L-type calcium channel blocker increases VEGF concentrations in retinal cells and human serum

Objective: Vascular endothelial growth factor (VEGF) plays a key role in diabetic retinopathy (DR). L-type calcium channel blockers (LTCCBs) have been widely used as antihypertensive medication (AHM), but their association with VEGF and DR is still unclear. Therefore, we explored the effect of LTCCBs compared to other AHMs on VEGF concentrations in retinal cells and human serum. Furthermore, we evaluated the association between the use of LTCCBs and the risk of severe diabetic eye disease (SDED). Research design and methods: Muller cells (MIO-M1) were cultured as per recommended protocol and treated with LTCCBs and other AHMs. VEGF secreted from cells were collected at 24 hours intervals. In an interventional study, 39 individuals received LTCCBs or other AHM for four weeks with a four-week wash-out placebo period between treatments. VEGF was measured during the medication and placebo periods. Finally, we evaluated the risk of SDED associated with LTCCB usage in 192 individuals from the FinnDiane Study in an oberservational setting. Results: In the cell cultures, medium VEGF concentration increased time-dependently after amlodipine (p<0.01) treatment, but not after losartan (p>0.01), or lisinopril (p>0.01). Amlodipine, but no other AHM, increased serum VEGF concentration (p<0.05) during the interventional clinical study. The usage of LTCCB was not associated with the risk of SDED in the observational study. Conclusions: LTCCB increases VEGF concentrations in retinal cells and human serum. However, the usage of LTCCBs does not appear to be associated with SDED in adults with type 1 diabetes.

Evaluation for Retinal Therapy for RPE65 Variation Assessed in hiPSC Retinal Pigment Epithelial Cells

Human induced pluripotent stem cells (hiPSCs) generated from patients and the derivative retinal cells enable the investigation of pathological and novel variants in relevant cell populations. Biallelic pathogenic variants in RPE65 cause early-onset severe retinal dystrophy (EOSRD) or Leber congenital amaurosis (LCA). Increasingly, regulatory-approved in vivo RPE65 retinal gene replacement therapy is available for patients with these clinical features, but only if they have biallelic pathological variants and sufficient viable retinal cells. In our cohort of patients, we identified siblings with early-onset severe retinal degeneration where genomic studies revealed compound heterozygous variants in RPE65, one a known pathogenic missense variant and the other a novel synonymous variant of uncertain significance. The synonymous variant was suspected to affect RNA splicing. Since RPE65 is very poorly expressed in all tissues except the retinal pigment epithelium (RPE), we generated hiPSC-derived RPE cells from the parental carrier of the synonymous variant. Sequencing of RNA obtained from hiPSC-RPE cells demonstrated heterozygous skipping of RPE65 exon 2 and the introduction of a premature stop codon in the mRNA. Minigene studies confirmed the splicing aberration. Results from this study led to reclassification of the synonymous variant to a pathogenic variant, providing the affected patients with access to RPE65 gene replacement therapy.

Detection of Mycoplasma Contamination in Transplanted Retinal Cells by Rapid and Sensitive Polymerase Chain Reaction Test

Contamination of cells/tissues by infectious pathogens (e.g., fungi, viruses, or bacteria, including mycoplasma) is a major problem in cell-based transplantation. In this study, we tested a polymerase chain reaction (PCR) method to provide rapid, simple, and sensitive detection of mycoplasma contamination in laboratory cultures for clinical use. This mycoplasma PCR system covers the Mycoplasma species (spp.) listed for testing in the 17th revision of the Japanese Pharmacopoeia, and we designed it for use in transplantable retinal cells. Here, we analyzed mycoplasma contamination in induced pluripotent stem cell (iPS cell)-derived transplantable retinal pigment epithelium (RPE) cells. In the spike tests to RPE cells with nine species of class Mollicutes bacteria, including seven Mycoplasma spp. and one of each Acholeplasma spp. and Ureaplasma spp., contamination at the concentration of 100 and 10 CFU/mL were detected with 100% probability in all cases, while 1 CFU/mL had a detection rate of 0–75%. DNA prepared from bacteria species other than class Mollicutes species was not detectable, indicating the specificity of this PCR. While iPS cells and iPS-RPE cells established in our laboratory were all negative by this PCR, some of the commercially available cell lines were positive. Cells for transplantation should never have infection, as once pathogens are implanted into the eyes, they can cause severe intraocular inflammation. Thus, it is imperative to monitor for infections in the transplants, although generally, mycoplasma infection is difficult to detect.

Starvation to Glucose Reprograms Development of Neurovascular Unit in Embryonic Retinal Cells

Perinatal exposure to starvation is a risk factor for development of severe retinopathy in adult patients with diabetes. However, the underlying mechanisms are not completely understood. In the present study, we shed light on molecular consequences of exposure to short-time glucose starvation on the transcriptome profile of mouse embryonic retinal cells. We found a profound downregulation of genes regulating development of retinal neurons, which was accompanied by reduced expression of genes encoding for glycolytic enzymes and glutamatergic signaling. At the same time, glial and vascular markers were upregulated, mimicking the diabetes-associated increase of angiogenesis—a hallmark of pathogenic features in diabetic retinopathy. Energy deprivation as a consequence of starvation to glucose seems to be compensated by upregulation of genes involved in fatty acid elongation. Results from the present study demonstrate that short-term glucose deprivation during early fetal life differentially alters expression of metabolism- and function-related genes and could have detrimental and lasting effects on gene expression in the retinal neurons, glial cells, and vascular elements and thus potentially disrupting gene regulatory networks essential for the formation of the retinal neurovascular unit. Abnormal developmental programming during retinogenesis may serve as a trigger of reactive gliosis, accelerated neurodegeneration, and increased vascularization, which may promote development of severe retinopathy in patients with diabetes later in life.

SARS-CoV-2 infects and replicates in photoreceptor and retinal ganglion cells of human retinal organoids

Several studies have pointed to retinal involvement in COVID 19 disease, yet many questions remain regarding the ability of SARS CoV 2 to infect and replicate in retinal cells and its effects on the retina. Here we have used human stem cell derived retinal organoids to study retinal infection by the SARS CoV 2 virus. Indeed, SARS CoV 2 can infect and replicate in retinal organoids, as it is shown to infect different retinal lineages, such as retinal ganglion cells and photoreceptors. SARS CoV 2 infection of retinal organoids also induces the expression of several inflammatory genes, such as interleukin 33, a gene associated with acute COVID 19 disease and retinal degeneration. Finally, we show that the use of antibodies to block the ACE2 receptor significantly reduces SARS CoV 2 infection of retinal organoids, indicating that SARS CoV 2 infects retinal cells in an ACE2 dependent manner. These results suggest a retinal involvement in COVID 19 and emphasize the need to monitor retinal pathologies as potential sequelae of long COVID.

Progressive protein aggregation in PRPF31 patient retinal pigment epithelium cells: the mechanism and its reversal through activation of autophagy

Mutations in pre-mRNA processing factor 31 (PRPF31), a core protein of the spliceosomal tri-snRNP complex, cause autosomal-dominant retinitis pigmentosa (adRP). It has remained an enigma why mutations in ubiquitously expressed tri-snRNP proteins result in retina-specific disorders, and so far, the underlying mechanism of splicing factors-related RP is poorly understood. Here, we used iPSC technology to generate retinal organoids and RPE models from three patients with severe and very severe PRPF31-adRP, normal individuals and a CRISPR/Cas9-corrected isogenic control. To fully assess the impacts of PRPF31 mutations, quantitative proteomics analyses of retinal organoids and RPE cells was carried out showing RNA splicing, autophagy and lysosome, unfolded protein response (UPR) and visual cycle-related pathways to be significantly affected. Strikingly, the patient-derived RPE and retinal cells were characterised by the presence of large amounts of cytoplasmic aggregates containing the mutant PRPF31 and misfolded, ubiquitin-conjugated proteins including key visual cycle proteins, which accumulated progressively with time. Mutant PRPF31 variant was not incorporated into splicing complexes, but reduction of PRPF31 wildtype levels led to tri-snRNP assembly defects in Cajal bodies of PRPF31 patient retinal cells with reduced U4/U6 snRNPs and accumulation of U5, smaller nuclear speckles and reduced formation of active spliceosomes giving rise to global splicing dysregulation. Moreover, the impaired waste disposal mechanisms further exacerbated aggregate formation, and targeting these by activating the autophagy pathway using Rapamycin resulted in reduction of cytoplasmic aggregates and improved cell survival. Our data demonstrate that it is the progressive aggregate accumulation that overburdens the waste disposal machinery rather than direct PRPF31-initiated mis-splicing, and thus relieving the RPE cells from insoluble cytoplasmic aggregates presents a novel therapeutic strategy that can be combined with gene therapy studies to fully restore RPE and retinal cell function in PRPF31-adRP patients.