Maribel Vazquez

Aim: This study evaluated the impact of retinal extracellular matrix (ECM) and key biomaterial substrates on the motility of transplantable retinal cells with genomic manipulation, using the therapeutic molecule, Topoisomerase II beta (Top2b), as a model. Methods: Tests first applied in ovo electroporation to examine the effects of a pharmacological Top2b inhibitor (ICRF-193) on progenitor motility and development of embryonic retina. Complementary qRT-PCR tests measured changes in select cadherin molecules in response to treatment. In vitro transfection produced cultured retinal progenitor cell groups with Top2b overexpression and Top2b knockdown. Differences in the adhesion and motility of Top2b altered groups, compared to wildtype cells, were measured upon biomaterial substrates used in emerging transplantation matrixes. Results: Data illustrated significant differences in the number and spacing of retinal ganglion cells when retina was treated with ICRF-193, as well as downregulation of several key cadherin molecules. Cultured retinal progenitors with Top2b knockdown and Top2b overexpression exhibited different expression of chemotactic receptors, adhesion parameters, and modalities of migration upon substrates of laminin, poly-L-lysine, and collagen IV. Significant changes in cell morphology and surface area were also measured compared to wildtype cells. Conclusions: Corroborating in vivo and in vitro data support Top2b as a therapeutic target for retinal progenitor motility but indicate significant differences in the migration of Top2b altered cells upon substrates used in transplantation. These data highlight the therapeutic advantages of bioinspired materials developed to aid the motility of replacement cells with modified genetic expression to improve transplantation outcomes across the nervous system.

Experiments demonstrate that individual cells that wander stochastically can migrate persistently as a cluster. We show by simulating cells and their interactions that collective migration by omnidirectional cells is a generic phenomenon that can be expected to arise whenever (a) leading and trailing cells migrate randomly, and (b) leading cells are more closely packed than trailing neighbors. The first condition implies that noise is essential to cluster motion, while the second implies that an internal cohesion gradient can drive external motion of a cluster. Unlike other swarming phenomena, we find that this effect is driven by cohesion asymmetry near the leading cell, and motion of interior cells contribute minimally – and in fact interfere with – a cluster’s persistent migration.

Microscale systems have been underexplored in contemporary regenerative therapies developed to treat vision loss. The pairing of in vitro cell systems with optical fluorescent imaging provides unique opportunities to examine the infiltration of donor stem cells needed for successful transplantation therapies. A parallel eye device was developed to provide electric field (EF) stimulation to guide the migration of cells within 3D eye facsimiles synthesized from different ocular biomaterials. Cell infiltration within facsimiles was rapidly resolved using confocal microscopy to eliminate dependence on the cryostat sectioning commonly used for cell study. Moreover, EF stimulated galvanotaxis of donor cells within different depths of eye facsimiles. Optical imaging provided rapid resolution of z-stack images at physiologically appropriate depths below 500 microns. This study demonstrates that paired microscale–optical systems can be developed to elucidate understudied transplantation processes and improve future outcomes in patients.

Neurodegeneration of retinal tissue leads to progressive vision loss in millions of working-age adults each year. Metabolic alterations caused by modern diets that are high in fats and sugars contribute to the development of diabetic retinopathy. Chronic, diet-induced metabolic changes are linked to high glucose and harmful, pro-inflammatory compounds in the blood, called advanced glycation end products (AGEs), that can alter the integrity of neurovascular barriers. AGEs-induced changes to the permeability of the inner blood-retinal barrier can lead to progressive vision loss with disparate impacts in patients with low estrogen, such as via natural aging processes, chronic illness, or aggressive medical interventions. Application of supplemental estradiol has been shown to ameliorate degeneration across the cardiovascular, intestinal, and nervous systems. This commentary describes the potential benefits of estradiol-based therapies on the visual health of older women with progressive diabetic retinopathy.

Diabetic retinopathy is a complex, microvascular disease that impacts millions of working adults each year. High blood glucose levels from Diabetes Mellitus lead to the accumulation of advanced glycation end-products (AGEs), which promote inflammation and the breakdown of the inner blood retinal barrier (iBRB), resulting in vision loss. This study used an in vitro model of hyperglycemia to examine how endothelial cells (ECs) and Müller glia (MG) collectively regulate molecular transport. Changes in cell morphology, the expression of junctional proteins, and the reactive oxygen species (ROS) of ECs and MG were examined when exposed to a hyperglycemic medium containing AGEs. Trans-endothelial resistance (TEER) assays were used to measure the changes in cell barrier resistance in response to hyperglycemic and inflammatory conditions, with and without an anti-VEGF compound. Both of the cell types responded to hyperglycemic conditions with significant changes in the cell area and morphology, the ROS, and the expression of the junctional proteins ZO-1, CX-43, and CD40, as well as the receptor for AGEs. The resistivities of the individual and dual ECs and MG barriers decreased within the hyperglycemia model but were restored to that of basal, normoglycemic levels when treated with anti-VEGF. This study illustrated significant phenotypic responses to an in vitro model of hyperglycemia, as well as significant changes in the expression of the key proteins used for cell–cell communication. The results highlight important, synergistic relationships between the ECs and MG and how they contribute to changes in barrier function in combination with conventional treatments.

Rapid prototyping has produced accessible manufacturing methods that offer faster and more cost-effective ways to develop microscale systems for cellular testing. Commercial 3D printers are now increasingly adapted for soft lithography, where elastomers are used in tandem with 3D-printed substrates to produce in vitro cell assays. Newfound abilities to prototype cellular systems have begun to expand fundamental bioengineering research in the visual system to complement tissue engineering studies reliant upon complex microtechnology. This project used 3D printing to develop elastomeric devices that examined the responses of retinal cells to flow. Our experiments fabricated molds for elastomers using metal milling, resin stereolithography, and fused deposition modeling via plastic 3D printing. The systems were connected to flow pumps to simulate different flow conditions and examined phenotypic responses of endothelial and neural cells significant to neurovascular barriers of the retina. The results indicated that microdevices produced using 3D-printed methods demonstrated differences in cell survival and morphology in response to external flow that are significant to barrier tissue function. Modern 3D printing technology shows great potential for the rapid production and testing of retinal cell responses that will contribute to both our understanding of fundamental cell response and the development of new therapies. Future studies will incorporate varied flow stimuli as well as different extracellular matrices and expanded subsets of retinal cells.

Diabetic retinopathy affects more than 100 million people worldwide and is projected to increase by 50% within 20 years. Increased blood glucose leads to the formation of advanced glycation end products (AGEs), which cause cellular and molecular dysfunction across neurovascular systems. These molecules initiate the slow breakdown of the retinal vasculature and the inner blood retinal barrier (iBRB), resulting in ischemia and abnormal angiogenesis. This project examined the impact of AGEs in altering the morphology of healthy cells that comprise the iBRB, as well as the effects of AGEs on thrombi formation, in vitro. Our results illustrate that AGEs significantly alter cellular areas and increase the formation of blood clots via elevated levels of tissue factor. Likewise, AGEs upregulate the expression of cell receptors (RAGE) on both endothelial and glial cells, a hallmark biomarker of inflammation in diabetic cells. Examining the effects of AGEs stimulation on cellular functions that work to diminish iBRB integrity will greatly help to advance therapies that target vision loss in adults.

Retinal degeneration is an escalating public health challenge, as diseases such as age-related macular degeneration, diabetic retinopathy, and retinitis pigmentosa cause irreversible vision loss in millions of adults each year. Regenerative medicine has pioneered the development of stem cell replacement therapies, which treat degeneration by replacing damaged retinal neurons with transplanted stem-like cells (SCs). While the collective migration of SCs plays critical roles during retinal development, our understanding of collective SC behaviors within biomaterials transplanted into adult tissue remains understudied. This project examines the potential therapeutic impacts of collective SC migration during transplantation by correlating the expression of cadherin, cell-cell cohesion molecules that maintain intercellular communication during development, with receptor proteins of chemoattractant molecules prevalent in degenerated adult tissue. Experiments examine these well-conserved biomechanisms by using two different model organisms: Drosophila melanogaster, a seminal model for retinal development, and Mus, an important preclinical model for transplantation. Results indicate that SCs from both animal models significantly upregulate cadherin expression to achieve more directed collective migration towards species-specific chemoattractants and exhibit longer distance motility upon different extracellular matrix substrates.

Retinal pathologies have been heavily studied in response to radiation and microgravity, including spaceflight-associated neuro-ocular syndrome (SANS), which is commonly developed in space flight. SANS has been characterized in clinical studies of astronauts returning to Earth and includes a range of symptoms, such as globe flattening, optic-disc edema, retinal folds, and retinal ischemia. In cases of retinal insult, Müller glia (MG) cells respond via neuroprotective gliotic responses that may become destructive to produce glial scarring and vison loss over time. Retinal pathology is further impacted by the production of excessive reactive oxygen species (ROS) that stimulate retinal inflammation and furthers the gliosis of MG. Neuroprotectants derived from natural products (NPs) able to scavenge excess ROS and mitigate long-term, gliotic responses have garnered recent interest, especially among mature and aging adults. The natural antioxidants aloin and ginkgolide A flavonoids, derived from Aloe vera and Ginkgo biloba species, respectively, have been of particular interest due to their recent use in other nervous-system studies. The current study examined MG behaviors in response to different doses of aloin and ginkgolide A over time by measuring changes in morphology, survival, and ROS production within microscale assays. The study was further enhanced by using galactic cosmic rays (GCR) at the Brookhaven NASA Space Radiation Laboratory to simulate ionizing radiation in low- and high-radiation parameters. Changes in the survival and ROS production of radiation-treated MG were then measured in response to varying dosage of NPs. Our study used in vitro systems to evaluate the potential of NPs to reduce oxidative stress in the retina, highlighting the underexplored interplay between NP antioxidants and MG endogenous responses both in space and terrestrially.