Akt-independent effects of triciribine on ACE2 expression in human lung epithelial cells: Potential benefits in restricting SARS-CoV2 infection.

The severe acute respiratory syndrome coronavirus 2 that causes coronavirus disease 2019 (COVID-19) binds to the angiotensin-converting enzyme 2 (ACE2) to gain cellular entry. Akt inhibitor triciribine (TCBN) has demonstrated promising results in promoting recovery from advanced-stage acute lung injury in preclinical studies. In the current study, we tested the direct effect of TCBN on ACE2 expression in human bronchial (H441) and lung alveolar (A549) epithelial cells. Treatment with TCBN resulted in the downregulation of both messenger RNA and protein levels of ACE2 in A549 cells. Since HMGB1 plays a vital role in the inflammatory response in COVID-19, and because hyperglycemia has been linked to increased COVID-19 infections, we determined if HMGB1 and hyperglycemia have any effect on ACE2 expression in lung epithelial cells and whether TCBN has any effect on reversing HMGB1- and hyperglycemia-induced ACE2 expression. We observed increased ACE2 expression with both HMGB1 and hyperglycemia treatment in A549 as well as H441 cells, which were blunted by TCBN treatment. Our findings from this study, combined with our previous reports on the potential benefits of TCBN in the treatment of acute lung injury, generate reasonable optimism on the potential utility of TCBN in the therapeutic management of patients with COVID-19.

Regulation of blood-retinal barrier cell-junctions in diabetic retinopathy.

Loss of the blood-retinal barrier (BRB) integrity and subsequent damage to the neurovascular unit in the retina are the underlying reasons for diabetic retinopathy (DR). Damage to BRB eventually leads to severe visual impairment in the absence of prompt intervention. Diabetic macular edema and proliferative DR are the advanced stages of the disease where BRB integrity is altered. Primary mechanisms contributing to BRB dysfunction include loss of cell-cell barrier junctions, vascular endothelial growth factor, advanced glycation end products-induced damage, and oxidative stress. Although much is known about the involvement of adherens and tight-junction proteins in the regulation of vascular permeability in various diseases, there is a significant gap in our knowledge on the junctional proteins expressed in the BRB and how BRB function is modulated in the diabetic retina. In this review article, we present our current understanding of the molecular composition of BRB, the changes in the BRB junctional protein turnover in DR, and how BRB functional modulation affects vascular permeability and macular edema in the diabetic retina.

Pharmacological Modulation of ß-Catenin Preserves Endothelial Barrier Integrity and Mitigates Retinal Vascular Permeability and Inflammation.

Compromised blood-retinal barrier (BRB) integrity is a significant factor in ocular diseases like uveitis and retinopathies, leading to pathological vascular permeability and retinal edema. Adherens and tight junction (AJ and TJ) dysregulation due to retinal inflammation plays a pivotal role in BRB disruption. We investigated the potential of ICG001, which inhibits ß-catenin-mediated transcription, in stabilizing cell junctions and preventing BRB leakage. In vitro studies using human retinal endothelial cells (HRECs) showed that ICG001 treatment improved ß-Catenin distribution within AJs post lipopolysaccharide (LPS) treatment and enhanced monolayer barrier resistance. The in vivo experiments involved a mouse model of LPS-induced ocular inflammation. LPS treatment resulted in increased albumin leakage from retinal vessels, elevated vascular endothelial growth factor (VEGF) and Plasmalemmal Vesicle-Associated Protein (PLVAP) expression, as well as microglia and macroglia activation. ICG001 treatment (i.p.) effectively mitigated albumin leakage, reduced VEGF and PLVAP expression, and reduced the number of activated microglia/macrophages. Furthermore, ICG001 treatment suppressed the surge in inflammatory cytokine synthesis induced by LPS. These findings highlight the potential of interventions targeting ß-Catenin to enhance cell junction stability and improve compromised barrier integrity in various ocular inflammatory diseases, offering hope for better management and treatment options.

A multi-arm, parallel, preclinical study investigating the potential benefits of acetazolamide, candesartan, and triciribine in combination with fluconazole for the treatment of cryptococcal meningoencephalitis.

Cryptococcus neoformans, an opportunistic fungal pathogen, primarily infects immunodeficient patients frequently causing cryptococcal meningoencephalitis (CM). Increased intracranial pressure (ICP) is a serious complication responsible for increased morbidity and mortality in CM patients. Non-invasive pharmacological agents that mitigate ICP could be beneficial in treating CM patients. The objective of the study was to investigate the efficacy of acetazolamide (AZA), candesartan (CAN), and triciribine (TCBN), in combination with the antifungal fluconazole, on C. neoformans-induced endothelial, brain, and lung injury in an experimental mouse model of CM. Our study shows that C. neoformans increases the expression of brain endothelial cell (BEC) junction proteins Claudin-5 (Cldn5) and VE-Cadherin to induce pathological cell-barrier remodeling and gap formation associated with increased Akt and p38 MAPK activation. All three agents inhibited C. neoformans-induced endothelial gap formation, only CAN and TCBN significantly reduced C. neoformans-induced Cldn5 expression, and only TCBN was effective in inhibiting Akt and p38MAPK. Interestingly, although C. neoformans did not cause brain or lung edema in mice, it induced lung and brain injuries, which were significantly reversed by AZA, CAN, or TCBN. Our study provides novel insights into the direct effects of C. neoformans on BECs in vitro, and the potential benefits of using AZA, CAN, or TCBN in the management of CM patients.

Claudin-17 Deficiency in Mice Results in Kidney Injury Due to Electrolyte Imbalance and Oxidative Stress.

The multi-gene claudin (CLDN) family of tight junction proteins have isoform-specific roles in blood-tissue barrier regulation. CLDN17, a putative anion pore-forming CLDN based on its structural characterization, is assumed to regulate anion balance across the blood-tissue barriers. However, our knowledge about CLDN17 in physiology and pathology is limited. The current study investigated how Cldn17 deficiency in mice affects blood electrolytes and kidney structure. Cldn17-/- mice revealed no breeding abnormalities, but the newborn pups exhibited delayed growth. Adult Cldn17-/- mice displayed electrolyte imbalance, oxidative stress, and injury to the kidneys. Ingenuity pathway analysis followed by RNA-sequencing revealed hyperactivation of signaling pathways and downregulation of SOD1 expression in kidneys associated with inflammation and reactive oxygen species generation, demonstrating the importance of Cldn17 in the maintenance of electrolytes and reactive oxygen species across the blood-tissue barrier.

Distinct Mechanisms of Human Retinal Endothelial Barrier Modulation In Vitro by Mediators of Diabetes and Uveitis.

Ocular diseases such as diabetic retinopathy (DR) and uveitis are associated with injury to the blood-retinal barrier (BRB). Whereas high glucose (HG) and advanced glycation end products (AGE) contribute to DR, bacterial infections causing uveitis are triggered by endotoxins such as lipopolysaccharide (LPS). It is unclear how HG, AGE, and LPS affect human retinal endothelial cell (HREC) junctions. Moreover, tumor necrosis factor-α (TNFα) is elevated in both DR and ocular infections. In the current study, we determined the direct effects of HG, AGE, TNFα, and LPS on the expression and intracellular distribution of claudin-5, VE-cadherin, and ß-catenin in HRECs and how these mediators affect Akt and P38 MAP kinase that have been implicated in ocular pathologies. In our results, whereas HG, AGE, and TNFα activated both Akt and P38 MAPK, LPS treatment suppressed Akt but increased P38 MAPK phosphorylation. Furthermore, while treatment with AGE and HG increased cell-junction protein expression in HRECs, LPS elicited a paradoxical effect. By contrast, when HG treatment increased HREC-barrier resistance, AGE and LPS stimulation compromised it, and TNFα had no effect. Together, our results demonstrated the differential effects of the mediators of diabetes and infection on HREC-barrier modulation leading to BRB injury.

Pharmacological Inhibition of Spermine Oxidase Reduces Neurodegeneration and Improves Retinal Function in Diabetic Mice.

Diabetic retinopathy (DR) is a significant cause of blindness in working-age adults worldwide. Lack of effective strategies to prevent or reduce vision loss is a major problem. Since the degeneration of retinal neurons is an early event in the diabetic retina, studies to characterize the molecular mechanisms of diabetes-induced retinal neuronal damage and dysfunction are of high significance. We have demonstrated that spermine oxidase (SMOX), a mediator of polyamine oxidation is critically involved in causing neurovascular damage in the retina. The involvement of SMOX in diabetes-induced retinal neuronal damage is completely unknown. Utilizing the streptozotocin-induced mouse model of diabetes, the impact of the SMOX inhibitor, MDL 72527, on neuronal damage and dysfunction in the diabetic retina was investigated. Retinal function was assessed by electroretinography (ERG) and retinal architecture was evaluated using spectral domain-optical coherence tomography. Retinal cryosections were prepared for immunolabeling of inner retinal neurons and retinal lysates were used for Western blotting. We observed a marked decrease in retinal function in diabetic mice compared to the non-diabetic controls. Treatment with MDL 72527 significantly improved the ERG responses in diabetic retinas. Diabetes-induced retinal thinning was also inhibited by the MDL 72527 treatment. Our analysis further showed that diabetes-induced retinal ganglion cell damage and neurodegeneration were markedly attenuated by MDL 72527 treatment. These results strongly implicate SMOX in diabetes-induced retinal neurodegeneration and visual dysfunction.