Targeted pharmacotherapy against neurodegeneration and neuroinflammation in early diabetic retinopathy.

Diabetic retinopathy (DR), the most frequent complication of diabetes, is one of the leading causes of irreversible blindness in working-age adults and has traditionally been regarded as a microvascular disease. However, increasing evidence has revealed that synaptic neurodegeneration of retinal ganglion cells (RGCs) and activation of glial cells may represent some of the earliest events in the pathogenesis of DR. Upon diabetes-induced metabolic stress, abnormal glycogen synthase kinase-3ß (GSK-3ß) activation drives tau hyperphosphorylation and ß-catenin downregulation, leading to mitochondrial impairment and synaptic neurodegeneration prior to RGC apoptosis. Moreover, glial cell activation triggers enhanced inflammation and oxidative stress, which may accelerate the deterioration of diabetic RGCs neurodegeneration. These findings have opened up opportunities for therapies, such as inhibition of GSK-3ß, glial cell activation, glutamate excitotoxicity and the use of neuroprotective drugs targeting early neurodegenerative processes in the retina and halting the progression of DR before the manifestation of microvascular abnormalities. Such interventions could potentially remedy early neurodegeneration and help prevent vision loss in people suffering from DR.

Experimental models of corneal endothelial cell therapy and translational challenges to clinical practice.

The human corneal endothelium (CE) is a post-mitotic monolayer of endothelial cells, thought to be incapable of in vivo regeneration. Dysfunction of the CE is a commonly cited indication for corneal transplantation, with corneal blindness being the fifth most common cause of blindness globally. In 2012 alone 184,576 corneal transplants were performed in 116 countries (Gain et al., 2016). Presently, outcomes following human corneal transplantation have been reported to have over 97% success rate in restoring the recipient's vision (Patel et al., 2019). However, the continuing demand for cadaveric human corneas has driven research into alternative sources of CE and with the advent of protocols to produce cultured hCECs there is now the potential for cell therapy to regenerate the damaged CE. This review aims to examine the merits and limitations of different types of human and animal models used so far to test the concept of CE cell therapy.

Feasibility Study of Human Corneal Endothelial Cell Transplantation Using an In Vitro Human Corneal Model.

PURPOSE: To test the feasibility of a cell therapy approach to treat corneal endothelial (CE) disorders using an in vitro model of human corneal decompensation. METHODS: A CE decompensation model was established by removal of the Descemet membrane/endothelium complex from cadaveric human corneas in an air interface organ culture system (group 2) and compared with normal corneas (group 1). The posterior stroma of decompensated corneas was seeded with immortalized human corneal endothelial cells (HCEC-12) in group 3 and passage 0 primary human CE cells in group 4 corneas. Functional effects on stromal thickness were determined with histological analysis 3 to 10 days after cell therapy treatment. RESULTS: Removal of the Descemet membrane/endothelium complex in group 2 corneas resulted in a stromal thickness of 903 ± 86 µm at 12 hours compared with 557 ± 72 µm in group 1 corneas. Stromal thickness reduced from 1218 ± 153 µm to 458 ± 90 µm (63% ± 6%, P = 0.001) after cell transplantation in group 3 and from 1100 ± 86 µm to 489 ± 94 µm (55% ± 7%, P = 0.00004) in group 4. Posttransplantation histology demonstrated formation of a monolayer of corneal endothelium attached to the posterior stromal surface. CONCLUSIONS: Direct transplantation of cultured human CE cells and immortalized HCEC-12 to bare posterior corneal stroma resulted in formation of an endothelial monolayer and restoration of stromal hydration to physiological thickness, demonstrating the feasibility of cell therapy in treatment of CE decompensation in a human in vitro model.

Identification of Prdm genes in human corneal endothelium.

Corneal endothelial cells (CECs) are essential for maintaining corneal stromal hydration and ensuring its transparency, which is necessary for normal vision. Dysfunction of CECs leads to stromal decompensation, loss of transparency and corneal blindness. Corneal endothelium has low proliferative potential compared to surface epithelial cells leading to poor regeneration of CEC following injury. Additionally, the tissue exhibits age related decline in endothelial cell density with re-organisation of the cell layer, but no regeneration. The mechanisms which control proliferation and differentiation of neural crest derived CEC progenitors are yet to be clearly elucidated. Prdm (Positive regulatory domain) family of transcriptional regulators and chromatin modifiers are important for driving differentiation of a variety of cellular types. Many Prdm proteins are expressed in specific precursor cell populations and are necessary for their progression to a fully differentiated phenotype. In the present work, we sought to identify members of the Prdm gene family which are specifically expressed in human (h) CECs with a view to begin addressing their potential roles in CEC biology, focussing especially on Prdm 4 and 5 genes. By performing semi-quantitative reverse transcription coupled to PCR amplification we found that in addition to Prdm4 and Prdm5, Prdm2 and Prdm10 genes are expressed in hCECs. We further found that cultured primary hCECs or immortalised HCEC-12 cells express all of the Prdm genes found in CECs, but also express additional Prdm transcripts. This difference is most pronounced between Prdm gene expression patterns of CECs isolated from healthy human corneas and immortalised HCEC-12 cells. We further investigated Prdm 4 and Prdm 5 protein expression in cultured primary hCECs and HCEC-12 cells as well as in a human cadaveric whole cornea. Both Prdm 4 and Prdm 5 are expressed in human corneal endothelium, primary hCECs and in HCECs-12 cells, characterised by expression of the Na+/K+-ATPase. We observed that both proteins exhibit cytosolic (intracellular, but non-nuclear and distinct from extracellular fluid) as well as nuclear localisation within the endothelial layer, with Prdm 5 being more concentrated in the nuclei of the endothelial cells than Prdm 4. Thus, our work identifies novel Prdm genes specifically expressed in corneal endothelial cells which may be important in the control of CEC differentiation and proliferation.