Marie Dennis Leo

Background and Purpose: Cerebral ischaemia is a leading cause of death in older adults and imposes a substantial global economic burden. Recent studies indicate that inhibiting the Gα _s ‐coupled EP ₂ receptor reduces ischaemic brain injury by modulating neuroinflammation. However, these findings derive from the transient middle cerebral artery occlusion (tMCAO) model, which represents only a small subset of large‐vessel occlusion (LVO) cases with successful recanalisation. Experimental Approach: We characterised the pharmacokinetic parameters of selective EP ₂ antagonists CJ‐18, CJ‐19 and CJ‐21 that we recently developed. We evaluated their therapeutic efficacy in mice subjected to permanent MCAO (pMCAO), a model that more accurately reflects the majority of LVO cases, and assessed the impact of EP ₂ inhibition on brain injury and inflammation. Finally, we examined the effects of EP ₂ inhibition in neuron–glia mixed cultures exposed to oxygen–glucose deprivation (OGD) without reoxygenation. Key Results: After systemic administration in mice, new compounds exhibited favourable plasma half‐lives and brain penetration. In the pMCAO model, treatment with EP ₂ antagonists significantly reduced brain infarction in both male and female mice, in a dose‐dependent manner, whilst improving sensorimotor performance without affecting muscle strength. These benefits were accompanied by reduced neuronal death, lowered pro‐inflammatory cytokines and attenuated reactive gliosis within the ischaemic penumbra. In addition, EP ₂ inhibition by CJ‐18 conferred marked neuroprotection in neuron–glia co‐cultures exposed to prolonged OGD, preserving neurite integrity and postsynaptic density. Conclusions and Implications: This proof‐of‐concept study supports our drug‐like EP ₂ receptor antagonists, particularly CJ‐18, as potential treatments for permanent focal cerebral ischaemia.

Background: Sickle cell disease (SCD) is a systemic disorder marked by chronic pain and neurocognitive deficits, yet the molecular drivers of these neurocognitive features remain poorly defined. Platelets, central to inflammation and vascular homeostasis, may reflect broad pathophysiologic processes in SCD. Methods: We performed high-resolution mass spectrometry on ultra-purified platelets from 16 adults with SCD and moderate to severe pain (self-reported ≥ 3/10 in the past year), identifying 4196 proteins, of which 1046 were significant (FDR < 0.05). Unsupervised clustering was used to stratify individuals into high- and low-pain phenotypes. Results: Contrary to expectations, canonical pain pathways were not enriched. Instead, significant alterations were observed in neurodegeneration, mitochondrial metabolism, ATP regulation, mitophagy, and tRNA aminoacylation pathways between high- and low-pain phenotypes. High-pain individuals exhibited elevated levels of proteins involved in proteostasis and neurodegenerative disease processes, whereas low-pain individuals showed increased expression of proteins linked to mitochondrial integrity, neuroprotection, and reduced oxidative stress. Protein-protein interaction networks revealed tightly connected clusters within neurodegenerative and central nervous system-related pathways. Disease association analysis ranked neurodegenerative and mitochondrial pathways above traditional hematologic and nociceptive mechanisms. Conclusions: These findings suggest that platelet proteomics may serve as a peripheral window into PNS or CNS vulnerability and cognitive risk in SCD. The enrichment of tRNA aminoacylation and mitochondrial regulation pathways underscores the metabolic complexity of SCD and highlights novel targets for biomarker development and therapeutic intervention.

Background: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality worldwide. Despite established guidelines recommending semiannual surveillance for high-risk patients, real-world adherence remains inconsistent. Gaps in healthcare personnel knowledge and care coordination are recognized contributors to this implementation failure. Methods: Under IRB review, a quality improvement project using the Plan-Do-Study-Act (PDSA) framework was conducted in an ambulatory liver clinic in the southwestern United States. One PDSA cycle was completed. Retrospective and prospective chart reviews (n = 50 charts each) were conducted to assess HCC surveillance ordering and scheduling rates. Thirty healthcare personnel completed investigator-developed pre- and postintervention surveys measuring knowledge and perceptions. Intervention: A structured educational session grounded in current American Association for the Study of Liver Diseases (AASLD) surveillance guidelines was delivered to the full interdisciplinary clinic team, incorporating clinic-specific compliance data and role-specific coordination responsibilities. Results: Knowledge scores improved significantly from a mean of 41.67% to 95.33% (t [29] = −20.27, p < 0.001, d = 3.70). Perception scores improved (Wilcoxon z = −4.30, p < 0.001). Surveillance ordering increased from 88% to 94% and scheduling from 60% to 80%. Conclusions: A single structured educational PDSA cycle was associated with significant improvements in healthcare personnel knowledge and perceptions and with improved ordering and scheduling of HCC surveillance imaging. Postintervention imaging completion and result review rates were not assessed within the available follow-up period. Run chart monitoring of surveillance metrics across subsequent PDSA cycles is planned to evaluate sustainment and guide iterative improvement.

Type 2 diabetes (T2D) features insulin resistance that promotes cerebrovascular injury, yet the immune signals linking metabolic stress to vascular dysfunction remain unclear. We tested the hypothesis that insulin resistance and soluble vascular cell adhesion molecule-1 (sVCAM1) act through complementary pathways in mast cells (MCs) to raise circulating histamine levels and impair cerebral vascular function. In a high-fat diet (HFD) plus low-dose streptozotocin (STZ) model, plasma histamine rose sharply after the onset of insulin resistance and remained elevated. Plasma sVCAM1 levels also increased after insulin resistance. In vitro, recombinant sVCAM1 upregulated histidine decarboxylase (HDC) in native MCs in a dose-dependent manner, indicating a shift toward histamine synthesis, but did not enhance degranulation. In contrast, pharmacological inhibition of Akt with MK2206 activated Glycogen Synthase Kinase 3 beta (GSK3β) and increased MC degranulation without affecting HDC expression. Diabetic endothelial cell monolayers exhibited a ~twofold reduction in transendothelial electrical resistance consistent with impaired blood–brain barrier (BBB) integrity. Diabetic cerebral arteries showed receptor remodeling that favored constriction with histamine H1 receptor (H1R) expression increasing in vascular smooth muscle, while endothelial H1R and histamine H2 receptor (H2R) decreased. Functionally, insulin treatment lowered HOMA2-IR in T2D mice but did not restore cerebral artery myogenic tone or improve stroke outcomes after distal middle cerebral artery occlusion (dMCAO). Neutralizing VCAM1 with a monoclonal antibody reduced circulating sVCAM1 and histamine levels, and, together with the GSK3β inhibitor Tideglusib, stabilized MCs, normalized cerebral artery tone, and reduced post-MCAO infarct size and edema. These findings identify two distinct yet complementary mast cell pathways in T2D, highlight an immune-vascular interface that drives cerebrovascular dysfunction, and propose sVCAM1 blockade plus GSK3β inhibition as rational strategies to protect cerebral vascular function in the diabetic brain.

Background: Type 2 diabetes (T2D), the most common form of diabetes, is associated with a significantly elevated risk of cardiovascular and cerebrovascular complications. However, circulating metabolic signatures that reliably predict the transition to insulin resistance, and are potentially linked to increased vascular risk, remain incompletely characterized. Rodent models, particularly those induced by a high-fat diet (HFD) combined with low-dose streptozotocin (STZ), are widely used to study the progression of T2D. However, the systemic metabolic shifts associated with this model, especially at the plasma level, are poorly defined. Methods: In this study, we performed untargeted liquid chromatography–mass spectrometry (LC-MS)-based metabolomic profiling on plasma samples from control, HFD-only (obese, insulin-sensitive), and HFD + STZ (obese, insulin-resistant) C57BL/6 mice. Results: In the HFD + STZ cohort, plasma profiles showed a global shift toward lipid classes; depletion of aromatic and branched-chain amino acids (BCAAs); accumulation of phenylalanine-derived co-metabolites, consistent with gut–liver axis dysregulation; elevations in glucose, fructose-6-phosphate, and nucleoside catabolites, indicating impaired glucose handling and heightened nucleotide turnover; increased free fatty acids, reflecting membrane remodeling and lipotoxic stress; and higher cAMP, thyroxine, hydrocortisone, and uric acid, consistent with endocrine and redox imbalance. By contrast, HFD-only mice exhibited elevations in aromatic amino acids and BCAAs relative to controls, a pattern compatible with early obesity-associated adaptation while insulin signaling remained partially preserved. KEGG analysis revealed disturbances in carbohydrate metabolism, amino acid degradation, nucleotide turnover, and hormone-related pathways, and HMDB mapping linked these changes to T2D, obesity, heart failure, and renal dysfunction. Conclusion: Collectively, these findings delineate insulin resistance-specific plasma signatures of metabolic inflexibility and inflammatory stress in the HFD + STZ model, distinguishing it from HFD alone and supporting its utility for mechanistic studies and biomarker discovery. Importantly, this plasma metabolomics study shows that insulin-sensitive and insulin-resistant states exhibit distinct variation in circulating metabolites and cardiovascular risk factors, underscoring the translational value of plasma profiling.

The calcium-activated chloride channel, TMEM16A, is upregulated in the diabetic vasculature to cause increased vasoconstriction. In this paper, we have uncovered that the TMEM16A gene expression is controlled by a Snail-mediated repressor complex that uncouples with both insulin-dependent and -independent pathways to allow for upregulated arterial protein expression thereby causing vasoconstriction. The paper highlights the effect of short- and long-term glucose-induced dysfunction of an ion channel expression as a causative factor in diabetic vascular disease.

Systemic blood pressure is acutely controlled by total peripheral resistance as determined by the diameter of small arteries and arterioles, the contractility of which is regulated by endothelial cells lining the lumen of blood vessels. We investigated the physiological functions of the chloride (Cl ⁻ ) channel TMEM16A in endothelial cells. TMEM16A channels generated calcium (Ca ²⁺ )–activated Cl ⁻ currents in endothelial cells from control ( TMEM16A ^fl/fl ) mice that were absent in those from mice with tamoxifen-inducible, endothelial cell–specific knockout of TMEM16A ( TMEM16A ecKO). TMEM16A currents in endothelial cells were activated by the muscarinic receptor agonist acetylcholine and an agonist of the Ca ²⁺ channel TRPV4, which localized in nanoscale proximity with TMEM16A as assessed by single-molecule localization imaging of endothelial cells. Acetylcholine stimulated TMEM16A currents by activating Ca ²⁺ influx through surface TRPV4 channels without altering the nanoscale properties of TMEM16A and TRPV4 surface clusters or their colocalization. In pressurized arteries, activation of TMEM16A channels in endothelial cells induced by acetylcholine; TRPV4 channel stimulation; or intraluminal ATP, another vasodilator, produced hyperpolarization and dilation. Furthermore, deficiency of TMEM16A channels in endothelial cells resulted in increased systemic blood pressure in conscious mice. These data indicate that vasodilators stimulate TRPV4 channels, leading to Ca ²⁺ -dependent activation of nearby TMEM16A channels in endothelial cells to produce arterial hyperpolarization, vasodilation, and reduced blood pressure. Thus, TMEM16A is an anion channel in endothelial cells that regulates arterial contractility and blood pressure.

Soluble cell adhesion molecules (sCAMs) are secreted ectodomain fragments of surface adhesion molecules, ICAM1 and VCAM1. sCAMs have diverse immune functions beyond their primary function, impacting immune cell recruitment and activation. Elevated sVCAM1 levels have been found to be associated with poor cardiovascular disease (CVD) outcomes, supporting VCAM1’s role as a potential diagnostic marker and therapeutic target. Inhibiting sVCAM1’s release or its interaction with immune cells could offer cardioprotection in conditions such as diabetes. Membrane-bound surface adhesion molecules are widely expressed in a wide variety of cell types with higher expression in endothelial cells (ECs). Still, the source of sCAMs in the circulation is not clear. Hypothesizing that endothelial cells (ECs) could be a potential source of sCAMs, this study investigated whether dysfunctional EC signaling mechanisms during diabetes cause VCAM1 ectodomain shedding. Our results from samples from an inducible diabetic mouse model revealed increased sVCAM1 plasma levels in diabetes. Protein analysis indicated upregulated VCAM1 expression and metalloproteases ADAM10 and ADAM17 in diabetic ECs. ADAMs are known for proteolytic cleavage of adhesion molecules, contributing to inflammation. GSK3β, implicated in EC VCAM1 expression, was found to be activated in diabetic ECs. GSK3β activation in control ECs increased ADAM10/17 and VCAM1. A GSK3β inhibitor reduced active GSK3β and VCAM1 ectodomain shedding. These findings suggest diabetic ECs with elevated GSK3β activity led to VCAM1 upregulation and ADAM10/17-mediated sVCAM1 shedding. This mechanism underscores the potential therapeutic role of GSK3β inhibition in reducing the levels of circulating sVCAM1. The complex roles of sCAMs extend well beyond CVD. Thus, unraveling the intricate involvement of sCAMs in the initiation and progression of vascular disease, particularly in diabetes, holds significant therapeutic potential.

Soluble cell adhesion molecules (sCAMs) are secreted ectodomain fragments of surface adhesion molecules, ICAM1 and VCAM1. sCAMs have diverse immune functions beyond their primary function, impacting immune cell recruitment and activation. Elevated sVCAM1 levels have been associated with poor cardiovascular disease (CVD) outcomes, supporting its role as a potential diagnostic marker and therapeutic target. Inhibiting sVCAM1 release or interaction with immune cells could offer cardioprotection in conditions such as diabetes. Membrane bound surface adhesion molecules are widely expressed in a wide variety of cell types with higher expression in endothelial cells (ECs), but the source of sCAMs in the circulation is not clear. Hypothesizing that endothelial cells (ECs) could be a potential source of sCAMs, this study investigated whether dysfunctional EC signaling mechanisms during diabetes cause VCAM1 ectodomain shedding. Our results from samples from a inducible diabetic mouse model revealed increased sVCAM1 plasma levels in diabetes. Protein analysis indicated upregulated VCAM1 expression and metalloproteases ADAM10 and ADAM17 in diabetic ECs. ADAMs are known for proteolytic cleavage of adhesion molecules, contributing to inflammation. GSK3β, implicated in EC VCAM1 expression, was found to be activated in diabetic ECs. GSK3β overexpression in control ECs increased ADAM10/17 and VCAM1. A GSK3β inhibitor reduced active GSK3β and VCAM1 ectodomain shedding. These findings suggest that diabetic ECs with elevated GSK3β activity led to VCAM1 upregulation and ADAM10/17-mediated sVCAM1 shedding. This mechanism underscores the potential therapeutic role of GSK3β inhibition in reducing the levels of circulating sVCAM1. The complex roles of sCAMs extend well beyond CVD. Thus, unraveling the intricate involvement of sCAMs in the initiation and progression of vascular disease particularly in diabetes, holds significant therapeutic potential.