Faculty Bibliography

After birth, tissues grow until they reach adult size, with each organ exhibiting unique cellular dynamics, growth patterns, and stem or non-stem cell sources. Using multiscale experimental and computational approaches, we found that aortic enlargement follows distinct growth principles, scaling with the vertebral column. Expansion proceeds via two temporally coordinated, spatially stochastic waves of proliferation aligned with blood flow, each with unique cell-cycle kinetics, with the first wave featuring cycles as short as 6 h. Single-cell RNA sequencing revealed increased fatty acid metabolism accompanying cell enlargement. Mathematical modeling and experiments showed that endothelial cell extrusion is essential for maintaining homeostatic aortic size as it adjusts for proliferation excess. Using a genetic model of achondroplasia, we mechanistically demonstrated that the aorta preserves proper scaling by increasing cell extrusion while keeping proliferation rates intact. These findings provide a blueprint of the principles orchestrating aortic growth, which relies entirely on the proliferation of resident differentiated cells. A record of this paper's transparent peer review process is included in the supplemental information.

AIM/OBJECTIVE:To investigate how psychosocial stress contributes to accelerated thrombosis, focusing on platelet activation and hyperreactivity. The specific objective was to identify novel platelet regulators involved in stress-mediated thrombosis, with a particular emphasis on the tetraspanin CD37. METHODS AND RESULTS/RESULTS:To explore how stress contributes to platelet hyperreactivity, platelets were isolated from (1) mice that experienced chronic variable stress and stress-free controls (n=8/group) and (2) human subjects with self-reported high and no stress levels (n=18/group), followed by RNA-sequencing. By comparing mutually expressed transcripts, a subset of genes differentially expressed following psychosocial stress was identified in both human and mouse platelets. In both mice and humans, platelet CD37 positively associates with platelet aggregation responses that underlie thrombosis, with Cd37-/- platelets exhibiting impaired integrin αIIbβ3 signaling, characterized by reduced platelet fibrinogen spreading and decreased agonist-induced αIIbβ3 activation. Consistent with a role for CD37 in regulating platelet activation responses, chimeric mice that received Cd37-/- bone marrow experienced a significantly increased time to vessel occlusion in the carotid artery FeCl3 model compared to mice reconstituted with wild-type bone marrow. CD37 deficiency did not alter hemostasis, as platelet count, coagulation metrics, prothrombin time, and partial thromboplastin time did not differ in Cd37-/- mice relative to wild-type mice. Consistent with this, bleeding time did not differ between wild-type and Cd37-/- mice following tail tip transection. CONCLUSIONS:This study provides new insights into the platelet-associated mechanisms underlying stress-mediated thrombosis. Identifying CD37 as a novel regulator of platelet activation responses offers potential therapeutic targets for reducing the thrombotic risk associated with psychosocial stress. The findings also contribute to understanding how psychosocial stress accelerates thrombotic events and underscore the importance of platelet activation in this process.

Atherosclerotic cardiovascular diseases (ASCVD) remain the leading cause of death globally. Animal and human studies link psychological stress-related disorders to ASCVD. Despite this accumulating evidence linking stress to increased cardiovascular disease (CVD) risk, it remains unclear whether stress impairs the benefits of standard risk-reduction therapies, of which lipid-lowering remains the most common, or whether this increased risk is driven by systemic inflammatory states. We tested the hypothesis that psychological stress limits the benefits of lipid lowering on resolving inflammation in atherosclerotic plaques by combining two established mouse models, namely one in which levels of atherogenic LDL cholesterol (LDL-C) can be lowered after plaques develop, and the other a model of chronic social defeat stress (CSDS). Here we show that mice susceptible to CSDS ("SUS") had attenuated benefits of LDL-C lowering compared to control (CON) or resilient (RES) mice. Moreover, in SUS mice (vs. CON or RES) there was heightened inflammation in the plaque macrophages, with evidence that this was a result of re-programming in the bone marrow (BM) of the precursors of macrophages, namely monocytes. Remarkably, these observations aligned with human imaging studies, in which LDL-C lowering therapy was not as effective in reducing either systemic or arterial inflammation in subjects with higher (vs. lower) neural imaging measures of psychological stress. In summary, the integration of the mouse model and human data provides important mechanistic and clinical insights into the crucial role of chronic stress in ASCVD, highlighting that this common risk factor impairs the anti-atherosclerotic benefits of lipid-lowering medications and may represent an important determinant of residual ASCVD risk.

The intestinal secretory lineage is thought to comprise four distinct cell types derived from one Atoh1⁺ progenitor, but the mechanisms that distinguish Paneth and goblet cells are unclear. Bhattacharya et al.¹ argue that these cells are instead phenotypic manifestations of a common terminal Atoh1⁺ cell, actively shaped by niche-derived signals.

BACKGROUND:/calmodulin-dependent protein kinase II and selected for probe collection. RESULTS:This approach significantly reduced the amount of FFPE tissue needed for robust single population RNA-seq. We demonstrate ~20 identified L3 or L5 pyramidal neurons or one lamina-specific cortical ribbon from a single 5µm thick section is sufficient to generate robust RNA-seq reads. Bioinformatic analysis of neurons and ribbons showed notable similarities and differences reflective of the single neuron and multiple admixed cell types within the former and latter, respectively. Comparison with existing methods Protocols exist for DSP of postmortem human FFPE brain tissue. However, this new approach enables profiling small groups of ~14-21 pyramidal neurons using the GeoMx DSP platform. CONCLUSIONS:This optimized DSP assay provides high resolution RNA-seq data demonstrating utility and versatility of the GeoMx platform for individually characterized neurons and isolated cortical ribbons within postmortem FFPE human brain tissue for downstream analyses.

Alzheimer's disease (AD) is characterized by the deposition of full-length and truncated amyloid beta (Aβ) species within brain parenchyma and cerebral vessels. However, Aβ truncated species remain understudied. Here, we present a protocol for culture and characterization of a mouse monoclonal antibody targeting N-terminally truncated proteoforms starting at position 4. We describe a detailed procedure for hybridoma culture, antibody collection, and isolation via affinity chromatography. We then describe steps for target validation via dot blot, as well as potential applications. For complete details on the use and execution of this protocol, please refer to Cabrera et al. and Rostagno et al.¹

BACKGROUND:Synovial fluid (SF) biomarkers demonstrate time-dependent variation after acute knee injury, and it is postulated that persistently elevated inflammatory markers may mediate worse long-term outcomes. PURPOSE/OBJECTIVE:This study investigated the relationship between biomarkers in SF at the time of meniscectomy and long-term patient-reported outcomes in patients with acute versus chronic meniscal injuries. STUDY DESIGN/METHODS:Cohort study; Level of evidence, 3. METHODS:This retrospective analysis included patients who underwent knee SF aspiration on the day of arthroscopic meniscectomy between October 2011 and October 2020 with minimum 4-year follow-up. SF aspirated from the operative knee was analyzed for 10 pro- and anti-inflammatory biomarkers. Patients completed the visual analog scale for pain, Lysholm Knee Questionnaire, Tegner Activity Scale, and Knee injury and Osteoarthritis Outcome Score-Physical Function Short-form (KOOS-PS) before surgery and at follow-up. Patients were categorized as having acute (<6 weeks) or chronic (>1 year) symptoms. K-means clustering analysis was performed using biomarker levels to group patients into distinct cohorts. RESULTS:= .020) than the low-inflammation cohort. CONCLUSION/CONCLUSIONS:In patients with chronic meniscal injury, those with a more proinflammatory SF biomarker profile at the time of meniscectomy had worse outcomes than those who had a low inflammatory profile. In acute meniscal injuries, most patients demonstrate a high inflammatory profile, which was not associated with a difference in long-term outcomes.

Epilepsy is a chronic condition characterized by unpredictable and recurrent spontaneous seizures. In a previous study, we reported that pharmacological inhibition of calpain prevented epileptogenesis in the rat pilocarpine model. In this study, we demonstrate that transgenic overexpression of calpastatin, the endogenous inhibitor of calpain, reduces calpain activation and lessens seizure burden in the mouse intrahippocampal kainate model. Blockade of calpain activation was evidenced by a reduction in the generation of spectrin breakdown products, a hallmark of calpain activation. CAST overexpression was associated with a significant reduction in seizure burden, further supporting the idea that blocking calpain overactivation prevents epilepsy. Moreover, a reduction in seizure burden was accompanied by a decrease in inflammatory markers but not cell death. Together, these observations corroborate the role of calpain overactivation in epileptogenesis and provide further support for the use of calpain inhibitors as a viable strategy to prevent epilepsy. PLAIN LANGUAGE SUMMARY: The mechanisms by which brain alterations lead to spontaneous seizures are not well understood. Acquired epilepsy often follows brain trauma. After a brain injury, the activation of the protease calpain has been associated with the development of spontaneous seizures. Our observations indicate that transgenic overexpression of calpastatin, an endogenous inhibitor of calpain, impacts epileptogenesis and reduces seizure burden. This suggests that inhibiting calpain could be a viable strategy to prevent epilepsy.

BACKGROUND:Endovascular aortic aneurysm repair (EVAR) is utilized to treat abdominal aortic aneurysms, while patients with short infrarenal necks can undergo fenestrated EVAR (FEVAR). Previous studies have demonstrated decreased aortic neck dilation for FEVAR compared to EVAR. Sac regression is a marker of success after EVAR; however, little is known regarding changes in sac volumetrics. This study compares aortic sac regression after EVAR versus FEVAR using volumetric analysis. METHODS:A retrospective review of prospectively collected data from 120 patients who underwent EVAR was performed. Thirty patients underwent FEVAR (Cook Medical Inc, Bloomington, IN) and 90 patients underwent EVAR (30 each with Endurant [Medtronic, Dublin, Ireland], Excluder [Gore, Flagstaff, AZ], and Zenith [Cook]). Demographic data were analyzed. Using 3-dimensional reconstruction software, preoperative and postoperative aneurysm sac volumes were measured, in addition to aneurysm characteristics. RESULTS:, P = 0.005). EVAR patients had greater number of lumbar arteries (7.26 ± 1.68 vs. 5.31 ± 1.93, P < 0.000001). On postoperative follow-up, FEVAR cases had greater sac regression compared to standard EVAR (-22.75 ± 25.7% vs. -5.98 ± 19.66%, P = 0.00031). The percentage of sac regression was greater when measured by volume compared to maximum diameter for FEVAR (-22.75 ± 25.7% vs. -13.90 ± 15.4%, P = 0.01) but not EVAR (-5.98 ± 19.7% vs. -4.51 ± 15.2%, P = 0.246). Those in the top tertile of percent volume of thrombus (>48.5%) were more likely to experience greater than 10% sac regression by volume (55% vs. 33.3%, P = 0.015). On multivariate analysis, FEVAR was associated with sac regression greater than 10% by volume (odds ratio [OR] 4.325, 95% confidence interval [CI] 1.346-13.901, P = 0.014), while endoleak (OR 0.162, 95% CI 0.055-0.479, P < 0.001) and 2 patent hypogastric arteries (OR 0.066, 95% CI 0.005-0.904, P = 0.042) were predictive against. CONCLUSIONS:Fenestrated EVAR is associated with greater sac regression compared to EVAR on volumetric analysis. This difference may be attributable to decreased endotension within the aneurysm resulting from less aortic neck dilatation, while the greater proportion of thrombus may be a protective factor from growth. Patients being evaluated for EVAR with borderline neck anatomy should be considered for FEVAR given increased sac regression.