Mechanical stretch upregulates connexin43 in human trabecular meshwork cells.

BACKGROUND: Primary open angle glaucoma (POAG) patients have hallmark increases in intraocular pressure (IOP) and noted dysfunction of the trabecular meshwork (TM). Connexin43 (Cx43) is a gap junction widely expressed on the TM that is important for intercellular communication. The human gene is known as gap junction alpha-1 (GJA1). Since the role of Cx43 in the TM is not fully understood, we set out to determine the effect of excess mechanical stretch on cultured human trabecular meshwork cells (hTMCs) and to specifically investigate the effect of stretch on Cx43 expression and function. METHODS: Primary hTMCs were cultured and subjected to 48 hours of 15% cyclic mechanical stretch at a frequency of 1 Hz. Levels of apoptosis and necrosis secondary to stretch were investigated using colorimetric assays. The effect of stretch on gap junction Cx43 and GJA1 was investigated by RT-PCR, immunoblotting and immunofluorescence. The migration of Lucifer Yellow dye was used to assess intercellular communication. RESULTS: Stretch significantly increased the rates of apoptosis and necrosis in hTMCs. The increased rate of injury in stretched hTMCs was further associated with significant upregulation of GJA1 mRNA and Cx43 protein. Upregulation of Cx43 protein was concomitant to increased intercellular communication. CONCLUSIONS: We have shown stretch to increase GJA1 gene and Cx43 protein expression, as well as intercellular communication. We have further shown stretch to be injurious to hTMCs. Upregulation of Cx43 in the hTM subsequent to stretch is a novel finding, which may be useful in elucidating the mechanism of TM injury in POAG patients.

Journey into the unknown: considering the international medical graduate perspective on the road to Canadian residency during the COVID-19 pandemic.

The COVID-19 pandemic has had a tremendous effect on education programs worldwide, including medical education. Particularly, International Medical Graduates (IMGs) planning to pursue residency training in Canada have been profoundly impacted. Cancellation of away electives, as well as changes to the format, timeline, and requirements of mandatory medical licensing exams has left IMG residency applicants in uncharted territory. Given that IMGs comprise up to 25% of the Canadian healthcare force, and often are based in underserviced areas, the licensure and eligibility of IMGs to continue to enter the Canadian healthcare force is of the utmost importance in the midst of the COVID-19 pandemic. As the pandemic evolves, it is imperative that key decision makers and stakeholders continue to consider the downstream effect for IMGs and their eligibility to practice in Canada.

La pandémie de la COVID-19 a fortement affecté les programmes d'éducation dans le monde entier, y compris l'éducation médicale. En particulier, les diplômés internationaux en médecine (DIM) qui prévoyaient s'inscrire à un programme de résidence au Canada ont été profondément touchés. L'annulation des stages à l'étranger, ainsi que les changements apportés au format, au calendrier et aux exigences des examens requis pour l'obtention du permis d'exercice de la médecine ont laissé en territoire inconnu les candidats internationaux aux programmes de résidence. Étant donné que les DIM représentent jusqu'à 25 % des professionnels de la santé au Canada et qu'ils exercent souvent dans les régions mal desservies, l'octroi de permis d'exercice et le recrutement de DIM dans le réseau de la santé canadien revêtent une importance capitale en contexte de pandémie de la COVID-19. À mesure que la pandémie évolue, il est impératif que les principaux décideurs et intervenants continuent de tenir compte des effets néfastes qu'elle peut avoir pour les DIM et leur admissibilité à l'exercice de la profession au Canada.

Administration of Nitric Oxide Through a Novel Copper-Chitosan Delivery System in Human Corneal and Limbal Epithelial Cell Injury.

Purpose: Nitric oxide (NO) has gained attention for its role in facilitating wound healing by promoting cell migration, while being cytoprotective in a variety of cell types. We determined the efficacy of NO, administered using a novel application of copper-chitosan treatments (Cu-Ch), in facilitating corneal epithelial wound healing using an in vitro model of corneal epithelial and limbal epithelial cell injury. Methods: Human corneal epithelial (HCE) and human limbal epithelial (HLE) cells were monitored under no-scratch (CON), untreated scratch (CS), scratch + plain chitosan composite (0%), scratch + 1% copper solution Cu-Ch (1%), and scratch + 2% copper solution Cu-Ch (2%) conditions. Cell migration, cytotoxicity, apoptosis, and total nitrate/nitrite concentrations were measured at 24, 48, and 72 hours after injury and treatment. iNOS expression in HLE cells also was determined using Western blot. Results: Wound closure significantly increased in HCE cells treated with Cu-Ch (1% and 2%) after 72 hours, while HLE cells showed a significant decrease in closure with Cu-Ch (1% and 2%) treatment compared to CS. Cytotoxic fragments decreased significantly with 1% and 2% Cu-Ch treatments in HCE cells. Nitrate/nitrite levels in HLE cells showed a significant increase with 2% Cu-Ch treatment compared to CS. This increase is complemented with an upregulation of iNOS. Conclusions: Overall, HCE wound healing was accelerated with administration of Cu-Ch treatment. Differences between HCE and HLE responses may be due to intrinsic differences in NO metabolism, as evidenced by differences in NO production, potentially caused by differences in iNOS expression with treatment.

TGF-ß induces phosphorylation of phosphatase and tensin homolog: implications for fibrosis of the trabecular meshwork tissue in glaucoma.

Fundamental cell signaling mechanisms that regulate dynamic remodeling of the extracellular matrix (ECM) in mechanically loaded tissues are not yet clearly understood. Trabecular meshwork (TM) tissue in the eye is under constant mechanical stress and continuous remodeling of ECM is crucial to maintain normal aqueous humor drainage and intraocular pressure (IOP). However, excessive ECM remodeling can cause fibrosis of the TM as in primary open-angle glaucoma (POAG) patients, and is characterized by increased resistance to aqueous humor drainage, elevated IOP, optic nerve degeneration and blindness. Increased levels of active transforming growth factor-ß2 (TGF-ß2) in the aqueous humor is the main cause of fibrosis of TM in POAG patients. Herein, we report a novel finding that, in TM cells, TGF-ß-induced increase in collagen expression is associated with phosphorylation of phosphatase and tensin homolog (PTEN) at residues Ser380/Thr382/383. Exogenous overexpression of a mutated form of PTEN with enhanced phosphatase activity prevented the TGF-ß-induced collagen expression by TM cells. We propose that rapid alteration of PTEN activity through changes in its phosphorylation status could uniquely regulate the continuous remodeling of ECM in the normal TM. Modulating PTEN activity may have high therapeutic potential to alleviating the fibrosis of TM in POAG patients.