Scholarly Works, Fralin Biomedical Research Institute at VTC

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Treatment and recovery from opioid use disorder: The role of pain severity in individuals with moderate to severe pain
Tegge, Allison N.; Ferreira, Marco A.R.; Garafola, Peter M.; Xu, Shuangshuang; Farrell, Michael; Marsden, John; Lee, Ken; Le Moigne, Anne; Gray, Frank; Bickel, Warren K. (Elsevier B.V., 2025-09-25)
Background: Pain is a frequent comorbidity among individuals with opioid use disorder (OUD), yet its impact on treatment outcomes is unclear. This study examined associations between pain severity and OUD treatment outcomes, including abstinence, craving, retention, and psychological functioning, in participants receiving longacting buprenorphine (BUP-XR). Methods: This secondary data analysis investigates participants from a BUP-XR phase 3 program: randomized clinical trial (NCT02357901; N = 192), open-label study (NCT02510014; N = 410); and a longitudinal observational follow-up (NCT03604861; N = 350). Pain was measured using the Brief Pain Inventory (BPI) at each treatment visit. Additional measures included demographics, opioid use, participant retention, opioid withdrawal, craving, depression, and quality of life. Analyses were performed on the full sample and the subgroup of individuals with moderate-to-severe pain (BPI≥4). Results: Participants averaged 40 years old, predominantly male (67%) and White (66%). Pain decreased after starting BUP-XR, and the reduction in pain continued throughout treatment (p-values<.001). For individuals with moderate-to-severe pain, greater concurrent pain severity was associated with lower abstinence rates (odds ratios: [0.801,0.852]; p-values<.001) in two datasets. Pain was not associated with participant retention. Lastly, greater pain severity was associated with worse physical quality of life (p-values<.001) and opioid withdrawal (pvalues<. 001), and greater depression (p-values<.001) and opioid craving (p-values<.001). Collectively, these findings are well replicated across three studies. Conclusions: Pain severity is a clinically relevant predictor of opioid use and psychosocial outcomes, but not treatment retention, in patients receiving BUP-XR. Routine pain severity monitoring may provide valuable insight into patient trajectories and support more tailored treatment approaches in OUD.
Comparing efficiency of patent production between US institutions using a hybrid NIH-USPTO dataset
DiSanto, Rocco M.; Van Dyke, Mark; Barker, Michael J.; Gourdie, Robert G. (Nature Portfolio, 2023-08)
A database that links patents to NIH awards enables evaluation of key milestones along the research translation pathway.
Degradation of the α-Carboxyl Terminus 11 Peptide: In Vivo and Ex Vivo Impacts of Time, Temperature, Inhibitors, and Gender in Rat
Tasdemiroglu, Yagmur; Council-Troche, McAlister; Chen, Miao; Ledford, Benjamin; Norris, Russell A.; Poelzing, Steven; Gourdie, Robert G.; He, Jia-Qiang (American Chemical Society, 2024-04-22)
In previous research, a synthetic α-carboxyl terminus 1 (αCT1) peptide derived from connexin 43 (Cx43) and its variant (αCT11) showed beneficial effects in an ex vivo ischemia-reperfusion (I/R) heart injury model in mouse. In an in vivo mouse model of cryo-induced ventricular injury, αCT1 released from adhesive cardiac patches reduced Cx43 remodeling and arrhythmias, as well as maintained cardiac conduction. Whether intravenous injection of αCT1 or αCT11 produces similar outcomes has not been investigated. Given the possibility of peptide degradation in plasma, this study utilized in vivo I/R cardiac injury and ex vivo blood plasma models to examine factors that may limit the therapeutic potential of peptide therapeutics in vivo. Following tail vein administration of αCT11 (100 μM) in blood, no effect on I/R infarct size was observed in adult rat hearts on day 1 (D1) and day 28 (D28) after injury (p > 0.05). There was also no difference in the echocardiographic ejection fraction (EF%) between the control and the αCT11 groups (p > 0.05). Surprisingly, αCT11 in blood plasma collected from these rats was undetectable within ∼10 min after tail vein injection. To investigate factors that may modulate αCT11 degradation in blood, αCT11 was directly added to blood plasma isolated from normal rats without I/R and peptide levels were measured under different experimental conditions. Consistent with in vivo observations, significant αCT11 degradation occurred in plasma within 10 min at 22 and 37 °C and was nearly undetectable by 30 min. These responses were reduced by the addition of protease/phosphatase (PTase/PPTase) inhibitors to the isolated plasma. Interestingly, no significant differences in αCT11 degradation in plasma were noted between male and female rats. We conclude that fast degradation of αCT11 is likely the reason that no beneficial effects were observed in the in vivo I/R model and inhibition or shielding from PTase/PPTase activity may be a strategy that will assist with the viability of peptide therapeutics.
Spontaneous Repolarization Alternans Causes VT/VF Rearrest That Is Suppressed by Preserving Gap Junctions
Laurita, Kenneth R.; Piktel, Joseph S.; Irish, Laken; Nassal, Michelle; Cheng, Aurelia; McCauley, Matthew; Pawlowski, Gary; Dennis, Adrienne T.; Suen, Yi; Almahameed, Sufian; Ziv, Ohaz; Gourdie, Robert G.; Wilson, Lance D. (Elsevier, 2024-07-01)
Background: Ventricular tachycardia (VT)/ventricular fibrillation (VF) rearrest after successful resuscitation is common, and survival is poor. A mechanism of VT/VF, as demonstrated in ex vivo studies, is when repolarization alternans becomes spatially discordant (DIS ALT), which can be enhanced by impaired gap junctions (GJs). However, in vivo spontaneous DIS ALT–induced VT/VF has never been demonstrated, and the effects of GJ on DIS ALT and VT/VF rearrest are unknown. Objectives: This study aimed to determine whether spontaneous VT/VF rearrest induced by DIS ALT occurs in vivo, and if it can be suppressed by preserving Cx43-mediated GJ coupling and/or connectivity. Methods: We used an in vivo porcine model of resuscitation from ischemia-induced cardiac arrest combined with ex vivo optical mapping in porcine left ventricular wedge preparations. Results: In vivo, DIS ALT frequently preceded VT/VF and paralleled its incidence at normal (37°C, n = 9) and mild hypothermia (33°C, n = 8) temperatures. Maintaining GJs in vivo with rotigaptide (n = 10) reduced DIS ALT and VT/VF incidence, especially during mild hypothermia, by 90% and 60%, respectively (P < 0.001; P < 0.013). Ex vivo, both rotigaptide (n = 5) and αCT11 (n = 7), a Cx43 mimetic peptide that promotes GJ connectivity, significantly reduced DIS ALT by 60% and 100%, respectively (P < 0.05; P < 0.005), and this reduction was associated with reduced intrinsic heterogeneities of action potential duration rather than changes in conduction velocity restitution. Conclusions: These results provide the strongest in vivo evidence to date suggesting a causal relationship between spontaneous DIS ALT and VT/VF in a clinically realistic scenario. Furthermore, our results suggest that preserving GJs during resuscitation can suppress VT/VF rearrest.
Development and characterization of the mode-of-action of inhibitory and agonist peptides targeting the voltage-gated sodium channel SCN1B beta-subunit
Williams, Zachary J.; Alvarez-Laviada, Anita; Hoagland, Daniel; Jourdan, L. Jane; Poelzing, Steven; Gorelik, Julia; Gourdie, Robert G. (Elsevier, 2024-09)
Cardiac arrhythmia treatment is a clinical challenge necessitating safer and more effective therapies. Recent studies have highlighted the role of the perinexus, an intercalated disc nanodomain enriched in voltage-gated sodium channels including both Nav1.5 and β1 subunits, adjacent to gap junctions. These findings offer insights into action potential conduction in the heart. A 19-amino acid SCN1B (β1/β1B) mimetic peptide, βadp1, disrupts VGSC beta subunit-mediated adhesion in cardiac perinexii, inducing arrhythmogenic changes. We aimed to explore βadp1's mechanism and develop novel SCN1B mimetic peptides affecting β1-mediated adhesion. Using patch clamp assays in neonatal rat cardiomyocytes and electric cell substrate impedance sensing (ECIS) in β1-expressing cells, we observed βadp1 maintained inhibitory effects for up to 5 h. A shorter peptide (LQLEED) based on the carboxyl-terminus of βadp1 mimicked this inhibitory effect, while dimeric peptides containing repeated LQLEED sequences paradoxically promoted intercellular adhesion over longer time courses. Moreover, we found a link between these peptides and β1-regulated intramembrane proteolysis (RIP) - a signaling pathway effecting gene transcription including that of VGSC subunits. βadp1 increased RIP continuously over 48 h, while dimeric agonists acutely boosted RIP for up to 6 h. In the presence of DAPT, an RIP inhibitor, βadp1's effects on ECIS-measured intercellular adhesion was reduced, suggesting a relationship between RIP and the peptide's inhibitory action. In conclusion, novel SCN1B (β1/β1B) mimetic peptides are reported with the potential to modulate intercellular VGSC β1-mediated adhesion, potentially through β1 RIP. These findings suggest a path towards the development of anti-arrhythmic drugs targeting the perinexus.
Acute adenoviral infection elicits an arrhythmogenic substrate prior to myocarditis
Padget, Rachel L.; Zeitz, Michael J.; Blair, Grace A.; Wu, Xiaobo; North, Michael D.; Tanenbaum, Mira T.; Stanley, Kari E.; Phillips, Chelsea M.; King, D. Ryan; Lamouille, Samy; Gourdie, Robert G.; Hoeker, Gregory S.; Swanger, Sharon A.; Poelzing, Steven; Smyth, James W. (American Heart Association, 2024-03-29)
BACKGROUND: Viral cardiac infection represents a significant clinical challenge encompassing several etiological agents, disease stages, complex presentation, and a resulting lack of mechanistic understanding. Myocarditis is a major cause of sudden cardiac death in young adults, where current knowledge in the field is dominated by later disease phases and pathological immune responses. However, little is known regarding how infection can acutely induce an arrhythmogenic substrate before significant immune responses. Adenovirus is a leading cause of myocarditis, but due to species specificity, models of infection are lacking, and it is not understood how adenoviral infection may underlie sudden cardiac arrest. Mouse adenovirus type-3 was previously reported as cardiotropic, yet it has not been utilized to understand the mechanisms of cardiac infection and pathology. METHODS: We have developed mouse adenovirus type-3 infection as a model to investigate acute cardiac infection and molecular alterations to the infected heart before an appreciable immune response or gross cardiomyopathy. RESULTS: Optical mapping of infected hearts exposes decreases in conduction velocity concomitant with increased Cx43^Ser368 phosphorylation, a residue known to regulate gap junction function. Hearts from animals harboring a phospho-null mutation at Cx43^Ser368 are protected against mouse adenovirus type-3–induced conduction velocity slowing. Additional to gap junction alterations, patch clamping of mouse adenovirus type-3–infected adult mouse ventricular cardiomyocytes reveals prolonged action potential duration as a result of decreased IK1 and IKs current density. Turning to human systems, we find human adenovirus type-5 increases phosphorylation of Cx43^Ser368 and disrupts synchrony in human induced pluripotent stem cell-derived cardiomyocytes, indicating common mechanisms with our mouse whole heart and adult cardiomyocyte data. CONCLUSIONS: Together, these findings demonstrate that adenoviral infection creates an arrhythmogenic substrate through direct targeting of gap junction and ion channel function in the heart. Such alterations are known to precipitate arrhythmias and likely contribute to sudden cardiac death in acutely infected patients.
Milk extracellular vesicles: A burgeoning new presence in nutraceuticals and drug delivery
Marsh, Spencer R.; Beard, Claire E.; Gourdie, Robert G. (Wiley, 2025-05-01)
Mammalian milk, a multifaceted developmental biofluid, has attracted new attention due to its diverse constituents and their implications for health and disease. Among these constituents, extracellular vesicles (EVs) have emerged as focal points of investigation. EVs, including exosomes and small EVs, have demonstrated biological activity in preclinical studies—including reports of enhancement of cognition and neural complexity, promotion of gastrointestinal development, barrier function and microbiome richness, the bolstering of immune response, and facilitation of musculoskeletal maturation in neonates. The richness of milk as a source of EVs is noteworthy, with hundreds of milliliters (at >10¹² EVs/mL) of nanovesicles extractable from a single liter of milk (>10¹⁴ EVs/starting liter of milk). Techniques such as tangential flow filtration hold promise for scalable production, potentially extending to thousands of liters. Together with the scale and increasing sophistication of the dairy industry, the abundance of EVs in milk underscores their commercial potential in various nutraceutical applications. Beyond natural bioactivity, milk EVs (mEVs) present intriguing possibilities as orally deliverable, non-immunogenic pharmaceutical carriers, with burgeoning interest in their utilization for heart disease and cancer chemotherapy and as vectors for gene-editing modules such as CrispR. This review synthesizes current knowledge on mEV biogenesis, characterization, isolation methodologies, and cargo contents. Moreover, it delves into the therapeutic potential of mEVs, both as inherently bioactive nanovesicles and as versatile platforms for drug delivery. As efforts progress toward large-scale implementation, rigorous attention to safe, industrial-scale production and robust assay development will be pivotal in harnessing the translational promise of small EVs from milk.
Tumor-derived extracellular vesicles disrupt the blood-brain barrier endothelium following high-frequency irreversible electroporation
Murphy, Kelsey R.; Aycock, Kenneth N.; Marsh, Spencer; Hay, Alayna N.; Athanasiadi, Ilektra; Bracha, Shay; Chang, Christine; Gourdie, Robert G.; Davalos, Rafael V.; Rossmeisl, John H. Jr.; Dervisis, Nikolaos G. (Nature Portfolio, 2024-11-18)
High-frequency irreversible electroporation (H-FIRE), a nonthermal brain tumor ablation therapeutic, generates a central tumor ablation zone while transiently disrupting the peritumoral blood–brain barrier (BBB). We hypothesized that bystander effects of H-FIRE tumor cell ablation, mediated by small tumor-derived extracellular vesicles (sTDEV), disrupt the BBB endothelium. Monolayers of bEnd.3 cerebral endothelial cells were exposed to supernatants of H-FIRE or radiation (RT)-treated LL/2 and F98 cancer cells. Endothelial cell response was evaluated microscopically and via flow cytometry for apoptosis. sTDEV were isolated following H-FIRE and RT, characterized via nanoparticle tracking analysis (NTA) and transmission electron microscopy, and applied to a Transwell BBB endothelium model to quantify permeability changes. Supernatants of H-FIRE-treated tumor cells, but not supernatants of sham- or RT-treated cells, disrupted endothelial cell monolayer integrity while maintaining viability. sTDEV released by glioma cells treated with 3000 V/cm H-FIRE increased permeability of the BBB endothelium model compared to sTDEV released after lower H-FIRE doses and RT. NTA revealed significantly decreased sTDEV release after the 3000 V/cm H-FIRE dose. Our results demonstrate that sTDEV increase permeability of the BBB endothelium after H-FIRE ablation in vitro. sTDEV-mediated mechanisms of BBB disruption may be exploited for drug delivery to infiltrative margins following H-FIRE ablation.
Interstitial fluid transport dynamics predict glioblastoma invasion and progression
Carman-Esparza, Cora M.; Stine, Caleb A.; Atay, Naciye; Kingsmore, Kathryn M.; Wang, Maosen Wang; Woodall, Ryan T.; Rockne, Russell C.; Cunningham, Jessica J.; Munson, Jennifer M. (Springer Nature, 2025-09-03)
Glioblastoma is characterized by aggressive infiltration into surrounding brain tissue, hindering complete surgical resection and contributing to poor patient outcomes. Identifying tumor-specific invasion patterns is essential for advancing our understanding of glioblastoma progression and improving surgical and radiotherapeutic strategies. Here, we leverage in vivo dynamic contrastenhanced magnetic resonance imaging (DCE-MRI) to noninvasively quantify interstitial fluid velocity, direction, and diffusion within and around glioblastomas. We introduce a novel vector-based pathline analysis to trace downstream accumulation of fluid flow originating from the tumor core, providing a spatially explicit perspective on local flow patterns. We find that localized fluid transport metrics predict glioblastoma invasion and progression, offering a new framework to non-invasively identify high-risk regions and guide targeted treatment approaches.
Caudate serotonin signaling during social exchange distinguishes essential tremor and Parkinson’s disease patients
Hartle, Alec E.; Kishida, Kenneth T.; Sands, L. Paul; Batten, Seth R.; Barbosa, Leonardo S.; Bang, Dan; Lohrenz, Terry; White, Jason P.; Sohrabi, Arian K.; Calafiore, Rebecca L.; DiFeliceantonio, Alexandra G.; Laxton, AdrianW.; Tatter, Stephen B.; Witcher, Mark R.; Montague, P. Read; Howe, W. Matt (Springer Nature, 2025-09-02)
Dynamic changes in dopamine, noradrenaline, and serotonin release are believed to causally contribute to the neural computations that support reward-based decision making. Accordingly, changes in signaling by these systems are hypothesized to underwrite multiple cognitive and behavioral symptoms observed in many neurological disorders. Here,we characterize the release of these neurotransmitters measured concurrently in the caudate of patients with Parkinson’s disease or essential tremor undergoing deep brain stimulation surgery as they played a social exchange game. We show that violations in the expected value of monetary offers are encoded by opponent patterns of dopamine and serotonin release in essential tremor, but not Parkinson’s disease, patients. We also demonstrate that these changes in serotonin signaling comprise a neurochemical boundary that subsegments these two neuromotor diseases. Our combined results point to a neural signature of altered reward processing that can be used to understand the signaling deficiencies that underwrite these diseases.
Elevated EGR1 binding at enhancers in excitatory neurons correlates with neuronal subtype-specific epigenetic regulation
Yin, Liduo; Xu, Xiguang; Conacher, Benjamin; Lin, Yu; Carrillo, Gabriela L.; Cun, Yupeng; Fox, Michael A.; Lu, Xuemei; Xie, Hehuang (2025-08-11)
Background: Brain development and neuronal cell specification are accompanied by epigenetic changes that enable the regulation of diverse gene expression patterns. During these processes, transcription factors interact with cell-type-specific epigenetic marks, binding to unique sets of cis-regulatory elements in different cell types. However, the detailed mechanisms through which cell-type-specific gene regulation is established in neurons remain to be explored. Results: In this study, we conducted a comparative histone modification analysis between excitatory and inhibitory neurons. Our results revealed that neuronal cell-type-specific histone modifications are enriched in super enhancer regions that contain abundant EGR1 motifs. Further CUT&RUN assay confirmed that excitatory neurons exhibit more EGR1 binding sites, primarily located in enhancers. Integrative analysis demonstrated that EGR1 binding is strongly correlated with various epigenetic markers of open chromatin regions and is linked to distinct gene pathways specific to neuronal subtypes. In inhibitory neurons, most genomic regions containing EGR1 binding sites become accessible during early embryonic stages, whereas super enhancers in excitatory neurons, which also host EGR1 binding sites, gain accessibility during postnatal stages. Conclusions: This study highlights the crucial role of transcription factor binding, such as EGR1, to enhancer regions, which may be key to establishing cell-type-specific gene regulation in neurons.
The impact of sulfatide loss on the progress of Alzheimer's disease
Finkielstein, Carla V.; Capelluto, Daniel G. S. (Wiley, 2023-08)
Metrics of glycemic control but not body weight influence flavor nutrient conditioning in humans
Baugh, Mary Elizabeth; Ahrens, Monica L.; Burns, Amber K.; Sullivan, Rhianna M.; Valle, Abigail N.; Hanlon, Alexandra L.; DiFeliceantonio, Alexandra G. (Elsevier, 2025-07)
The modern food landscape, marked by a rising prevalence of highly refined, ultra-processed, and highly palatable foods, combined with genetic and environmental susceptibilities, is widely considered a key factor driving obesity at the population level. Gaining insight into the physiological and behavioral mechanisms that shape food preferences and choices is crucial for understanding obesity's development and informing prevention strategies. One factor influencing habitual eating patterns, which may impact body weight, is flavor-nutrient learning. Research suggests that post-oral signaling is diminished in both animals and humans with obesity, potentially affecting flavor-nutrient learning. By analyzing pooled data from two similar preliminary studies, we found that markers of glycemic control-specifically fasting glucose and HbA1C-rather than BMI, were negatively correlated with changes in flavor liking in our flavor-nutrient learning task. These findings contribute to the expanding body of research on flavor-nutrient learning and underscore the variability in individual responses to these paradigms. Obesity is increasingly recognized as a complex and heterogeneous condition with diverse underlying mechanisms. Together, our findings and existing evidence emphasize the importance of further investigating how phenotypic factors interact to shape food preferences and eating behaviors.
Single-cell spatial transcriptomics reveals immunotherapy-driven bone marrow niche remodeling in AML
Gui, Gege; Bingham, Molly A.; Herzog, Julius R.; Wong-Rolle, Abigail; Dillon, Laura W.; Goswami, Meghali; Martin, Eddie; Reeves, Jason; Kim, Sean; Bahrami, Arya; Degenhardt, Hermann F.; Zaki, George; Divakar, Prajan; Schrom, Edward C.; Calvo, Katherine R.; Hourigan, Christopher S.; Hansen, Kasper D.; Zhao, Chen (American Association for the Advancement of Science, 2025-07-09)
Given the graft-versus-leukemia effect observed with allogeneic hematopoietic stem cell transplantation in refractory or relapsed acute myeloid leukemia (AML), immunotherapies have been explored in nontransplant settings. We applied a multiomic approach to examine bone marrow interactions in patients with AML treated with pembrolizumab and decitabine. Using extensively trained nuclear and membrane segmentation models, we achieved precise transcript assignment and deep learning–based image analysis. To address read-depth limitations, we integrated single-cell RNA sequencing with single-cell spatial transcriptomics from the same sample. Quantifying cell-cell distances at the edge level enabled more accurate tumor microenvironment analysis, revealing global and local immune cell enrichment near leukemia cells postpembrolizumab treatment, potentially linked to clinical response. Furthermore, ligand-receptor analysis indicated potential alterations in specific signaling pathways between leukemia and immune cells following immunotherapy treatment. These findings provide insights into immune interactions in AML and may inform therapeutic strategies.
Single-Cell and Spatial Transcriptomics Reveal a Tumor-Associated Macrophage Subpopulation that Mediates Prostate Cancer Progression and Metastasis
Mei, Shenglin; Zhang, Hanyu; Hirz, Taghreed; Jeffries, Nathan Elias; Xu, Yanxin; Baryawno, Ninib; Wu, Shulin; Wu, Chin-Lee; Patnaik, Akash; Saylor, Philip J.; Sykes, David B.; Dahl, Douglas M. (American Association for Cancer Research, 2025-07-02)
Tumor-associated macrophages (TAM) are a transcriptionally heterogeneous population, and their abundance and function in prostate cancer is poorly defined. We integrated parallel datasets from single-cell RNA sequencing, spatial transcriptomics, and multiplex immunofluorescence to reveal the dynamics of TAMs in primary and metastatic prostate cancer. Four TAM subpopulations were identified. Notably, one of these TAM subsets was defined by the co-expression of SPP1+ and TREM2+ and was significantly enriched in metastatic tumors. The SPP1+/TREM2+ TAMs were enriched in the metastatic tumor microenvironment in both human patient samples and murine models of prostate cancer. The abundance of these SPP1+/TREM2+ macrophages was associated with patient progression-free survival. Spatially, TAMs within prostate cancer bone metastases were highly enriched within the tumor region, consistent with their protumorigenic role. Blocking SPP1 in the RM1 prostate cancer mouse model led to improved efficacy of anti-PD-1 treatment and increased CD8+ T-cell infiltration in tumor. These findings suggest that targeting SPP1+ TAMs may offer a promising therapeutic strategy and potentially enhance the effects of immune checkpoint inhibition in advanced prostate cancer.
Implications
This study expands our understanding of the diverse roles of macrophage populations in prostate cancer metastases and highlights new therapeutic targets.
Interstitial fluid flow in an engineered human lymph node stroma model modulates T cell egress and stromal change
Hammel, Jennifer H.; Anerja, Abhinav; Cunningham, Jessica; Wang, Maosen; Schumaecker, Sophia; Orihuela, Yamilet Macias; Ozulumba, Tochukwu; Zatorski, Jonathan M.; Braciale, Thomas J.; Luckey, Chance John; Pompano, Rebecca R.; Munson, Jennifer M. (AIP Publishing, 2025-04-04)
The lymph node (LN) performs essential roles in immunosurveillance throughout the body. Developing in vitro models of this key tissue is of great importance to enhancing physiological relevance in immunoengineering. The LN consists of stromal populations and immune cells, which are highly organized and bathed in constant interstitial fluid flow (IFF). The stroma, notably the fibroblastic reticular cells (FRCs) and the lymphatic endothelial cells (LECs), play crucial roles in guiding T cell migration and are known to be sensitive to fluid flow. During inflammation, interstitial fluid flow rates drastically increase in the LN. It is unknown how these altered flow rates impact crosstalk and cell behavior in the LN, and most existing in vitro models focus on the interactions between T cells, B cells, and dendritic cells rather than with the stroma. To address this gap, we developed a human engineered model of the LN stroma consisting of FRC-laden hydrogel above a monolayer of LECs in a tissue culture insert with gravity-driven interstitial flow. We found that FRCs had enhanced coverage and proliferation in response to high flow rates, while LECs experienced decreased barrier integrity. We added CD4+ and CD8+ T cells and found that their egress was significantly decreased in the presence of interstitial flow, regardless of magnitude. Interestingly, 3.0 lm/s flow, but not 0.8 lm/s flow, correlated with enhanced inflammatory cytokine secretion in the LN stroma. Overall, we demonstrate that interstitial flow is an essential consideration in the lymph node for modulating LN stroma morphology, T cell migration, and inflammation.
Cytoplasmic connexin43-microtubule interactions promote glioblastoma stem-like cell maintenance and tumorigenicity
Smyth, James W.; Guo, Sujuan; Chaunsali, Lata; O’Rourke, Laurie; Dahlka, Jacob; Deaver, Stacie; Lunski, Michael; Nurmemmedov, Elmar; Sontheimer, Harald; Sheng, Zhi; Gourdie, Robert G.; Lamouille, Samy (Springer, 2025-05-16)
Glioblastoma (GBM) is the most common primary tumor of the central nervous system. One major challenge in GBM treatment is the resistance to chemotherapy and radiotherapy observed in subpopulations of cancer cells, including GBM stem-like cells (GSCs). These cells have the capacity to self-renew and differentiate and as such, GSCs participate in tumor recurrence following treatment. The gap junction protein connexin43 (Cx43) has complex roles in oncogenesis and we have previously demonstrated an association between Cx43 and GBM chemotherapy resistance. Here, we report, for the first time, increased direct interaction between non-junctional Cx43 and microtubules in the cytoplasm of GSCs. We hypothesize that non-junctional Cx43/microtubule complexing is critical for GSC maintenance and survival and sought to specifically disrupt this interaction while maintaining other Cx43 functions, such as gap junction formation. Using a Cx43 mimetic peptide of the carboxyl terminal tubulin-binding domain of Cx43 (JM2), we successfully disrupted Cx43 interaction with microtubules in GSCs. Importantly, administration of JM2 significantly decreased GSC survival in vitro, and limited GSC-derived and GBM patient-derived xenograft tumor growth in vivo. Together, these results identify JM2 as a novel peptide drug to ablate GSCs in GBM treatment.
AEOL-induced NRF2 activation and DWORF overexpression mitigate myocardial I/R injury
Asensio-Lopez, Maria del Carmen; Ruiz-Ballester, Miriam; Pascual-Oliver, Silvia; Bastida-Nicolas, Francisco J.; Sassi, Yassine; Fuster, Jose J.; Pascual-Figal, Domingo; Soler, Fernando; Lax, Antonio (2025-05-15)
Background: The causal relationship between the activation of nuclear factor erythroid 2-related factor 2 (NRF2) and the preservation of SERCA2a function in mitigating myocardial ischemia–reperfusion (mI/R) injury, along with the associated regulatory mechanisms, remains incompletely understood. This study aims to unravel how NRF2 directly or indirectly influences SERCA2a function and its regulators, phospholamban (PLN) and Dwarf Open Reading Frame (DWORF), by testing the pharmacological repositioning of AEOL-10150 (AEOL) in the context of mI/R injury. Methods C57BL6/J, Nrf2 knockout (Nrf2−/−), and wild-type (Nrf2+/+) mice, as well as human induced pluripotent stem cell-derived cardiomyocytes (hiPSCMs) were subjected to I/R injury. Gain/loss of function techniques, RT-qPCR, western blotting, LC/MS/MS, and fluorescence spectroscopy were utilized. Cardiac dimensions and function were assessed by echocardiography. Results: In the early stages of mI/R injury, AEOL administration reduced mitochondrial ROS production, decreased myocardial infarct size, and improved cardiac function. These effects were due to NRF2 activation, leading to the overexpression of the micro-peptide DWORF, consequently enhancing SERCA2a activity. The cardioprotective effect induced by AEOL was diminished in Nrf2−/− mice and in Nrf2/Dworf knockdown models in hiPSCMs subjected to simulated I/R injury. Our data show that AEOL-induced NRF2-mediated upregulation of DWORF disrupts the phospholamban-SERCA2a interaction, leading to enhanced SERCA2a activation and improved cardiac function. Conclusions: Taken together, our study reveals that AEOL-induced NRF2-mediated overexpression of DWORF enhances myocardial function through the activation of the SERCA2a offering promising therapeutic avenues for mI/R injury.
Red blood cell aggregation within a blood clot causes platelet-independent clot shrinkage
Peshkova, Alina; Rednikova, Ekaterina; Khismatullin, Rafael; Kim, Oleg; Muzykantov, Vladimir; Purohit, Prashant; Litvinov, Rustem; Weisel, John (American Society of Hematology, 2025-04-16)
Platelet-driven blood clot contraction (retraction) is important for hemostasis and thrombosis. RBCs occupy about half of the clot volume, but their possible active contribution to contraction is unknown. The work was aimed at elucidating the ability of RBCs to promote clot shrinkage. To distinguish effects of platelets and RBCs, we formed thrombin-induced clots from reconstituted human samples containing platelet-free plasma and platelet-depleted RBCs, followed by tracking the clot size. The clots before and after RBC-induced shrinkage were analyzed using histology and scanning electron microscopy. Tension developed in the RBC-containing plasma clots was measured with rheometry and theoretical modeling was used to elucidate the clot shrinkage mechanisms. Platelet-depleted clots formed in the presence of RBCs exhibited >20% volume shrinkage within one hour. This process was insensitive to blebbistatin, latrunculin A, and abciximab. At a higher RBC count clot shrinkage increased, whereas in the absence of RBCs no plasma clot shrinkage was observed. At low platelet counts RBCs stimulated clot contraction proportionately to the platelet level. Inside the shrunken clots, RBCs formed aggregates. The average tensile force per one RBC was ~120±100 pN. Clots from purified fibrinogen formed in the presence of RBCs did not change in size, but underwent shrinkage in the presence of osmotically active dextran. Blood clot shrinkage can be caused by RBCs alone and this effect is due to the RBC aggregation driven mainly by osmotic depletion. The RBC-induced clot shrinkage may reinforce platelet-driven blood clot contraction and promote clot compaction when there are few and/or dysfunctional platelets.
Transient Lymphatic Remodeling Follows Sub‑Ablative High‑Frequency Irreversible Electroporation Therapy in a 4T1 Murine Model
Esparza, Savieay; Jacobs, Edward; Hammel, Jennifer H.; Michelhaugh, Sharon K.; Alinezhadbalalami, Nastaran; Nagai‑Singer, Margaret; Imran, Khan Mohammad; Davalos, Rafael V.; Allen, Irving C.; Verbridge, Scott S.; Munson, Jennifer M. (Springer, 2025-02-25)
High-frequency irreversible electroporation (H-FIRE) is a minimally invasive local ablation therapy known to activate the adaptive immune system and reprogram the tumor microenvironment. Its predecessor, irreversible electroporation (IRE), transiently increases microvascular density and immune cell infiltration within the surviving non-ablated and non-necrotic tumor region, also known as the viable tumor region. However, the impact of pulse electric field therapies on lymphatic vessels, crucial for T-cell fate and maturation, remains unclear. This study investigates how sub-ablative H-FIRE (SA-HFIRE) affects lymphatic and blood microvascular remodeling in the 4T1 mammary mouse model. We conducted a temporal and spatial analysis to evaluate vascular changes in the viable tumor, peritumoral fat pad, and tumor-draining lymph node posttreatment. Histological examination showed a transient increase in blood vessel density on Day 1 post-treatment, followed by a spike in lymphatic vessel density in the viable tumor region on Day 3 post-treatment, increased lymphatic vessel density in the peripheral fat pad, and minimal remodeling of the tumor-draining lymph node within 3 days following treatment. Gene expression analysis indicated elevated levels of CCL21 and CXCL2 on Day 1 post-treatment, while VEGFA and VEGFC did not appear to contribute to vascular remodeling. Likewise, CCL21 protein content in tumor-draining axillary lymph nodes correlated with gene expression data from the viable tumor region. These findings suggest a dynamic shift in lymphatic and blood microvascular structures post-SA-HFIRE, potentially enhancing the adaptive immune response through CCL21- mediated lymphatic homing and subsequent lymph node microvascular remodeling. Future work will assess the immune and transport function of the microvasculature to inform experiments aimed at the application of adjuvant therapies during scenarios of tumor partial ablation.

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