Mullins Lab Publications
Retinal Pigment Epithelium-Secreted VEGF-A Induces Alpha-2-Macroglobulin Expression in Endothelial Cells
Cells. 2022 Sep 24;11(19):2975. doi: 10.3390/cells11192975.
Alpha-2-macroglobulin (A2M) is a protease inhibitor that regulates extracellular matrix (ECM) stability and turnover. Here, we show that A2M is expressed by endothelial cells (ECs) from human eye choroid. We demonstrate that retinal pigment epithelium (RPE)-conditioned medium induces A2M expression specifically in ECs. Experiments using chemical inhibitors, blocking antibodies, and recombinant proteins revealed a key role of VEGF-A in RPE-mediated A2M induction in ECs. Furthermore, incubation of ECs with RPE-conditioned medium reduces matrix metalloproteinase-2 gelatinase activity of culture supernatants, which is partially restored after A2M knockdown in ECs. We propose that dysfunctional RPE or choroidal blood vessels, as observed in retinal diseases such as age-related macular degeneration, may disrupt the crosstalk mechanism we describe here leading to alterations in the homeostasis of choroidal ECM, Bruch's membrane and visual function.
Characterization of a novel Pde6b-deficient rat model of retinal degeneration and treatment with adeno-associated virus (AAV) gene therapy
Gene Ther. 2022 Sep 29. doi: 10.1038/s41434-022-00365-y. Online ahead of print.
In humans, mutations in the beta subunit of cGMP-phosphodiesterase type 6 (PDE6B) cause autosomal recessive retinitis pigmentosa (RP), which typically has an aggressive clinical course of early-onset severe vision loss due to rapid photoreceptor degeneration. In this study, we describe the generation of a novel Pde6b-deficient rat model using CRISPR-Cas9 genome editing. We characterize the model at multiple time points using clinical imaging modalities as well as histology with immunohistochemistry to show rapid photoreceptor degeneration compared to wild-type and heterozygous animals. We describe the manufacture of two different adeno-associated viral (AAV) vectors (AAV2/1, AAV2/5) under current Good Manufacturing Practices (cGMP) and demonstrate their ability to drive human PDE6B expression in vivo. We further demonstrate the ability of AAV-mediated subretinal gene therapy to delay photoreceptor loss in Pde6b-deficient rats compared to untreated controls. However, severe progressive photoreceptor loss was noted even in treated eyes, likely due to the aggressive nature of the disease. These data provide useful preclinical data to guide the development of potential human gene therapy for PDE6B-associated RP. In addition, the rapid photoreceptor degeneration of the Pde6b-deficient rat with intact inner retina may provide a useful model for the study of cell replacement strategies.
Vascular Findings in the Choriocapillaris in a Case of Radiation Retinopathy Secondary to Choroidal Melanoma
Case Rep Ophthalmol. 2022 Aug 15;13(2):589-598. doi: 10.1159/000525568. eCollection 2022 May-Aug.
The effects of radiation retinopathy on the retinal vasculature have been well established; however, the literature describing the pathologic changes in the choriocapillaris is relatively lacking. In this report, we describe the histologic findings of a donor eye with a choroidal melanoma with special attention to the choriocapillaris. Clinical and histological findings, including immunohistochemistry and transmission electron microscopy, are described for the retina and choroid of a donor eye affected by radiation retinopathy secondary to treatment of choroidal melanoma. Cells within the tumor exhibited an epithelioid structure and balloon melanosomes. Notable infiltration of macrophages with elongated morphology was also observed. Atrophy of photoreceptors, retinal pigmented epithelium, and choriocapillaris was observed on the inferior edge of the lesion and extending past the tumor. The choriocapillaris endothelium showed more severe dropout at the periphery of the lesion where loss of fenestration, thickened cytosol, and degenerated pericytes were observed. Morphologic analysis revealed choriocapillaris loss with pronounced degeneration of choroidal pericytes. Understanding the differences in sensitivity to radiation injury between different cell types and different patients will provide better insight into radiation retinopathy.
Exp Eye Res. 2022 Sep 12:109248. doi: 10.1016/j.exer.2022.109248. Online ahead of print.
Genomic studies in age-related macular degeneration (AMD) have identified genetic variants that account for the majority of AMD risk. An important next step is to understand the functional consequences and downstream effects of the identified AMD-associated genetic variants. Instrumental for this next step are 'omics' technologies, which enable high-throughput characterization and quantification of biological molecules, and subsequent integration of genomics with these omics datasets, a field referred to as systems genomics. Single cell sequencing studies of the retina and choroid demonstrated that the majority of candidate AMD genes identified through genomic studies are expressed in non-neuronal cells, such as the retinal pigment epithelium (RPE), glia, myeloid and choroidal cells, highlighting that many different retinal and choroidal cell types contribute to the pathogenesis of AMD. Expression quantitative trait locus (eQTL) studies in retinal tissue have identified putative causal genes by demonstrating a genetic overlap between gene regulation and AMD risk. Linking genetic data to complement measurements in the systemic circulation has aided in understanding the effect of AMD-associated genetic variants in the complement system, and supports that protein QTL (pQTL) studies in plasma or serum samples may aid in understanding the effect of genetic variants and pinpointing causal genes in AMD. A recent epigenomic study fine-mapped AMD causal variants by determing regulatory regions in RPE cells differentiated from induced pluripotent stem cells (iPSC-RPE). Another approach that is being employed to pinpoint causal AMD genes is to produce synthetic DNA assemblons representing risk and protective haplotypes, which are then delivered to cellular or animal model systems. Pinpointing causal genes and understanding disease mechanisms is crucial for the next step towards clinical translation. Clinical trials targeting proteins encoded by the AMD-associated genomic loci C3, CFB, CFI, CFH, and ARMS2/HTRA1 are currently ongoing, and a phase III clinical trial for C3 inhibition recently showed a modest reduction of lesion growth in geographic atrophy. The EYERISK consortium recently developed a genetic test for AMD that allows genotyping of common and rare variants in AMD-associated genes. Polygenic risk scores (PRS) were applied to quantify AMD genetic risk, and may aid in predicting AMD progression. In conclusion, genomic studies represent a turning point in our exploration of AMD. The results of those studies now serve as a driving force for several clinical trials. Expanding to omics and systems genomics will further decipher function and causality from the associations that have been reported, and will enable the development of therapies that will lessen the burden of AMD.
The Essential Role of the Choriocapillaris in Vision: Novel Insights from Imaging and Molecular Biology
Annu Rev Vis Sci. 2022 Sep 15;8:33-52. doi: 10.1146/annurev-vision-100820-085958.
The choriocapillaris, a dense capillary network located at the posterior pole of the eye, is essential for supporting normal vision, supplying nutrients, and removing waste products from photoreceptor cells and the retinal pigment epithelium. The anatomical location, heterogeneity, and homeostatic interactions with surrounding cell types make the choroid complex to study both in vivo and in vitro. Recent advances in single-cell RNA sequencing, in vivo imaging, and in vitro cell modeling are vastly improving our knowledge of the choroid and its role in normal health and in age-related macular degeneration (AMD). Histologically, loss of endothelial cells (ECs) of the choriocapillaris occurs early in AMD concomitant with elevated formation of the membrane attack complex of complement. Advanced imaging has allowed us to visualize early choroidal blood flow changes in AMD in living patients, supporting histological findings of loss of choroidal ECs. Single-cell RNA sequencing is being used to characterize choroidal cell types transcriptionally and discover their altered patterns of gene expression in aging and disease. Advances in induced pluripotent stem cell protocols and 3D cultures will allow us to closely mimic the in vivo microenvironment of the choroid in vitro to better understand the mechanism leading to choriocapillaris loss in AMD.