Vulnerability to environmental and climatic health provocations among women and men hospitalized with chronic heart disease: insights from the RESILIENCE TRIAL cohort.

AIMS: We aimed to recruit a representative cohort of women and men with multi-morbid chronic heart disease as part of a trial testing an innovative, nurse-co-ordinated, multi-faceted intervention to lower rehospitalization and death by addressing areas of vulnerability to external challenges to their health. METHODS AND RESULTS: The prospective, randomized open, blinded end-point RESILIENCE Trial recruited 203 hospital inpatients (mean age 75.7 ± 10.2 years) of whom 51% were women and 94% had combined coronary artery disease, heart failure, and/or atrial fibrillation. Levels of concurrent multi-morbidity were high (mean Charlson Index of Comorbidity Score 6.5 ± 2.7), and 8.9% had at least mild frailty according to the Rockwood Clinical Frailty Scale. Including the index admission, 19-20% of women and men had a pre-existing pattern of seasonally linked hospitalization (seasonality). Detailed phenotyping revealed that 48% of women and 40% of men had ≥3 physiological factors, and 15% of women and 16% of men had ≥3 behavioural factors likely to increase their vulnerability to external provocations to their health. Overall, 61-62% of women and men had ≥4 combined factors indicative of such vulnerability. Additional factors such as reliance on the public health system (63 vs. 49%), lower education (30 vs. 14%), and living alone (48 vs. 29%) were more prevalent in women. CONCLUSION: We successfully recruited women and men with multi-morbid chronic heart disease and bio-behavioural indicators of vulnerability to external provocations to their health. Once completed, the RESILIENCE TRIAL will provide important insights on the impact of addressing such vulnerability (promoting resilience) on subsequent health outcomes. REGISTRATION: ClinicalTrials.org: NCT04614428.

Doxycycline treatment of high-risk COVID-19-positive patients with comorbid pulmonary disease.

Infection with novel SARS-CoV-2 carries significant morbidity and mortality in patients with pulmonary compromise, such as lung cancer, autoimmune disease, and pneumonia. For early stages of mild to moderate disease, care is entirely supportive.Antiviral drugs such as remdesivir may be of some benefit but are reserved for severe cases given limited availability and potential toxicity. Repurposing of safer, established medications that may have antiviral activity is a possible approach for treatment of earlier-stage disease. Tetracycline and its derivatives (e.g. doxycycline and minocycline) are nontraditional antibiotics with a well-established safety profile, potential efficacy against viral pathogens such as dengue fever and chikungunya, and may regulate pathways important in initial infection, replication, and systemic response to SARS-CoV-2. We present a series of four high-risk, symptomatic, COVID-19+ patients, with known pulmonary disease, treated with doxycycline with subsequent rapid clinical improvement. No safety issues were noted with use of doxycycline.Doxycycline is an attractive candidate as a repurposed drug in the treatment of COVID-19 infection, with an established safety profile, strong preclinical rationale, and compelling initial clinical experience described here.The reviews of this paper are available via the supplemental material section.

Myh11+ microvascular mural cells and derived mesenchymal stem cells promote retinal fibrosis.

Retinal diseases are frequently characterized by the accumulation of excessive scar tissue found throughout the neural retina. However, the pathophysiology of retinal fibrosis remains poorly understood, and the cell types that contribute to the fibrotic response are incompletely defined. Here, we show that myofibroblast differentiation of mural cells contributes directly to retinal fibrosis. Using lineage tracing technology, we demonstrate that after chemical ocular injury, Myh11+ mural cells detach from the retinal microvasculature and differentiate into myofibroblasts to form an epiretinal membrane. Inhibition of TGFßR attenuates Myh11+ retinal mural cell myofibroblast differentiation, and diminishes the subsequent formation of scar tissue on the surface of the retina. We demonstrate retinal fibrosis within a murine model of oxygen-induced retinopathy resulting from the intravitreal injection of adipose Myh11-derived mesenchymal stem cells, with ensuing myofibroblast differentiation. In this model, inhibiting TGFßR signaling does not significantly alter myofibroblast differentiation and collagen secretion within the retina. This work shows the complexity of retinal fibrosis, where scar formation is regulated both by TGFßR and non-TGFßR dependent processes involving mural cells and derived mesenchymal stem cells. It also offers a cautionary note on the potential deleterious, pro-fibrotic effects of exogenous MSCs once intravitreally injected into clinical patients.

Pericyte Bridges in Homeostasis and Hyperglycemia.

Diabetic retinopathy is a potentially blinding eye disease that threatens the vision of one-ninth of patients with diabetes. Progression of the disease has long been attributed to an initial dropout of pericytes that enwrap the retinal microvasculature. Revealed through retinal vascular digests, a subsequent increase in basement membrane bridges was also observed. Using cell-specific markers, we demonstrate that pericytes rather than endothelial cells colocalize with these bridges. We show that the density of bridges transiently increases with elevation of Ang-2, PDGF-BB, and blood glucose; is rapidly reversed on a timescale of days; and is often associated with a pericyte cell body located off vessel. Cell-specific knockout of KLF4 in pericytes fully replicates this phenotype. In vivo imaging of limbal vessels demonstrates pericyte migration off vessel, with rapid pericyte filopodial-like process formation between adjacent vessels. Accounting for off-vessel and on-vessel pericytes, we observed no pericyte loss relative to nondiabetic control retina. These findings reveal the possibility that pericyte perturbations in location and process formation may play a role in the development of pathological vascular remodeling in diabetic retinopathy.

REAVER: A program for improved analysis of high-resolution vascular network images.

Alterations in vascular networks, including angiogenesis and capillary regression, play key roles in disease, wound healing, and development. The spatial structures of blood vessels can be captured through imaging, but effective characterization of network architecture requires both metrics for quantification and software to carry out the analysis in a high-throughput and unbiased fashion. We present Rapid Editable Analysis of Vessel Elements Routine (REAVER), an open-source tool that researchers can use to analyze high-resolution 2D fluorescent images of blood vessel networks, and assess its performance compared to alternative image analysis programs. Using a dataset of manually analyzed images from a variety of murine tissues as a ground-truth, REAVER exhibited high accuracy and precision for all vessel architecture metrics quantified, including vessel length density, vessel area fraction, mean vessel diameter, and branchpoint count, along with the highest pixel-by-pixel accuracy for the segmentation of the blood vessel network. In instances where REAVER's automated segmentation is inaccurate, we show that combining manual curation with automated analysis improves the accuracy of vessel architecture metrics. REAVER can be used to quantify differences in blood vessel architectures, making it useful in experiments designed to evaluate the effects of different external perturbations (eg, drugs or disease states).

Comparison of amyloid PET measured in Centiloid units with neuropathological findings in Alzheimer's disease.

BACKGROUND: The Centiloid scale was developed to standardise the results of beta-amyloid (Aß) PET. We aimed to determine the Centiloid unit (CL) thresholds for CERAD sparse and moderate-density neuritic plaques, Alzheimer's disease neuropathologic change (ADNC) score of intermediate or high probability of Alzheimer's Disease (AD), final clinicopathological diagnosis of AD, and expert visual read of a positive Aß PET scan. METHODS: Aß PET results in CL for 49 subjects were compared with post-mortem findings, visual read, and final clinicopathological diagnosis. The Youden Index was used to determine the optimal CL thresholds from receiver operator characteristic (ROC) curves. RESULTS: A threshold of 20.1 CL (21.3 CL when corrected for time to death, AUC 0.97) yielded highest accuracy in detecting moderate or frequent plaque density while < 10 CL was optimal for excluding neuritic plaque. The threshold for ADNC intermediate or high likelihood AD was 49.4 CL (AUC 0.98). Those cases with a final clinicopathological diagnosis of AD yielded a median CL result of 87.7 (IQR ± 42.2) with 94% > 45 CL. Positive visual read agreed highly with results > 26 CL. CONCLUSIONS: Centiloid values < 10 accurately reflected the absence of any neuritic plaque and > 20 CL indicated the presence of at least moderate plaque density, but approximately 50 CL or more best confirmed both neuropathological and clinicopathological diagnosis of Alzheimer's disease.

Myh11 Lineage Corneal Endothelial Cells and ASCs Populate Corneal Endothelium.

Purpose: To establish Myh11 as a marker of a subset of corneal endothelial cells (CECs), and to demonstrate the feasibility of restoring the corneal endothelium with Myh11-lineage (Myh11-Lin[+]) adipose-derived stromal cells (ASCs). Methods: Intraperitoneal administration of tamoxifen and (Z)-4-hydroxytamoxifen eyedrops were used to trace the lineage of Myh11-expressing cells with the Myh11-Cre-ERT2-flox-tdTomato mouse model. Immunostaining and Western blot characterized marker expression and spatial distribution of Myh11-Lin(+) cells in the cornea, and administration of 5-ethynyl-2'-deoxyuridine labeled proliferating cells. ASCs were isolated from epididymal adipose Myh11+ mural cells and treated with cornea differentiation media to evaluate corneal endothelial differentiation potential. Differentiated ASCs were injected into the anterior chamber to test for incorporation into corneal endothelium following scratch injury. Results: A subset of CECs express Myh11, a marker previously thought restricted to only mural cells. Myh11-Lin(+) CECs marked a stable subpopulation of cells in the cornea endothelium. Myh11-Lin(+) ASCs undergo CEC differentiation in vitro and incorporate into injured corneal endothelium. Conclusions: Dystrophy and dysfunction of the corneal endothelium accounts for almost half of all corneal transplants, the maintenance of the cornea endothelium is poorly understood, and there are a lack of mouse models to study specific CEC populations. We establish a mouse model that can trace the cell fate of a subpopulation of CECs based on Myh11 expression. A subset of ASCs that share this Myh11 transcriptional lineage are capable of differentiating into CECs that can incorporate into injured corneal endothelium, revealing a potential cell source for creating engineered transplant material.

APOE and cortical superficial siderosis in CAA: Meta-analysis and potential mechanisms.

OBJECTIVE: To assess potential mechanisms of cortical superficial siderosis (cSS), a central MRI biomarker in cerebral amyloid angiopathy (CAA), we performed a collaborative meta-analysis of APOE associations with cSS presence and severity. METHODS: We pooled data from published studies reporting APOE genotype and MRI assessment of cSS in 3 distinct settings: (1) stroke clinic patients with symptomatic CAA (i.e., lobar intracerebral hemorrhage, transient focal neurologic episodes) according to the Boston criteria; (2) memory clinic patients; and (3) population-based studies. We compared cSS presence and severity (focal or disseminated vs no cSS) in participants with ε2+ or ε4+ genotype vs the ε3/ε3 genotype, by calculating study-specific and random effects pooled, unadjusted odds ratios (ORs). RESULTS: Thirteen studies fulfilled inclusion criteria: 7 memory clinic cohorts (n = 2,587), 5 symptomatic CAA cohorts (n = 402), and 1 population-based study (n = 1,379). There was no significant overall association between APOE ε4+ and cSS presence or severity. When stratified by clinical setting, APOE ε4+ was associated with cSS in memory clinic (OR 2.10; 95% confidence interval [CI] 1.11-3.99) but not symptomatic CAA patients. The pooled OR showed significantly increased odds of having cSS for APOE ε2+ genotypes (OR 2.42, 95% CI 1.48-3.95) in both patient populations. This association was stronger for disseminated cSS in symptomatic CAA cohorts. In detailed subgroup analyses, APOE ε2/ε2 and APOE ε2/ε4 genotypes were most consistently and strongly associated with cSS presence and severity. CONCLUSION: CAA-related vasculopathic changes and fragility associated with APOE ε2+ allele might have a biologically meaningful role in the pathophysiology and severity of cSS.

Sexual Dimorphism of Resting-State Network Connectivity in Healthy Ageing.

OBJECTIVES: The onset of many illnesses is confounded with age and sex. Increasing age is a risk factor for the development of many illnesses, and sexual dimorphism influences brain anatomy, function, and cognition. Here, we examine frequency-specific connectivity in resting-state networks in a large sample (n = 406) of healthy aged adults. METHOD: We quantify frequency-specific connectivity in three resting-state networks known to be implicated in age-related decline: the default mode, dorsal attention, and salience networks, using multiband functional magnetic resonance imaging. Frequency-specific connectivity was quantified in four bands: low (0.015-0.027 Hz), moderately low (0.027-0.073 Hz), moderately high (0.073-0.198 Hz), and high (0.198-0.5 Hz) frequency bands, using mean intensity and spatial extent. Differences in connectivity between the sexes in each of the three networks were examined. RESULTS: Each network showed the largest intensity and spatial extent at low frequencies and smallest extent at high frequencies. Males showed greater connectivity than females in the salience network. Females showed greater connectivity than males in the default mode network. DISCUSSION: Results in this healthy aged cohort are compatible with those obtained in young samples, suggesting that frequency-specific connectivity, and differences between the sexes, are maintained into older age. Our results indicate that sex should be considered as an influencing factor in studies of resting-state connectivity.

CIRCOAST: a statistical hypothesis test for cellular colocalization with network structures.

Motivation: Colocalization of structures in biomedical images can lead to insights into biological behaviors. One class of colocalization problems is examining an annular structure (disk-shaped such as a cell, vesicle or molecule) interacting with a network structure (vascular, neuronal, cytoskeletal, organellar). Examining colocalization events across conditions is often complicated by changes in density of both structure types, confounding traditional statistical approaches since colocalization cannot be normalized to the density of both structure types simultaneously. We have developed a technique to measure colocalization independent of structure density and applied it to characterizing intercellular colocation with blood vessel networks. This technique could be used to analyze colocalization of any annular structure with an arbitrarily shaped network structure. Results: We present the circular colocalization affinity with network structures test (CIRCOAST), a novel statistical hypothesis test to probe for enriched network colocalization in 2D z-projected multichannel images by using agent-based Monte Carlo modeling and image processing to generate the pseudo-null distribution of random cell placement unique to each image. This hypothesis test was validated by confirming that adipose-derived stem cells (ASCs) exhibit enriched colocalization with endothelial cells forming arborized networks in culture and then applied to show that locally delivered ASCs have enriched colocalization with murine retinal microvasculature in a model of diabetic retinopathy. We demonstrate that the CIRCOAST test provides superior power and type I error rates in characterizing intercellular colocalization compared to generic approaches that are confounded by changes in cell or vessel density. Availability and implementation: CIRCOAST source code available at: https://github.com/uva-peirce-cottler-lab/ARCAS. Supplementary information: Supplementary data are available at Bioinformatics online.

Dynamic, heterogeneous endothelial Tie2 expression and capillary blood flow during microvascular remodeling.

Microvascular endothelial cell heterogeneity and its relationship to hemodynamics remains poorly understood due to a lack of sufficient methods to examine these parameters in vivo at high resolution throughout an angiogenic network. The availability of surrogate markers for functional vascular proteins, such as green fluorescent protein, enables expression in individual cells to be followed over time using confocal microscopy, while photoacoustic microscopy enables dynamic measurement of blood flow across the network with capillary-level resolution. We combined these two non-invasive imaging modalities in order to spatially and temporally analyze biochemical and biomechanical drivers of angiogenesis in murine corneal neovessels. By stimulating corneal angiogenesis with an alkali burn in Tie2-GFP fluorescent-reporter mice, we evaluated how onset of blood flow and surgically-altered blood flow affects Tie2-GFP expression. Our study establishes a novel platform for analyzing heterogeneous blood flow and fluorescent reporter protein expression across a dynamic microvascular network in an adult mammal.

ASPREE-NEURO study protocol: A randomized controlled trial to determine the effect of low-dose aspirin on cerebral microbleeds, white matter hyperintensities, cognition, and stroke in the healthy elderly.

Rationale Cerebral microbleeds seen on brain magnetic resonance imaging are markers of small vessel disease, linked to cognitive dysfunction and increased ischemic and hemorrhagic stroke risk. Observational studies suggest that aspirin use may induce cerebral microbleeds, and associated overt intracranial hemorrhage, but this has not been definitively resolved. Aims ASPREE-NEURO will determine the effect of aspirin on cerebral microbleed development over three years in healthy adults aged 70 years and over, participating in the larger 'ASPirin in Reducing Events in the Elderly (ASPREE)' primary prevention study of aspirin. Sample size Five hundred and fifty-nine participants provide 75% power (two-sided p value of 0.05) to determine an average difference of 0.5 cerebral microbleed per person after three years. Methods and design A multi-center, randomized placebo-controlled trial of 100 mg daily aspirin in participants who have brain magnetic resonance imaging at study entry, one and three years after randomization and who undergo cognitive testing at the same time points. Study outcomes The primary outcome is the number of new cerebral microbleeds on magnetic resonance imaging after three years. Secondary outcomes are the number of new cerebral microbleeds after one year, change in volume of white matter hyperintensity, cognitive function, and stroke. Discussion ASPREE-NEURO will resolve whether aspirin affects the presence and number of cerebral microbleeds, their relationship with cognitive performance, and indicate whether consideration of cerebral microbleeds alters the risk-benefit profile of aspirin in primary prevention for older people. Trial registration Australian New Zealand Clinical Trials Registry ACTRN12613001313729.

Adipose-derived stem cells from diabetic mice show impaired vascular stabilization in a murine model of diabetic retinopathy.

Diabetic retinopathy is characterized by progressive vascular dropout with subsequent vision loss. We have recently shown that an intravitreal injection of adipose-derived stem cells (ASCs) can stabilize the retinal microvasculature, enabling repair and regeneration of damaged capillary beds in vivo. Because an understanding of ASC status from healthy versus diseased donors will be important as autologous cellular therapies are developed for unmet clinical needs, we took advantage of the hyperglycemic Akimba mouse as a preclinical in vivo model of diabetic retinopathy in an effort aimed at evaluating therapeutic efficacy of adipose-derived stem cells (mASCs) derived either from healthy, nondiabetic or from diabetic mice. To these ends, Akimba mice received intravitreal injections of media conditioned by mASCs or mASCs themselves, subsequent to development of substantial retinal capillary dropout. mASCs from healthy mice were more effective than diabetic mASCs in protecting the diabetic retina from further vascular dropout. Engrafted ASCs were found to preferentially associate with the retinal vasculature. Conditioned medium was unable to recapitulate the vasoprotection seen with injected ASCs. In vitro diabetic ASCs showed decreased proliferation and increased apoptosis compared with healthy mASCs. Diabetic ASCs also secreted less vasoprotective factors than healthy mASCs, as determined by high-throughput enzyme-linked immunosorbent assay. Our findings suggest that diabetic ASCs are functionally impaired compared with healthy ASCs and support the utility of an allogeneic injection of ASCs versus autologous or conditioned media approaches in the treatment of diabetic retinopathy.

Incidence of cerebral microbleeds in preclinical Alzheimer disease.

OBJECTIVE: We sought to determine the incidence and associations of lobar microbleeds (LMBs) in a longitudinal cohort with (11)C-Pittsburgh compound B (PiB) PET imaging. METHODS: One hundred seventy-four participants from the observational Australian Imaging, Biomarkers and Lifestyle Study of Ageing (97 with normal cognition [NC], 37 with mild cognitive impairment [MCI], and 40 with Alzheimer disease [AD] dementia) were assessed at 3 time points over 3 years with 3-tesla susceptibility-weighted MRI and (11)C-PiB PET. MRIs were inspected for microbleeds, siderosis, infarction, and white matter hyperintensity severity, blind to clinical and PiB findings. Neocortical PiB standardized uptake value ratio, normalized to cerebellar cortex, was dichotomized as positive or negative (PiB+/-, standardized uptake value ratio >1.5). Annualized LMB incidence was calculated, and logistic regression was used to determine the association of incident LMBs with PiB, APOE ε4+ status, and cerebrovascular disease. RESULTS: LMBs were present in 18.6% of NC, 24.3% of MCI, and 40% of AD participants (p < 0.05 vs NC). LMB incidence was 0.2 ± 0.6 per year in NC participants, 0.2 ± 0.5 in MCI, and 0.7 ± 1.4 in AD (p < 0.03 vs NC) and was 6-fold higher in PiB+ than PiB-NC. Incident LMBs were associated with age, APOE ε4+, PiB+, and baseline LMBs. Incidence of multiple LMBs was also associated with lacunar infarction and white matter hyperintensity severity. CONCLUSIONS: Older age, baseline LMBs, higher ß-amyloid burden, and concomitant cerebrovascular disease may all confer higher risk of incident LMBs. This should be considered when designing protocols for amyloid-modifying clinical trials.

Cerebral microbleeds: a review of clinical, genetic, and neuroimaging associations.

Cerebral microbleeds (microbleeds) are small, punctuate hypointense lesions seen in T2* Gradient-Recall Echo (GRE) and Susceptibility-Weighted (SWI) Magnetic Resonance Imaging (MRI) sequences, corresponding to areas of hemosiderin breakdown products from prior microscopic hemorrhages. They occur in the setting of impaired small vessel integrity, commonly due to either hypertensive vasculopathy or cerebral amyloid angiopathy. Microbleeds are more prevalent in individuals with Alzheimer's disease (AD) dementia and in those with both ischemic and hemorrhagic stroke. However they are also found in asymptomatic individuals, with increasing prevalence with age, particularly in carriers of the Apolipoprotein (APOE) ε4 allele. Other neuroimaging findings that have been linked with microbleeds include lacunar infarcts and white matter hyperintensities on MRI, and increased cerebral ß-amyloid burden using (11)C-PiB Positron Emission Tomography. The presence of microbleeds has been suggested to confer increased risk of incident intracerebral hemorrhage - particularly in the setting of anticoagulation - and of complications of immunotherapy for AD. Prospective data regarding the natural history and sequelae of microbleeds are currently limited, however there is a growing evidence base that will serve to inform clinical decision-making in the future.

Pericytes derived from adipose-derived stem cells protect against retinal vasculopathy.

BACKGROUND: Retinal vasculopathies, including diabetic retinopathy (DR), threaten the vision of over 100 million people. Retinal pericytes are critical for microvascular control, supporting retinal endothelial cells via direct contact and paracrine mechanisms. With pericyte death or loss, endothelial dysfunction ensues, resulting in hypoxic insult, pathologic angiogenesis, and ultimately blindness. Adipose-derived stem cells (ASCs) differentiate into pericytes, suggesting they may be useful as a protective and regenerative cellular therapy for retinal vascular disease. In this study, we examine the ability of ASCs to differentiate into pericytes that can stabilize retinal vessels in multiple pre-clinical models of retinal vasculopathy. METHODOLOGY/PRINCIPAL FINDINGS: We found that ASCs express pericyte-specific markers in vitro. When injected intravitreally into the murine eye subjected to oxygen-induced retinopathy (OIR), ASCs were capable of migrating to and integrating with the retinal vasculature. Integrated ASCs maintained marker expression and pericyte-like morphology in vivo for at least 2 months. ASCs injected after OIR vessel destabilization and ablation enhanced vessel regrowth (16% reduction in avascular area). ASCs injected intravitreally before OIR vessel destabilization prevented retinal capillary dropout (53% reduction). Treatment of ASCs with transforming growth factor beta (TGF-ß1) enhanced hASC pericyte function, in a manner similar to native retinal pericytes, with increased marker expression of smooth muscle actin, cellular contractility, endothelial stabilization, and microvascular protection in OIR. Finally, injected ASCs prevented capillary loss in the diabetic retinopathic Akimba mouse (79% reduction 2 months after injection). CONCLUSIONS/SIGNIFICANCE: ASC-derived pericytes can integrate with retinal vasculature, adopting both pericyte morphology and marker expression, and provide functional vascular protection in multiple murine models of retinal vasculopathy. The pericyte phenotype demonstrated by ASCs is enhanced with TGF-ß1 treatment, as seen with native retinal pericytes. ASCs may represent an innovative cellular therapy for protection against and repair of DR and other retinal vascular diseases.

Construction of an inexpensive, hand-held fundus camera through modification of a consumer "point-and-shoot" camera.

PURPOSE: To construct a low-cost, easy-to-use, high-image-quality mydriatic fundus camera with "point-and-shoot" operation, and to evaluate the efficacy of this camera to accurately document retinal disease. METHODS: A prototype portable fundus camera was designed by interfacing a novel optical module with a Panasonic Lumix G2 consumer camera. Low-cost, commercially available optics were used to create even illumination of the fundus, providing a 50° retinal field of view. A comparative study assessing the image quality of the prototype camera against a traditional tabletop fundus camera was conducted under an Institutional Review Board (IRB)-approved study. RESULTS: A stand-alone, mydriatic camera prototype was successfully developed at a parts cost of less than $1000. The prototype camera was capable of operating in a point-and-shoot manner with automated image focusing and exposure, and the image quality of fundus photos was comparable to that of existing commercial cameras. Pathology related to both nonproliferative and proliferative diabetic retinopathy and age-related macular degeneration was easily identified from fundus images obtained from the low-cost camera. CONCLUSIONS: Early prototype development and clinical testing have shown that a consumer digital camera can be inexpensively modified to image the fundus with professional diagnostic quality. The combination of low cost, portability, point-and-shoot operation, and high image quality provides a foundational platform on which one can design an accessible fundus camera to screen for eye disease.

Attenuation of ephrinB2 reverse signaling decreases vascularized area and preretinal vascular tuft formation in the murine model of oxygen-induced retinopathy.

PURPOSE: EphB4 and ephrinB2 are known key regulators of retinal vascular development, but due to their capacity for bidirectional signaling, delineation of their individual roles in this process remains unclear. To better dissect out individual contributions, a model of proliferative retinopathy in mice with attenuated ephrinB2 reverse signaling was studied. It was hypothesized that endothelial ephrinB2 reverse signaling regulates hypoxia-induced capillary sprouting, as well as the pathologic formation of neovascular tufts in postnatal retinal microvascular networks. METHODS: Genetically manipulated mice with attenuated ephrinB2 reverse signaling (ephrinB2(lacZ/+)), along with wild-type (WT) controls, were exposed to oxygen-induced retinopathy (OIR), a postnatal model of proliferative retinopathy. At peak disease (postnatal day 18), microvascular networks were analyzed to examine intraretinal revascularization, capillary sprouting, and pathologic neovascularization responses. EphB4 and phosphorylated ephrinB protein expression patterns along retinal microvessels were also assessed. RESULTS: EphrinB2(lacZ/+) mice exhibited reduced hypoxia-induced revascularization (P ≤ 0.04) and reduced formation of neovascular tufts (P < 0.001), as compared with WT controls. Corresponding to the observed inhibition of retinal angiogenesis, ephrinB2(lacZ/+) retinas displayed an increased number of blind-ended capillary sprout tips (P < 0.02) and endothelial filopodial processes (P = 0.001). In WT and ephrinB2(lacZ/+) OIR-exposed retinas, ephrinB was confined to endothelial cells, with expression detected along angiogenic vascular processes including neovascular tufts and blind-ended capillary sprouts. CONCLUSIONS: EphrinB2 reverse signaling is a regulator of key processes during retinal vascularization and controls pathologic retinal angiogenesis through direct effects on capillary sprouting and endothelial filopodia formation.

Chronic whole-body hypoxia induces intussusceptive angiogenesis and microvascular remodeling in the mouse retina.

Currently, little is known about the response of the adult retinal microvasculature to hypoxia. To test the hypothesis that chronic systemic hypoxia induces angiogenesis and microvascular remodeling in the adult mouse retina, adult 10-week old female C57Bl/6 mice were exposed to 10% O(2) for 2 or 3 weeks. After hypoxia exposure, retinas were harvested, whole-mounted, and processed for immunohistochemistry. Retinas were stained with lectin, anti-smooth muscle alpha-actin antibody, and anti-NG2 antibody to visualize microvascular networks and their cellular components. Confocal microscopy was used to obtain images of superficial retinal networks. Images were analyzed to assess vessel diameter, vascular length density, branch point density, and the presence of vascular loops, a hallmark of intussusceptive angiogenesis. Both 2 and 3 weeks of hypoxia exposure resulted in a significant increase in the diameters of arterioles and post-arteriole capillaries (p<0.003). After 3 weeks of hypoxia, vascular length density and branch point density were significantly increased in retinas exposed to hypoxia as compared to normoxic controls (p<0.001). The number of vascular loops in the superficial retinal networks was significantly greater in hypoxia-exposed retinas (p < or = 0.001). Our results demonstrate, for the first time, intussusceptive angiogenesis as a tissue-level mechanism of vascular adaptation to chronic systemic hypoxia in the adult mouse retina and contribute to our understanding of hypoxia-induced angiogenesis and microvascular remodeling in the adult animal.