Manipulating cerebral endothelial cell autophagy impacts outcomes of acute ischemic stroke in mice

Physiology · 2026-05-01

Outcomes of acute ischemic stroke (AIS) are worsened by aging, but precise mechanisms are unclear. Earlier we reported that autophagic flux in non-cerebral endothelial cells (ECs) is blunted in mice and humans. EC autophagy is an intrinsic cytoprotective process that is heightened in response to nutrient-stress, and the accrual of damaged or dysregulated intracellular organelles, both of which occur in response to a sudden reduction in cerebral perfusion. Here we tested the hypothesis that repressed cerebral EC autophagy contributes to worse AIS outcomes in older vs. adult mice. Adult (~4-mo) and older (~24-mo) male mice completed 60-min transient middle cerebral artery occlusion (tMCAO) + 23-h reperfusion (R) to evoke AIS. Ipsilesional reperfusion blood flow measured via laser speckle contrast imaging (LSCI), neurobehavioral (Bederson test) and motor (Grip test) performance, and neuronal viability (NeuN staining) were worse (p< 0.05) in older vs. adult mice (n=10 per group), substantiating results from others. In additional cohorts that completed tMCAO+R we isolated cerebral ECs from ipsilesional hemispheres and confirmed their purity via immunoblotting i.e., robust CD31 yet sparse α-SMA, NeuN in the EC-enriched fraction and reciprocal results from the non-EC fraction. Indexes of autophagy assessed via immunoblotting (e.g., LC3-II/GAPDH) and immunofluorescence (e.g., Atg3:CD31 colocalization) were lower (p< 0.05) in cerebral EC fractions isolated from older vs. adult mice (n=3 per group). Next, we reasoned that if repressed cerebral EC autophagy is responsible for worse stroke outcomes in older mice, then adult male mice with inducible depletion of cerebral autophagy related gene 3 (Atg3EC-/-) should be more vulnerable to AIS. In support, AIS -induced reductions in reperfusion blood flow, tissue injury, neurobehavioral and motor performance, and neuronal death were more severe in Atg3EC-/- vs. wild-type (WT) mice (p< 0.05, n=10 per group). Underscoring the important role for cerebral EC autophagy, adult male Atg3EC-/- mice were refractory to benefits (p< 0.05) afforded by mTORC1 inhibition using rapamycin, observed in WT mice concerning AIS outcomes (n=5 per group). Because results to this point indicate defective cerebral EC autophagy contributes to worse AIS outcomes in older mice, we reasoned that boosting cerebral EC autophagy in older mice might be protective. Older male mice received a cerebral EC-specific adeno-associated viral vector (AAV-BR1) to deliver Atg3 or Null virus. In one cohort we verified increased (p< 0.05) Atg3 gene expression in cerebral EC but not non-EC -enriched fractions from mice that received AAV-BR1-Atg3 vs. Null virus (n=3 per group). A second cohort of older male mice completed tMCAO+R 21-days after receiving the respective virus. Ipsilesional reperfusion blood flow was greater, neuronal viability heightened, and infarct volume and neurobehavioral and motor deficits were less severe (p< 0.05) in older mice that received AAV-BR1-Atg3 vs. Null virus. (n=10 per group). Collectively, these results support that cerebral EC autophagy confers intrinsic cytoprotection in the context of AIS and should be considered further as a therapeutic target for treating AIS. Funding: NIH R21AG081780-01A1 to JDS and RC, AHA23PRE1025910 and University of Utah Graduate Research Fellowship to SM This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Brush-mediated angular constraints reshape structure, rigidity, and percolation in colloidal depletion gels

arXiv (Cornell University) · 2026-03-13

Colloidal gels, like many other soft and disordered solids derive their mechanical properties not only from the strength of interparticle attraction, but also from the symmetry of the forces that constrain particle motion. While non-central interactions are known to profoundly alter rigidity and elasticity, they are typically introduced through particle anisotropy, surface roughness, or patchy interactions, obscuring their independent role. Here we demonstrate a minimal and geometry-preserving route to emergent non-central forces in colloidal gels by reducing the density of surface-grafted polymer brushes. At low brush density, partial brush interpenetration introduces an effective angular bending rigidity at particle contacts, despite fully isotropic particle geometry. This emergent constraint suppresses local densification, stabilizes low-coordination networks, and produces highly ramified gel structures with enhanced elasticity. Combining experiments, simulations, and mean-field theory, we show that these non-central constraints reorganize structure and mechanics across length scales, shifting gelation boundaries and increasing the elastic modulus by nearly a factor of three. Our results establish surface brush density as a generic control parameter for programming interaction symmetry in soft particulate matter, with implications for rigidity, percolation, and mechanical design in disordered systems.

Proteomic profiling identifies systemic drivers of blood-brain barrier injury in sickle cell disease

Blood · 2026-05-13

Sickle cell disease (SCD) causes brain injury and cognitive disability. Systemic inflammation and endothelial injury are central to SCD pathophysiology, yet the relationship between systemic drivers of blood-brain barrier (BBB) disruption and brain injury remains understudied. This cross-sectional study assessed whole-brain and regional BBB permeability (Ktrans) using dynamic contrast-enhanced magnetic resonance imaging in 37 adults with SCD in steady-state and 37 non-SCD adults. Cerebral oxygen extraction fraction (OEF) and white matter mean diffusivity (MD) measured tissue hypoxia and microstructural injury, respectively. The SCD cohort showed elevated Ktrans compared with controls (3.6 x10-4min-1 vs. 2.58x10-4min-1, 95% CI median difference [0.36, 1.30]x10-4min-1, P< 0.001), indicating BBB disruption. In SCD, white matter Ktrans was associated with MD (β [95% Cl]: 6.25 [1.72, 10.77], P=0.008), independent of OEF (β [95% Cl]: 0.22 [0.09, 0.35]) and silent cerebral infarcts (β [95% Cl]: 0.01 [0.00, 0.02]). The interaction (P=0.037) between Ktrans and OEF on MD suggested a combined, deleterious effect of BBB disruption and hypoxia on microstructural injury. High-throughput plasma proteomics followed by differential expression analysis, and weighted gene correlation network analysis in a subset of 61 participants revealed 79 proteins associated with BBB permeability belonged to iron homeostasis, response to hypoxia, immune dysregulation, extracellular matrix degradation, lipoprotein homeostasis, and arginine-proline metabolism pathways. All pathways were independently associated with microstructural injury. BBB permeability is a mediator of brain injury for all pathways except extracellular matrix degradation. Targeting specific systemic pathways to protect the BBB may represent a therapeutic approach to preserve brain health in SCD.

Brush-mediated angular constraints reshape structure, rigidity, and percolation in colloidal depletion gels

arXiv (Cornell University) · 2026-03-13

Colloidal gels, like many other soft and disordered solids derive their mechanical properties not only from the strength of interparticle attraction, but also from the symmetry of the forces that constrain particle motion. While non-central interactions are known to profoundly alter rigidity and elasticity, they are typically introduced through particle anisotropy, surface roughness, or patchy interactions, obscuring their independent role. Here we demonstrate a minimal and geometry-preserving route to emergent non-central forces in colloidal gels by reducing the density of surface-grafted polymer brushes. At low brush density, partial brush interpenetration introduces an effective angular bending rigidity at particle contacts, despite fully isotropic particle geometry. This emergent constraint suppresses local densification, stabilizes low-coordination networks, and produces highly ramified gel structures with enhanced elasticity. Combining experiments, simulations, and mean-field theory, we show that these non-central constraints reorganize structure and mechanics across length scales, shifting gelation boundaries and increasing the elastic modulus by nearly a factor of three. Our results establish surface brush density as a generic control parameter for programming interaction symmetry in soft particulate matter, with implications for rigidity, percolation, and mechanical design in disordered systems.

Enhancing validation of case-control omics signatures through “minimalist” single-subject analysis (N-of-1 trials): proof of concept in sepsis

Journal of the American Medical Informatics Association · 2026-04-12

OBJECTIVE: To evaluate if a single-subject study (S3) design, utilizing paired transcriptome samples from the same patient (eg, "sepsis" vs "recovered"), can replicate transcriptomic signatures from small case-control studies, addressing challenges in patient accrual for rare or sub-stratified diseases. METHODS: We generated a sepsis gene signature (SGS) comprising 300 differentially expressed genes (DEGs; FDR < 5%) from a human sepsis case-control cohort using general linear models (GLMs). Reproducibility of SGS was assessed through three approaches applied to sub-sampled independent datasets: single-subject analyses (N-of-1-MixEnrich), anticipated to perform better; conventional paired-sample GLM analyses; and a traditional case-control GLM analysis. RESULTS: SGS reproducibility in GLM analyses was inconsistent at smaller cohort sizes (∼80% reproducibility; n = 5) but stabilized at cohort sizes >6. Remarkably, the single-subject-study approach consistently reproduced SGS in each of the 18 subjects individually (100% reproducibility; n = 1). DISCUSSION: Conventional GLMs are not designed for single-subject or small cohort analyses due to their dependence on larger samples to mitigate variable dispersion and human heterogeneity. In contrast, S3 methods enhance statistical power by: reducing multiple testing through gene set aggregation, emphasizing concordant changes in pathway activity rather than exact molecular consistency, and exploiting paired samples from the same individual. CONCLUSION: This proof-of-concept demonstrates that S3 designs effectively validate gene expression signatures derived from case-control studies, highlighting their potential in research or clinical trials constrained by small sample sizes. However, further validation and computational simulation are needed to demonstrate scalability to other conditions and sensitivity to validation subject variations from the "average subject" of discovery cohorts.

Association of rurality with reduced shunt survival in child and young adult hydrocephalus: a prospective nationwide cohort from the Australasian Shunt Registry

Journal of Neurosurgery Pediatrics · 2026-01-16 · 1 citations

OBJECTIVE: Patients with ventriculoperitoneal shunts have a lifelong risk of requiring revision procedures that may be time critical. Many of these patients reside distantly from neurosurgical services; however, the influence of this key sociodemographic factor on the outcomes of patients with ventriculoperitoneal shunts has not been previously studied. The Australasian Shunt Registry provides a rare opportunity to study the association of rurality with ventriculoperitoneal shunt outcomes across a large landmass with a broadly distributed population. METHODS: Data were extracted from the Australasian Shunt Registry for all patients aged ≤ 25 years who underwent first-time ventriculoperitoneal shunt insertion from December 2016 to October 2024. The patient's postcode of residence was matched to the corresponding Modified Monash Model (MM) rurality index for that region. The metropolitan cohort was defined as all patients with MM 1, and the rural/regional cohort included all patients with MM ≥ 2. The primary outcome of interest was time from ventriculoperitoneal shunt insertion to first shunt revision (shunt survival). RESULTS: At the conclusion of the study period, the Australasian Shunt Registry contained records for 5023 unique patients. After application of exclusion criteria, the resulting sample included 930 patients. The majority of these patients reside in a metropolitan MM1 area (617/930 [66%]). The cohorts were similar for demographic and clinical variables. The estimated mean shunt survival was 5.04 (95% CI 4.64-5.43) years for rural patients and 5.69 (95% CI 5.40-5.98) years for metropolitan patients (Wilcoxon test, p = 0.01). That effect remained statistically significant in the Cox proportional hazards model that included all variables that were significant on univariate analysis: a metropolitan address exerted a protective effect on overall shunt survival (HR 0.75, 95% CI 0.59-0.95, p = 0.02). CONCLUSIONS: Rural/regional patients with ventriculoperitoneal shunts experienced reduced overall shunt survival when compared to metropolitan patients.

Reciprocal stabilization of coagulation factor XIII-A and -B subunits is a determinant of plasma FXIII concentration

UNC Libraries · 2026-04-24

Transglutaminase factor XIII (FXIII) is essential for hemostasis, wound healing, and pregnancy maintenance. Plasma FXIII is composed of A and B subunit dimers synthesized in cells of hematopoietic origin and hepatocytes, respectively. The subunits associate tightly in circulation as FXIII-A2B2. FXIII-B2 stabilizes the (pro)active site-containing FXIII-A subunits. Interestingly, people with genetic FXIII-A deficiency have decreased FXIII-B2, and therapeutic infusion of recombinant FXIII-A2 (rFXIII-A2) increases FXIII-B2, suggesting FXIII-A regulates FXIII-B secretion, production, and/or clearance. We analyzed humans and mice with genetic FXIII-A deficiency and developed a mouse model of rFXIII-A2 infusion to define mechanisms mediating plasma FXIII-B levels. Like humans with FXIII-A deficiency, mice with genetic FXIII-A deficiency had reduced circulating FXIII-B2, and infusion of FXIII-A2 increased FXIII-B2. FXIII-A-deficient mice had normal hepatic function and did not store FXIII-B in liver, indicating FXIII-A does not mediate FXIII-B secretion. Transcriptional analysis and polysome profiling indicated similar F13b levels and ribosome occupancy in FXIII-A-sufficient and -deficient mice and in FXIII-A-deficient mice infused with rFXIII-A2, indicating FXIII-A does not induce de novo FXIII-B synthesis. Unexpectedly, pharmacokinetic/pharmacodynamic modeling of FXIII-B antigen after rFXIII-A2 infusion in humans and mice suggested FXIII-A2 slows FXIII-B2 loss from plasma. Accordingly, comparison of free FXIII-B2 vs FXIII-A2-complexed FXIII-B2 (FXIII-A2B2) infused into mice revealed faster clearance of free FXIII-B2. These data show FXIII-A2 prevents FXIII-B2 loss from circulation and establish the mechanism underlying FXIII-B2 behavior in FXIII-A deficiency and during rFXIII-A2 therapy. Our findings reveal a unique, reciprocal relationship between independently synthesized subunits that mediate an essential hemostatic protein in circulation. This trial was registered at www.ClinicalTrials.com as #NCT00978380.

Heterozygous <i>Eif4nif1</i> Stop-Gain Mice Replicate the Primary Ovarian Insufficiency Phenotype in Women

Endocrinology · 2025-01-17 · 1 citations

We created the c.1286C>G stop-gain mutation found in a family with primary ovarian insufficiency (POI) at age 30 years. The Eif4enif1 C57/Bl6 transgenic mouse model contained a floxed exon 10-19 cassette with a conditional knock-in cassette containing the c.1286C>G stop-gain mutation in exon 10. The hybrid offspring of CMV-Cre mice with Eif4enif1WT/flx mice were designated Eif4enif1WT/Δ for simplicity. A subset of female heterozygotes (Eif4enif1WT/Δ) had no litters. In those with litters, the final litter was earlier (5.4 ± 2.6 vs 10.5 ± 0.7 months; P = .02). Heterozygous breeding pair (Eif4enif1WT/Δ × Eif4enif1WT/Δ) litter size was 60% of WT litter size (3.9 ± 2.0 vs 6.5 ± 3.0 pups/litter; P < .001). The genotypes were 35% Eif4enif1WT/flx and 65% Eif4enif1WT/Δ, with no homozygotes. Homozygote embryos did not develop beyond the 4- to 8-cell stage. The number of follicles in ovaries from Eif4enif1WT/Δ mice was lower starting at the primordial (499 ± 290 vs 1445 ± 381) and primary follicle stage (1069 ± 346 vs 1450 ± 193) on day 10 (P < .05). The preantral follicle number was lower starting on day 21 (213 ± 86 vs 522 ± 227; P < .01). Examination of ribosome protected mRNAs demonstrated altered mRNA expression. The Eif4enif1 stop-gain mice replicate the POI phenotype in women based on an earlier end to reproduction due to oocyte loss. The unique mouse model provides a platform to study regulation of protein translation across oocyte and embryo development in mammals.

Neutrophil extracellular traps drive myeloproliferation and immune dysfunction in chronic myelomonocytic leukemia

Blood · 2025-11-03

Abstract Background: Chronic myelomonocytic leukemia (CMML) is an aggressive hematologic malignancy characterized by myelodysplastic and myeloproliferative features, with high risk of transformation to acute myeloid leukemia. CMML is associated with systemic inflammation, clonal monocytosis, and susceptibility to life-threatening cytokine release in response to stressors. We hypothesized that clonal neutrophils contribute to chronic inflammation, excess myeloproliferation and decreased T cell immunosurveillance in CMML via dysregulated neutrophil extracellular trap (NET) formation (NETosis), and use a novel inhibitor of NETosis, neonatal NET-inhibitory factor (nNIF), to therapeutically target NET formation in CMML. Methods: Neutrophils (PMNs) were isolated via magnetic negative selection from the peripheral blood of healthy donors (HD) and CMML patients. NET formation by PMNs +/- nNIF was assessed in vitro under both basal and stimulated (phorbol myristate acetate; PMA) conditions and analyzed by myeloperoxidase (MPO)-DNA ELISA and confocal microscopy. Colony-forming assays with primary CD34+ hematopoietic stem and progenitor cells (HSPCs) from HD and CMML patients were performed in semisolid media supplemented with primary patient plasma or cell-free NET assay supernatant (NET-S) +/- nNIF. PMNs from CMML and HD samples were further analyzed for distinct transcriptional profiles using bulk RNA-seq. T-cell differentiation and exhaustion markers were assessed in primary T-cells from HD and CMML patients by flow cytometry. Results: We found that CMML patient-derived PMNs show elevated spontaneous NET formation compared to HD PMNs. CMML NET formation did not increase significantly upon PMA stimulation, indicating that CMML PMNs are at maximal basal NET induction. Preincubation of CMML PMNs with nNIF significantly inhibited both baseline and PMA-stimulated NET formation. Further, plasma from CMML patients induced NETosis in HD PMNs, an effect completely reversed by the nNIF preincubation. To assess the ability of NET components to drive myeloproliferation, we performed colony-forming assays using HD and CMML-derived HSPCs cultured with HD or CMML plasma or NET-S. HD HSPCs cultured with CMML plasma or CMML NET-S showed significantly increased colony number and size compared to controls. Preincubation of CMML PMNs with nNIF prior to NET-S collection abrogated this pro-proliferative effect and reduced HD colony formation. Similarly, CMML HSPCs cultured with CMML NET-S demonstrated increased colony formation that was reversed by preincubation of CMML PMNs with nNIF, confirming that soluble factors released during CMML NETosis promote myeloproliferation. These findings were recapitulated in an in vivo CMML mouse model. We next investigated CMML-associated immune dysfunction. Flow cytometric analysis of peripheral blood T-cells from HD and CMML patients revealed significantly increased expression of exhaustion markers PD-1 and TIGIT on CD4+ T cells and PD-1, TIGIT, and KLRG1 on CD8+ T cells in CMML patients versus HD. Bulk RNA-seq of CMML versus HD PMNs showed distinct gene expression profiles, with CMML PMNs demonstrating upregulated expression of multiple NETosis-related genes, including neutrophil elastase (ELANE), MPO, neutrophil collagenase (MMP8), and lactoferrin (LTF), and downregulation of gene terms associated with T-cell activation and co-stimulation. These transcriptomic data suggest that chronic NET activation in CMML may impair T-cell priming and immunosurveillance. Conclusion: Our data suggest dysregulated NETosis as a mechanism linking inflammation, myeloproliferation, and immune dysfunction in CMML. CMML PMNs are primed for constitutive NETosis, and NET-derived factors promote both healthy and malignant HSPC proliferation while impairing T-cell function. Inhibition of NETosis using nNIF suppresses augmented NETosis at baseline and under stimulated conditions in CMML PMNs and hinders the proliferative capacity of CMML HSPCs. These findings provide a compelling rationale for targeting NETs as a novel therapeutic strategy in CMML.

Linking thromboembolism to the pathogenesis of long COVID

Nature Cardiovascular Research · 2025-12-16