MACUSTAR: Development and Clinical Validation of Functional, Structural, and Patient-Reported Endpoints in Intermediate Age-Related Macular Degeneration.

PURPOSE: Currently, no outcome measures are clinically validated and accepted as clinical endpoints by regulatory agencies for drug development in intermediate age-related macular degeneration (iAMD). The MACUSTAR Consortium, a public-private research group funded by the European Innovative Medicines Initiative intends to close this gap. PROCEDURES: Development of study protocol and statistical analysis plan including predictive modelling of multimodal endpoints based on a review of the literature and expert consensus. RESULTS: This observational study consists of a cross-sectional and a longitudinal part. Functional outcome measures assessed under low contrast and low luminance have the potential to detect progression of visual deficit within iAMD and to late AMD. Structural outcome measures will be multimodal and investigate topographical relationships with function. Current patient-reported outcome measures (PROMs) are not acceptable to regulators and may not capture the functional deficit specific to iAMD with needed precision, justifying development of novel PROMs for iAMD. The total sample size will be n = 750, consisting mainly of subjects with iAMD (n = 600). CONCLUSIONS: As clinical endpoints currently accepted by regulators cannot detect functional loss or patient-relevant impact in iAMD, we will clinically validate novel candidate endpoints for iAMD.

G-CSF induces E-selectin ligand expression on human myeloid cells.

Clinical use of G-CSF can result in vascular and inflammatory complications. To investigate the molecular basis of these effects, we analyzed the adherence of G-CSF-mobilized human peripheral blood leukocytes (ML) to inflamed (TNF-alpha-stimulated) vascular endothelium. Studies using parallel plate assays under physiologic flow conditions and intravital microscopy in a mouse inflammation model each showed that ML take part in heightened adhesive interactions with endothelium compared to unmobilized (native) blood leukocytes, mediated by markedly increased E-selectin receptor-ligand interactions. Biochemical studies showed that ML express the potent E-selectin ligand HCELL (ref. 8) and another, previously unrecognized approximately 65-kDa E-selectin ligand, and possess enhanced levels of transcripts encoding glycosyltransferases (ST3GalIV, FucT-IV and FucT-VII) conferring glycan modifications associated with E-selectin ligand activity. Enzymatic treatments and physiologic binding assays showed that HCELL and the approximately 65-kDa E-selectin ligand contribute prominently to the observed G-CSF-induced myeloid cell adhesion to inflamed endothelium. Treatment of normal human bone marrow cells with a pharmacokinetically relevant concentration of G-CSF in vitro resulted in increased expression of these two molecules, coincident with increased transcripts encoding pertinent glycosyltransferases and heightened E-selectin binding. These findings provide direct evidence for a role of G-CSF in the induction of E-selectin ligands on myeloid cells, thus providing mechanistic insight into the pathobiology of G-CSF complications.

Clinical study protocol for a low-interventional study in intermediate age-related macular degeneration developing novel clinical endpoints for interventional clinical trials with a regulatory and patient access intention-MACUSTAR.

BACKGROUND: There is an unmet need for treatment options in intermediate age-related macular degeneration (iAMD). However, for any new interventions to be tested in clinical trials, novel currently unavailable clinical endpoints need to be developed. Thus, the MACUSTAR study aims to develop and evaluate functional, structural, and patient-reported candidate endpoints for use in future iAMD trials. METHODS: The protocol describes a low-interventional clinical multicenter study employing a novel two-part design. The cross-sectional part (total duration, 1 month) and the longitudinal part (total duration, 36 months) include participants with iAMD and control groups with early/late/no AMD. The cross-sectional part's primary objective is a technical evaluation of functional, structural, and patient-reported candidate outcomes. The longitudinal part's primary objective is to assess the prognostic power of changes in functional, structural, and patient-reported outcomes for progression from iAMD to late AMD. All data will be used to support a biomarker qualification procedure by regulatory authorities. DISCUSSION: The MACUSTAR study characterizes and evaluates much needed novel functional, structural, and patient-reported endpoints for future clinical trials in iAMD and will improve our understanding of the natural history and prognostic markers of this condition. TRIAL REGISTRATION: ClinicalTrials.gov NCT03349801 . Registered on 22 November 2017.

Assessment of Novel Genome-Wide Significant Gene Loci and Lesion Growth in Geographic Atrophy Secondary to Age-Related Macular Degeneration.

IMPORTANCE: Age-related macular degeneration (AMD) is a common threat to vision loss in individuals older than 50 years. While neovascular complications in AMD are treatable, there is currently no therapy for geographic atrophy secondary to AMD. Geographic atrophy lesion progression over time shows considerable interindividual variability, but little is known about prognostic factors. OBJECTIVE: To elucidate the contribution of common genetic variants to geographic atrophy lesion growth. DESIGN, SETTING, AND PARTICIPANTS: This pooled analysis combined 4 independent studies: the Fundus Autofluorescence Imaging in Age-Related Macular Degeneration (FAM) study, the Directional Spread in Geographic Atrophy (DSGA) study, the Age-Related Eye Disease Study (AREDS), and the Geographic Atrophy Treatment Evaluation (GATE) study. Each provided data for geographic atrophy lesion growth in specific designs. Patients with geographic atrophy secondary to AMD were recruited to these studies. Genotypes were retrieved through the database of Genotypes and Phenotypes (for AREDS) or generated at the Cologne Center for Genomics (for FAM, DSGA, and GATE). MAIN OUTCOMES: The correlation between square root-transformed geographic atrophy growth rate and 7 596 219 genetic variants passing quality control was estimated using linear regression. The calculations were adjusted for known factors influencing geographic atrophy growth, such as the presence of bilateral geographic atrophy as well as the number of lesion spots and follow-up times. MAIN OUTCOMES AND MEASURES: Slopes per allele, 95% CIs, and P values of genetic variants correlated with geographic atrophy lesion growth. RESULTS: A total of 935 patients (mean [SD] age, 74.7 [7.8] years; 547 female participants [59.0%]) were included. Two gene loci with conservative genome-wide significance were identified. Each minor allele of the genome-wide associated variants increased the geographic atrophy growth rate by a mean of about 15% or 0.05 mm per year. Gene prioritization within each locus suggests the protein arginine methyltransferase 6 gene (PRMT6; chromosome 1; slope, 0.046 [95% CI, 0.026-0.066]; P = 4.09 × 10-8) and the lanosterol synthase gene (LSS; chromosome 21; slope, 0.105 [95% CI, 0.068-0.143]; P = 4.07 × 10-7) as the most likely progression-associated genes. CONCLUSIONS AND RELEVANCE: These data provide further insight into the genetic architecture of geographic atrophy lesion growth. Geographic atrophy is a clinical outcome with a high medical need for effective therapy. The genes PRMT6 and LSS are promising candidates for future studies aimed at understanding functional aspects of geographic atrophy progression and also for designing novel and targeted treatment options.

Immunosuppressive properties of the pigmented epithelial cells and the subretinal space.

The immune privilege of the anterior chamber of the eye has been recognized for over 100 years. However, the unique immunological properties of the pigmented epithelial (PE) cells of the eye and the subretinal space (SRS) have only recently been appreciated. The PE cells of the iris, ciliary body, and retina reside in anatomically disparate locations and serve distinctly different functions, yet share interesting immunomodulatory properties that contribute to ocular immune privilege. PE cells in the ciliary body and retina elaborate a variety of soluble factors that either directly or indirectly dampen immune-mediated inflammation; these include transforming growth factor-Beta, somatostatin, thrombospondin and pigment epithelial derived factor (PEDF). The constitutive expression of the immune co-stimulatory molecule, CD86, on iris PE cells not only inhibits T cell proliferation, but also promotes the generation of regulatory T cells. The SRS is now recognized as an immune-privileged site that shares many, but not all, of the properties ascribed to the anterior chamber, including the induction of systemic immune deviation. The prospect of therapeutic retinal transplantation and the possible immunologic etiology for some forms of age-related macular degeneration provides new impetus for gaining a better understanding of ocular immune privilege in the posterior regions of the eye.

Pigment epithelial growth factor suppresses inflammation by modulating macrophage activation.

PURPOSE: To study the contribution of murine retinal pigment epithelial (RPE) cells to the innate immune-privilege status of the subretinal space as determined by the ability of pigment epithelial-derived factor (PEDF) and somatostatin (SOM), produced by RPE, to regulate macrophage-mediated inflammation. METHODS: Serum-free medium was added to RPE eyecups (a healthy monolayer of RPE resting on choroid and sclera) and the supernatants were removed after 24 hours (RPE SN). The RPE SN was assayed for the presence of PEDF and SOM and for its ability to regulate interleukin (IL)-12, IL-10, and nitric oxide (NO) production by resting and activated macrophages. A group of mice received intradermal injection of lipopolysaccharide (LPS) and PEDF in one ear and LPS alone in the other ear. Ear thickness was measured before- and 24 hours after ear injections. RESULTS: Soluble factors present in the RPE SN inhibited IL-12 production and substantially increased IL-10 while having minimal effects on NO production by activated macrophages. The message for PEDF, SOM, and IL-10 was detected in RPE cells, and the protein for these factors was found in the RPE SN. The stimulation of IL-10 and suppression of IL-12 production by RPE-SN-treated macrophages was neutralized by anti-PEDF antibodies. Neutralization of SOM in the RPE SN, suppressed NO production by activated macrophages. Intradermal injection of PEDF substantially inhibited LPS-induced inflammatory response. CONCLUSIONS: PEDF inhibits LPS-driven macrophage activation in vitro and in vivo. By producing PEDF, the RPE contributes to innate immune privilege of the eye.

Retinal progenitor cell xenografts to the pig retina: immunological reactions.

We evaluated the host response to murine retinal progenitor cells (RPCs) following transplantation to the subretinal space (SRS) of the pig. RPCs from GFP mice were transplanted subretinally in 18 nonimmunosuppressed normal or laser-treated pigs. Evaluation of the SRS was performed on hematoxylin-eosin (H&E)-stained sections. Serum samples were taken from naive and RPC-grafted pigs and mouse-reactive antibody responses were assessed. At 1 week, histology showed a few perivascular lymphocytes consistent with a mild retinal vasculitis, and depigmentation of the RPE with large numbers of mononuclear inflammatory cells in the choroid near the transplantation site. Large choroidal infiltrates were evident at 2-5 weeks. Serum from naive and RPC-xenografted pigs contained significant levels of preformed IgG and IgM antibodies against murine antigens. Xenogeneic RPCs transplanted to the porcine SRS induced mononuclear infiltration in the choroid with graft rejection occurring over 2-5 weeks. Serum analysis confirmed that mice and pigs are discordant species; however, a cell-mediated acute mechanism appears to be responsible, rather than an antibody-mediated rejection.

Thrombospondin plays a vital role in the immune privilege of the eye.

PURPOSE: The role of thrombospondin (TSP)-1 in TGF-beta activation and T-cell suppression was studied in the retinal pigment epithelial (RPE) cells, a monolayer of pigmented cells that line the subretinal space, an immune-privileged site in the eye. METHODS: Posterior eyecups were prepared by excising the anterior segment, lens, and retina from enucleated eyes of C57BL/6, thrombospondin-1 knockout (TSP-1KO), and TGF-beta2 receptor II double-negative (TGF-beta2 RII DN) mice, leaving behind a healthy monolayer of RPE resting on choroid and sclera. Serum-free medium was added to these RPE eyecups, and, after various time intervals, supernatants (SNs) were removed and tested. RESULTS: SNs of an ex vivo culture of RPE cells from C57BL/6 mice were shown to inhibit both antigen and anti-CD3 activation of T cells, partially due to constitutive production of TGF-beta and to the ability of RPE to activate the latent form of TGF-beta. Activation of TGF-beta was entirely dependent on TSP-1, also produced by RPE. SNs of RPE from TSP-1KO mice failed to inhibit T-cell activation. Ovalbumin (OVA)-specific delayed hypersensitivity (DH) was not impaired when OVA was injected either into the subretinal space or into the anterior chamber of TSP-1KO mice before OVA immunization. Moreover, experimental autoimmune uveoretinitis was significantly more intense in eyes of TSP-1KO mice and failed to undergo spontaneous resolution unlike wild-type mice. CONCLUSIONS: Production of both TSP-1 and active TGF-beta by RPE is essential to the creation and maintenance of immune privilege in the subretinal space and that the immune privilege limits the severity and duration of retinal inflammation due to autoimmunity.

Vulnerability of allogeneic retinal pigment epithelium to immune T-cell-mediated damage in vivo and in vitro.

PURPOSE: Because retinal pigment epithelium (RPE) constitutively expresses class I major histocompatibility complex (MHC) molecules, and CD95 ligand and secretes immunosuppressive factors, the vulnerability of these cells to attack by immune T cells is open to question. This study was conducted to determine the vulnerability of allogeneic RPE to damage by specifically sensitized T cells, both in vivo within the subretinal space, and in vitro. METHOD: BALB/c lymphocytes presensitized to C57BL/6 antigens were injected into the subretinal space of eyes of C57BL/6 and gld/gld mice, and the eyes were examined clinically and histologically. RPE eyecups were produced from mouse eyes by removing the anterior segment and neuronal retina, leaving an intact monolayer of RPE. Sensitized BALB/c lymphocytes were placed in the RPE eyecup and incubated for 4 hours. The RPE layer of the eyecups was assessed by confocal microscopy for viability, after staining with propidium iodide and acridine orange. RESULT: Eyes that received T cells sensitized to C57BL/6 antigens displayed a circumscribed patch of persistent choroidal "whitening" clinically and a disrupted RPE cell layer histologically at the injection site at 5 days after injection. By 14 days, only RPE cells at the injection site were lost. RPE in eyecup preparations was relatively resistant in vitro to cytolysis by sensitized T cells, whether the eyecups were obtained from CD95-deficient or wild-type mice. CONCLUSIONS: RPE monolayers, both in vivo and in vitro, are relatively resistant to immune-mediated attack by specifically sensitized T cells. This relative lack of vulnerability is independent of the expression of CD95 ligand by target RPE cells and implies that immune barriers to acceptance of allogeneic RPE transplants may be less than if transplanted cells are from nonocular tissues.

Immunobiology and privilege of neuronal retina and pigment epithelium transplants.

Despite the existence of ocular immune privilege, immune rejection may be a barrier to successful retinal transplantation. We have examined in mice the extent to which the subretinal space (SRS) is an immune privileged site, and whether retinal pigment epithelium and neuronal retinal tissue have properties of immune privileged tissues. We report that (1) The SRS is an immune privileged site; (2) Neonatal RPE is an immune privileged tissue; (3) Neuronal retina is a partially immune privileged tissue; and (4) Microglia within neonatal neural retina grafts promote photoreceptor differentiation, become activated, and induce sensitization of the recipient and serve as targets of immune rejection.

IKKß regulates essential functions of the vascular endothelium through kinase-dependent and -independent pathways.

Vascular endothelium provides a selective barrier between the blood and tissues, participates in wound healing and angiogenesis, and regulates tissue recruitment of inflammatory cells. Nuclear factor (NF)-κB transcription factors are pivotal regulators of survival and inflammation, and have been suggested as potential therapeutic targets in cancer and inflammatory diseases. Here we show that mice lacking IKKß, the primary kinase mediating NF-κB activation, are smaller than littermates and born at less than the expected Mendelian frequency in association with hypotrophic and hypovascular placentae. IKKß-deleted endothelium manifests increased vascular permeability and reduced migration. Surprisingly, we find that these defects result from loss of kinase-independent effects of IKKß on activation of the serine-threonine kinase, Akt. Together, these data demonstrate essential roles for IKKß in regulating endothelial permeability and migration, as well as an unanticipated connection between IKKß and Akt signalling.

In vivo fluorescent imaging of the mouse retina using adaptive optics.

In vivo imaging of the mouse retina using visible and near infrared wavelengths does not achieve diffraction-limited resolution due to wavefront aberrations induced by the eye. Considering the pupil size and axial dimension of the eye, it is expected that unaberrated imaging of the retina would have a transverse resolution of 2 microm. Higher-order aberrations in retinal imaging of human can be compensated for by using adaptive optics. We demonstrate an adaptive optics system for in vivo imaging of fluorescent structures in the retina of a mouse, using a microelectromechanical system membrane mirror and a Shack-Hartmann wavefront sensor that detects fluorescent wavefront.

Imaging molecular expression on vascular endothelial cells by in vivo immunofluorescence microscopy.

Molecular expression on the vascular endothelium is critical in regulating the interaction of circulating cells with the blood vessel wall. Leukocytes as well as circulating cancer cells gain entry into tissue by interacting with adhesion molecules on the endothelial cells (EC). Molecular targets on the EC are increasingly being explored for tissue-specific delivery of therapeutic and imaging agents. Here we use in vivo immunofluorescence microscopy to visualize the endothelial molecular expression in the vasculature of live animals. High-resolution images are obtained by optical sectioning through the intact skin using in vivo confocal and multiphoton microscopy after in situ labeling of EC surface markers with fluorescent antibodies. Other vascular beds such as the bone marrow and ocular blood vessels can be imaged with little or no tissue manipulation. Live imaging is particularly useful for following the dynamic expression of inducible molecules such as E-selectin during an inflammatory response.