in outer nuclear layer (ONL) volume (p = 0.002) and an expansion in outer plexiform layer volume (p = 0.015) in eyes that developed occult CNV. CONCLUSION: Our study shows that rate of increase in drusen load and reduction in ONL are significant features seen in eyes developing occult CNV, highlighting new imaging markers that need to be replicated in larger studies. These markers provide insight into the pathogenesis of CNV and may serve as prognostic indicators, as classic CNV carries a poorer prognosis compared to occult CNV.

Four-week outcomes of vascular endothelial growth factor inhibitors for neovascular age-related macular degeneration.

IMPORTANCE: To assess early outcomes of intravitreal vascular endothelial growth factor (VEGF) inhibitor treatment in neovascular age-related macular degeneration (nAMD) before patients have a chance to miss or discontinue treatment. BACKGROUND: Intravitreal VEGF inhibitors used to treat nAMD have been compared in various ways. The present study compared the 4-week responses to the first injection of either aflibercept, bevacizumab, or ranibizumab. DESIGN: Observational study. PARTICIPANTS: Treatment-naïve nAMD patients with visual acuity (VA) taken 22 to 48 days after the first treatment with an intravitreal VEGF inhibitor. METHODS: An observational study from a prospectively designed database. MAIN OUTCOME MEASURES: VA change from baseline and proportion of eyes judged active 22 to 48 days after the first treatment. RESULTS: The overall mean (95% confidence interval [CI]) VA change at 4 weeks was +3.7 (3.3, 4.0) letters. No pairwise comparisons in crude VA change or VA change after multivariate adjustment between the three agents were significant. However, after multivariate adjustment, more eyes treated with bevacizumab (90%) had active disease 4 weeks after the first injection than ranibizumab (84%; P = .013) and aflibercept (82%; P = .004). Older age, higher baseline vision and larger lesions were associated with lower VA change. CONCLUSION: There was no significant difference in VA gains amongst all three drugs but ranibizumab and aflibercept seemed to be more efficacious in quelling disease activity 4 weeks after the first treatment. VA change after the first injection was driven largely by baseline characteristics such as age, baseline VA and lesion size.

Comparison of different morphologies of choroidal neovascularization evaluated by ocular coherence tomography angiography in age-related macular degeneration.

IMPORTANCE: The clinical implications of different morphologies of choroidal neovascularization (CNV), as evaluated by ocular coherence tomography angiography (OCTA) in neovascular age-related macular degeneration (nAMD), are lacking. BACKGROUND: To describe the morphology of CNV in nAMD using OCTA, and to compare the visual prognosis and other structural OCT biomarkers between different morphologic patterns. DESIGN: Retrospective cohort study. PARTICIPANTS: One hundred and forty eyes with nAMD treated with anti-vascular endothelial growth factor (VEGF). METHODS: Patients were examined using OCTA prior to and at 3, 6 and 12 months after receiving anti-VEGF therapy. MAIN OUTCOME MEASURES: Best-corrected visual acuity (BCVA) and morphologic retinal features. RESULTS: Organized CNV was identified in 110/140 eyes (78.6%) using OCTA. These CNV complexes could be divided into three OCTA patterns: the 'medusa' pattern (n = 41), characterized by branching vessels radiating in all directions; the 'seafan' pattern (n = 43), characterized by branching vessels radiating to one side of the lesion; and the 'tangled' pattern (n = 26), characterized by globular entwined vessels without a main trunk. At baseline, the eyes with the tangled pattern were from younger patients (P = .031) with better BCVA (P = .007). There were also fewer intraretinal cysts (P = .021), less fibrovascular pigment epithelial detachment (P = .009), and more pachychoroid (P = .007) in eyes with the tangled pattern on OCT. At 12 months post-treatment, patients with the tangled CNV pattern also showed greater visual improvement than patients with the other two patterns (P = .049). CONCLUSIONS AND RELEVANCE: Using OCTA, distinct morphologies of CNV in nAMD patients were identified. These different patterns might be useful predictors for the prognosis of nAMD patients after anti-VEGF therapy.