Stan Gee Louie

Severe full-thickness corneal lacerations disrupt the tight cellular and extracellular matrix (ECM) organization required for corneal transparency. Following injury, an influx of transforming growth factor beta (TGFβ) into the corneal stroma signals the formation of haze-inducing myofibroblasts, resulting in excessive stromal remodeling and corneal haze. We hypothesized that MasR activation using NorLeu³Angiotensin (1-7) (NLE) engages the pro-resolving arm of the renin–angiotensin system (RAS) to minimize fibrotic corneal repair. In this study, 6 mm stellate-shaped, full-thickness corneal lacerations were induced in New Zealand Black (NZB) rabbits and treated with topical vehicle, or 0.1%, 0.3%, or 0.45% NLE. Corneal healing was evaluated using noninvasive corneal imaging, histology, and the gene expression of RAS- and fibrosis-related targets (MasR, AT1R, TGFβR1). Corneal imaging revealed significantly decreased corneal haze (p < 0.05) and increased keratocyte density with 0.1% NLE treatment (p < 0.05). Immunofluorescence showed significantly reduced α-smooth muscle actin (αSMA), indicating decreased myofibroblast formation (p < 0.05). Additionally, 0.1% NLE reduced stromal TGFβR1, suggesting that NLE mediates its activity by disrupting the TGFβ/TGFβR axis. MasR and AT1R gene expression were downregulated, which contributes to a reduction in fibrosis. Collectively, these findings suggest that the NLE activation of MasR modulates RAS and TGFβ/TGFβR signaling to reduce myofibroblast activity and fibrosis following severe corneal trauma.

Chronic neuroinflammation plays a key role in the progression of Alzheimer’s disease (AD), and the cytosolic calcium-dependent phospholipase A₂ (cPLA₂) enzyme is a critical mediator of inflammatory lipid signaling pathways. Here we investigate the therapeutic potential of novel cPLA₂ inhibitors in modulating neuroinflammation in AD. By leveraging the giga-scale V-SYNTHES2 virtual screening in on-demand chemical space and conducting two rounds of optimization for potency and selectivity, we have identified BRI-50460, achieving an IC₅₀ of 0.88 nM in cellular assays that measure cPLA₂-mediated arachidonic acid release. In vivo studies revealed favorable brain-to-plasma ratios, highlighting the ability of BRI-50460 to penetrate the central nervous system, modulating neuroinflammatory pathways, and restoring lipid homeostasis. In astrocytes and neurons derived from human induced pluripotent stem cells, BRI-50460 mitigates the effects of amyloid beta 42 oligomers on cPLA₂ activation, tau hyperphosphorylation, and synaptic loss. Our results support that small molecule inhibitors of cPLA₂ can modulate the downstream inflammatory signaling, offering a promising therapeutic strategy for neurodegenerative diseases.

Many studies have examined the acute effects of chemotherapies and radiation on the ovary; however, few address the impact of long-term chemotherapeutic agents on future reproduction for cancer survivors. The purpose of our study was to determine the effect of long-term tamoxifen (TAM) treatment on follicle development, ovarian reserve, and embryogenesis in a murine model. Adult female mice were treated with TAM (250 mg/kg fed ad libitum in mouse chow) or control chow for three weeks, followed by 0-, 1-, or 3-weeks of TAM washout. Mice were then superovulated, mated, and their oviducts flushed to determine ovulation, fertilization, and in vitro embryo development rates. At specific timepoints, one ovary from each animal was harvested, sectioned, and stained with H&E, while the other ovary was evaluated for gene expression of targeted genes. Ovarian size was calculated based on average surface area over six slices. Follicles (primordial, primary, secondary, antral / pre-ovulatory), and corpora lutea (CL) were counted and averaged for each slice. All data were analyzed using mixed-effects analysis of variance (ANOVA), with treatment group as the fixed effect and ovary treated as a random effect nested within treatment. Ovaries of TAM treated mice were 36% smaller on average than control ovaries (P = 0.01). Primordial follicle counts were 35% lower than control ovaries (P = 0.007) and there were 3.4x more atretic follicles in the ovaries of TAM treated mice (P < 0.001). There were no significant differences in the number of primary, secondary, antral or pre-ovulatory follicles. The number of oocytes and embryos flushed from control mice (total ovulated) was 1.65x greater than TAM treated mice. However, fertilization rates and in vitro embryo blastulation rates were not significantly different between controls and TAM groups. In summary, for this murine model, long-term TAM negatively impacted ovarian reserve (primordial follicle number) without overtly affecting the in vitro embryo development to the blastocyst.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a respiratory viral infection that disrupts renin angiotensin system (RAS) peptide metabolism by downregulating angiotensin converting enzyme-II (ACE2), leading to accumulation of pro-inflammatory angiotensin II (AngII). We posit that angiotensin receptor blockers (ARBs) like telmisartan (TEM) can activate the pro-resolving arm of RAS (MasR/ACE2/Ang1–7), reducing lung inflammation in COVID-19 patients. In this randomized, double-blinded, placebo-controlled pilot clinical trial, outpatient SARS-CoV-2-infected study participants received either 40 mg TEM or placebo once daily. Plasma inflammatory biomarker levels revealed a reduction in global systemic inflammation with TEM treatment, corresponding with an increase in MasR. We corroborated these clinical findings with in vitro analysis. A549-ACE2 lung epithelial cells treated with TEM showed increased ACE2 and MasR, as well as decreased angiotensin II receptor type I (AT1R) and angiotensin II receptor type II (AT2R) expression levels. Additionally, AngII peptide levels decreased, while Ang(1–9) and Ang(1–7) increased. TEM treatment at physiologically achievable concentration reduced SARS-CoV-2 viral load. Taken collectively, these results show TEM mediated activation of the Ang(1–7)/MasR/ACE2 pro-resolving arm of RAS, which activates anti-inflammatory events that reduce global inflammation. These findings support the use of ARBs like TEM in mitigating COVID-19 driven alterations in RAS.

Retinal degenerative diseases, including age-related macular degeneration and retinitis pigmentosa, significantly contribute to adult blindness. The Royal College of Surgeons (RCS) rat is a well-established disease model for studying these dystrophies; however, molecular investigations remain limited. We conducted a comprehensive analysis of retinal degeneration in RCS rats, including an immunodeficient RCS (iRCS) sub-strain, using ocular coherence tomography, electroretinography, histology, and molecular dissection using transcriptomics and immunofluorescence. No significant differences in retinal degeneration progression were observed between the iRCS and immunocompetent RCS rats, suggesting a minimal role of adaptive immune responses in disease. Transcriptomic alterations were primarily in inflammatory signaling pathways, characterized by the strong upregulation of Tnfa, an inflammatory signaling molecule, and Nox1, a contributor to reactive oxygen species (ROS) generation. Additionally, a notable decrease in Alox15 expression was observed, pointing to a possible reduction in anti-inflammatory and pro-resolving lipid mediators. These findings were corroborated by immunostaining, which demonstrated increased photoreceptor lipid peroxidation (4HNE) and photoreceptor citrullination (CitH3) during retinal degeneration. Our work enhances the understanding of molecular changes associated with retinal degeneration in RCS rats and offers potential therapeutic targets within inflammatory and oxidative stress pathways for confirmatory research and development.

Retinitis pigmentosa (RP) is a retinal degenerative disease associated with a diversity of genetic mutations. In a natural progression study (NPS) evaluating the molecular changes in Royal College of Surgeons (RCS) rats using lipidomic profiling, RNA sequencing, and gene expression analyses, changes associated with retinal degeneration from p21 to p60 were evaluated, where reductions in retinal ALOX15 expression corresponded with disease progression. This important enzyme catalyzes the formation of specialized pro-resolving mediators (SPMs) such as lipoxins (LXs), resolvins (RvDs), and docosapentaenoic acid resolvins (DPA RvDs), where reduced ALOX15 corresponded with reduced SPMs. Retinal DPA RvD2 levels were found to correlate with retinal structural and functional decline. Retinal RNA sequencing comparing p21 with p60 showed an upregulation of microglial inflammatory pathways accompanied by impaired damage-associated molecular pattern (DAMP) clearance pathways. This analysis suggests that ALXR/FPR2 activation can ameliorate disease progression, which was supported by treatment with an LXA4 analog, NAP1051, which was able to promote the upregulation of ALOX12 and ALOX15. This study showed that retinal inflammation from activated microglia and dysregulation of lipid metabolism were central to the pathogenesis of retinal degeneration in RP, where ALXR/FPR2 activation was able to preserve retinal structure and function.

The emergence and mutation of pathogenic viruses have been occurring at an unprecedented rate in recent decades. The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has developed into a global public health crisis due to extensive viral transmission. In situ RNA mapping has revealed angiotensin-converting enzyme 2 (ACE2) expression to be highest in the nose and lower in the lung, pointing to nasal susceptibility as a predominant route for infection and the cause of subsequent pulmonary effects. By blocking viral attachment and entry at the nasal airway using a cyclodextrin-based formulation, a preventative therapy can be developed to reduce viral infection at the site of entry. Here, we assess the safety and antiviral efficacy of cyclodextrin-based formulations. From these studies, hydroxypropyl beta-cyclodextrin (HPBCD) and hydroxypropyl gamma-cyclodextrin (HPGCD) were then further evaluated for antiviral effects using SARS-CoV-2 pseudotypes. Efficacy findings were confirmed with SARS-CoV-2 Delta variant infection of Calu-3 cells and using a K18-hACE2 murine model. Intranasal pre-treatment with HPBCD-based formulations reduced viral load and inflammatory signaling in the lung. In vitro efficacy studies were further conducted using lentiviruses, murine hepatitis virus (MHV), and influenza A virus subtype H1N1. These findings suggest HPBCD may be used as an agnostic barrier against transmissible pathogens, including but not limited to SARS-CoV-2.

Background: Neuroprotective nutritional interventions involving greater consumption of polyunsaturated fats (Mediterranean diet, fish consumption or DHA supplements) are less effective in APOE4 carriers with dementia compared to non‐carriers. We recently demonstrated greater activation of calcium dependent phospholipase A2 (cPLA2) in APOE4 compared with non‐APOE4 using both postmortem human brains and in brain tissues from APOE4 animal models. Activation of cPLA2 is associated with lower omega‐3 brain levels and lower measures of blood‐brain barrier (BBB) integrity as assessed using 7T DCE MRI to derive contrast transfer values (K trans).Method: Here, we present new data on aging (18 months) old APOE mouse models on a low DHA diet, APOE4‐TR mice treated with LPS and APOE knock out mice as a model of lower BBB integrity. All models were treated with the brain penetrant cPLA2 inhibitor ASB14780 at 10 mg/kg IP daily over 8 weeks in the aging model and acutely (7 days) in the younger LPS and APOE KO models.Result: cPLA2 inhibition was associated with increased brain omega‐3 levels in both the aging APOE4 model and in the younger APOE4 mice treated acutely with LPS. In the aging APOE4 mouse model, cPLA2 inhibition associated with better performance on novel object recognition, improved synaptic markers, and lower microglial activation. In the APOE KO model, treatment was associated with improved measures of BBB with lower K trans following treatment (Figure).Conclusion: The results here demonstrate how neuroinflammation associated with APOE4 depletes brain omega‐3s via activation of cPLA2 and how reducing cPLA2 activity can rescue this phenotype. The development of specific and potent brain penetrant cPLA2 inhibitors promises to enhance the brain effects of omega‐3 enriched diets by limiting the effects of neuroinflammation on the aging APOE4 brain.