Biology subjects

Grande-Allen, K. J.

Publications and source records attributed to Grande-Allen, K. J..

13 recordsLinked to original sources

bioRxiv2026

Loss of Syndecan-3 Drives a Pro-inflammatory, EndMT-prone Phenotype in Aortic Valve Endothelial Cells

The earliest events in calcific aortic valve disease (CAVD) occur at the valve endothelium, where the cells lining the leaflet undergo NF-kappaB (Nuclear Factor kappa B)-driven inflammatory activation and endothelial-to-mesenchymal transition (EndMT). The endothelial glycocalyx (EG) sets the threshold at which endothelial cells (ECs) respond to such cues, yet its composition and function in the aortic valve remain almost entirely uncharacterized. Here we define the cell-surface proteoglycan (CPG) landscape of human aortic valve endothelial cells (HAVECs) under inflammatory stimulation and identify syndecan-3 (SDC3) as a regulator of valve endothelial activation. Among the proteoglycans examined, SDC3 was highly expressed and inflammation-responsive. Silencing SDC3 in unstimulated HAVECs was sufficient to upregulate NF-kappaB pathway components and the leukocyte adhesion molecules vascular cell adhesion molecule-1 (VCAM1), intercellular adhesion molecule-1 (ICAM1), and E-selectin, and it amplified their induction upon TNF-alpha challenge. Pharmacological inhibition of IKKbeta; abolished this difference, placing SDC3 upstream of NF-kappaB, while SDC3 overexpression attenuated tumor necrosis factor-alpha (TNF-alpha)-induced VCAM1, the inverse of the knockdown effect. Loss of SDC3 further primed HAVECs toward a mesenchymal, EndMT-prone phenotype under transforming growth factor-beta 2 (TGF-beta2) stimulation, marked by increased smooth muscle protein 22-alpha (SM22alpha) and reduced platelet endothelial cell adhesion molecule (PECAM1/CD31). Together, these findings identify SDC3 as an endogenous restraint on the inflammatory and mesenchymal programs that characterize the endothelial phase of CAVD and establish the valve endothelial glycocalyx as a previously unappreciated yet potent regulator of aortic valve endothelial dysfunction. Preserving or augmenting SDC3 may warrant investigation as a means of holding valve endothelial cells in a quiescent state.

pathology↗

bioRxiv2025

Shear-Induced Macrophage Secretome Promotes Endothelial Permeability

BackgroundDiscrete subaortic stenosis (DSS) is a pediatric cardiovascular disease marked by fibrotic growth within the left ventricular outflow tract (LVOT), leading to severe complications, including left ventricular hypertrophy, aortic regurgitation, and arrhythmias. Despite surgical intervention, a 20-30% recurrence rate suggests a complex underlying pathophysiology. Elevated flow and resultant hemodynamic shear stress within the LVOT are key factors in DSS development. While effects of shear stress on endothelial cells have been studied, the impact on macrophages and their interactions with endothelial cells remains unclear. MethodsIn this study, human monocyte-derived macrophages (MDMs) and human aortic endothelial cells (HAECs) were subjected to shear using a cone-and-plate viscometer. Cellular crosstalk was evaluated through conditioned media (CM) transfers. Gene expression, permeability and chemotaxis assays, immunofluorescent staining, and ELISAs assessed cellular responses. ResultsMDMs exposed to shear stress exhibited a pro-inflammatory response with upregulated TNF and CXCL8 genes. HAECs exposed to MDM-CM showed increased expression of inflammatory markers (VCAM-1, ICAM-1) and decreased VE-Cadherin and CD31, indicating increased permeability. Permeability assays confirmed that HAECs became more permeable when exposed to MDM-CM. Chemotaxis assays showed time-dependent monocyte migration in both MDM-CM and HAEC-CM. Immunofluorescent staining revealed diminished VE-Cadherin and CD31 in HAECs exposed to MDM-CM. ConclusionsOverall, pathological shear stress induced macrophages to secrete factors that increased endothelial permeability and perpetuated an inflammatory response. This interaction likely exacerbates fibrosis in DSS, promoting recurrence post-surgery. Understanding these mechanisms opens potential therapeutic avenues targeting inflammatory crosstalk between macrophages and endothelial cells, which could mitigate fibrosis and improve patient outcomes.

bioengineering↗

bioRxiv2025

Shear Stress Conditioning Promotes a Pro-Inflammatory Response in Porcine Endocardial Endothelial Cells

PURPOSEDiscrete subaortic stenosis (DSS) is a congenital heart disease characterized by a narrowing of the passage below the aortic valve in the left ventricular outflow tract (LVOT) [1]. While endocardial endothelial cells (EECs) are known to play a role in DSS, the response of these cells to shear stress is not known. In this study, we hypothesize that the response of EECs to shear stress in the LVOT is a mediator of DSS. METHODSTo test this hypothesis, we conditioned porcine EECs to controlled shear stress regimes using cone and plate bioreactors. Subsequently, we quantified the concentration of proinflammatory cytokine in the conditioned media using the Luminex assay. Bulk-RNA sequencing was used to quantify changes in the genotype of the shear stress conditioned EECs. RESULTSThe expression of CD31 was knocked down and subsequently, the changes in release of shear stress induced proinflammatory cytokines released by EECs quantified using the Luminex assay. The results of these studies show that the inflammatory cytokines were highly selected in the conditioning medium, and under bioreactor treatment the cell activated the PI3K-AKT and TNF-a signaling, which also triggered the other immune cell responses though Th1, Th2 and Th17 cell differentiation pathways. Furthermore, CD31 was identified as a mediator of the pro-inflammatory response of shear stress conditioned EECs. CONCLUSIONSThe studies provide a clear link between shear stress, and the subsequent proinflammatory response of EECs as a mediator of DSS.

bioengineering↗

bioRxiv2025

Shear Stress Modulates Inflammatory Responses in Porcine Endothelial Cells Contributing to the Fibrotic Response of Porcine Cardiac Fibroblasts

This study is focused on discrete subaortic stenosis (DSS) which is a congenital heart disease resulting in the formation of fibrotic tissue in the left ventricular outflow track of pediatric patients [1]. The pathogenesis of the molecular mechanism resulting in the formation of the fibrotic tissue remains unknown and is the focus of this study. We hypothesized the cytokine released in response to wall shear stress act on the fibroblasts to promote the formation of the fibrotic membrane and subsequently, DSS. To test this study hypothesis, porcine endocardial endothelial cells were cultured within cone and plate bioreactors, designed to replicate the wall shear stress observed in the left ventricular outflow track of DSS patients. Conditioned media was collected as a function of shear stress magnitude and used to condition porcine cardiac fibroblasts. We used an extensive set of end-point metrics to characterize fibroblast phenotype to include bulk-RNA sequencing, RT-qPCR, cell viability and immunofluorescence. The results of this study demonstrate that shear stress-conditioned media from porcine EEC releases a defined cocktail of cytokines that provide the trigger to program the fibrotic response during DSS. These results provide specific molecular targets that can be developed into a therapeutic strategy for patients with DSS and provide a solution to an otherwise challenging disease to manage.

bioengineering↗

bioRxiv2024

Assessing Drivers of Gender Balance and Racial Makeup of Editorial Board Members in Biomedical Engineering

Scientific journal editors serve as gatekeepers with the decision-making power of assigning reviewers to manuscripts. Serving as an editor is also an important stage in a young academics career progression, and an indicator of high regard/acceptance within ones academic field. For both of these reasons, it is important to have representation with members of underrepresented groups serving in these roles. In this paper, we explore the gender and racial distribution among editorial boards for peer-reviewed scientific journals that are relevant to the field of biomedical engineering (BME). Further, we examine changes in these distributions from 2016 to 2021, amidst societal shifts catalyzed by movements such as #metoo and Black Lives Matter (BLM). Despite BMEs reputation for a relatively high percentage of female degree-earners, this study reveals stark disparities in gender and racial representation among editorial leadership positions. Through meticulous data collection and analysis of 75 BME journals-- including 44 for which data from both 2016 and 2021 were analyzed--it was found that while the proportion of female editors increased over time, this proportion consistently fell short of the expected standard, which was based on current BME degree awardee values. Moreover, the percentage of Black editors remained stagnant. Correlation analyses between gender, race, and changes in journal impact factor ({Delta}JIF) revealed nuanced trends, in some cases showing that increasing {Delta}JIFs were associated with decreasing proportions of female editors. The study underscores the urgent need for changes in social and policy standards to address gender and racial inequities in BME [editorial] leadership, both of which will be necessary to foster greater diversity and inclusivity throughout the field.

bioengineering↗

bioRxiv2024

Tunable Macroscopic Alignment of Self-Assembling Peptide Nanofibers

Fibrous proteins that comprise the extracellular matrix (ECM) guide cellular growth and tissue organization. A lack of synthetic strategies able to generate aligned, ECM-mimetic biomaterials has hampered bottom-up tissue engineering of anisotropic tissues and led to a limited understanding of cell-matrix interactions. Here, we present a facile extrusion-based fabrication method to produce anisotropic, nanofibrous hydrogels using self-assembling peptides. The application of shear force coinciding with ion-triggered gelation is used to kinetically trap supramolecular nanofibers into aligned, hierarchical structures. We establish how modest changes in phosphate buffer concentration during peptide self-assembly can be used to tune their alignment and packing. In addition, increases in the nanostructural anisotropy of fabricated hydrogels are found to enhance their strength and stiffness under hydrated conditions. To demonstrate their utility as an ECM-mimetic biomaterial, aligned nanofibrous hydrogels are used to guide directional spreading of multiple cell types, but strikingly, increased matrix alignment is not always correlated with increased cellular alignment. Nanoscale observations reveal differences in cell-matrix interactions between variably aligned scaffolds and implicate the need for mechanical coupling for cells to understand nanofibrous alignment cues. In total, innovations in the supramolecular engineering of self-assembling peptides allow us to generate a gradient of anisotropic nanofibrous hydrogels, which are used to better understand directed cell growth.

bioengineering↗

bioRxiv2023

Technique for rapidly forming networks of microvessel-like structures

Modelling organ-blood barriers through the inclusion of microvessel networks within in vitro tissue models could lead to more physiologically accurate results, especially since organ-blood barriers are crucial to the normal function, drug transport, and disease states of vascularized organs. Microvessel networks are difficult to form, since they push the practical limit of most fabrication methods, and it is difficult to coax vascular cells to self-assemble into structures larger than capillaries. Here we present a method for rapidly forming networks of microvessel-like structures using sacrificial, alginate structures. Specifically, we encapsulated endothelial cells within short alginate threads, then embedded them in collagen gel. Following enzymatic degradation of the alginate, the collagen gel contained a network of hollow channels seeded with cells, all surrounding a perfusable central channel. This method uses a 3D printed coaxial extruder and syringe pumps to generate short threads in a way that is repeatable and easily transferrable to other labs. The cell-laden, sacrificial alginate threads can be frozen after fabrication and thawed before embedding without significant loss of cell viability. The ability to freeze the threads enables future scale up and ease of use. Within millifluidic devices that restrict access to media, the threads enhance cell survival under static conditions. These results indicate the potential for use of this method in a range of tissue engineering applications. Impact StatementGenerating microvascular networks is a challenge in tissue engineering. Popular 3D bioprinting techniques use sacrificial structures to generate vascular networks, but those approaches can require expensive equipment and extensive troubleshooting. In this article, a method is presented for easy and rapid generation of centimeter-scale microvascular networks using endothelial cells frozen within sacrificial structures. The method for fabricating and freezing the sacrificial threads of alginate containing cells using a 3D printed coaxial extruder is also described. Experimental results indicate that these microstructures have the potential to enhance cell survival in environments with restricted access to media.

bioengineering↗

bioRxiv2023

Genomic and Proteomic Analysis of Patients with Reoccurring Discrete Subaortic Stenosis

Discrete subaortic stenosis (DSS) is a pediatric condition in which a fibrotic membrane forms within the left ventricular outflow track. The fibrotic membrane is removed surgically; however, there is a high rate of reoccurrence which requires a second surgery. There are currently no tools available to predict the risk of reoccurrence in DSS patients, a limitation addressed by this study. In this study, we analyzed resected fibrotic membranes for DSS patients at the time of first surgery for non-recurrent and recurrent patients, and at the time of second surgery for recurrent patients. RNA-sequencing was conducted to obtain a global screen of changes in RNA expression while mass spectrometry was used to obtain a global screen of changes in protein expression. The results from the RNA-sequencing and mass spectrometry provide valuable insight into genes and the proteins that are differentially regulated in recurrent vs non-recurrent DSS patients.

molecular biology↗

bioRxiv2022

Shear Stress Induces a Pro-Inflammatory Phenotype in Human Monocyte-Derived Macrophages

Macrophages are innate immune cells that are known for their extreme plasticity, enabling diverse phenotypes that lie on a continuum. In a simplified model, they switch between pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes depending on surrounding microenvironmental cues, which have been implicated in disease outcomes. Although considerable research has been focused on macrophage response to biochemical cues and mechanical signals, there is a scarcity of knowledge surrounding their behavior in response to shear stress. In this study, we applied varying magnitudes of shear stress on human monocyte-derived macrophages (MDMs) using a cone-and-plate viscometer and evaluated changes in morphology, gene expression, protein expression, and cytokine secretion over time. MDMs exposed to shear stress exhibited a rounder morphology compared to statically-cultured controls. RT-qPCR results showed significant upregulation of TNF-, and analysis of cytokine release revealed increased secretion of IL-8, IL-18, fractalkine, and other chemokines. The upregulation of pro-inflammatory factors was evident with both increasing magnitudes of shear and time. Taken together, these results indicate that prolonged shear exposure induced a pro-inflammatory phenotype in human MDMs. These findings have implications for medical technology development, such as in situ vascular graft design wherein macrophages are exposed to shear and have been shown to affect graft resorption, and in delineating disease pathophysiology, for example to further illuminate the role of macrophages in atherosclerosis where shear is directly related to disease outcome.

bioengineering↗

bioRxiv2022

Application of Physiological and Pathological Wall Shear Stress to Endocardial Endothelial Cells Triggers Complex Signaling Pathways

Discrete subaortic stenosis (DSS) is a congenital heart disease in which a fibrotic membrane forms below the aortic valve; the underlying cellular mechanisms are currently unknown. Since an elevated pressure gradient in the left ventricular outflow tract (LVOT) is a distinguishing feature of DSS, it is hypothesized that the membrane formation is caused by elevated wall shear stress applied to the endocardial endothelial cells (EECs) that line the LVOT, triggering fibrosis. To correlate shear stress to an EEC fibrotic phenotype, we applied fluid shear stress to EECs at physiological and pathological shear rates using a cone-and-plate device, designed to recapitulate physiological wall shear stress in a controlled in vitro environment. Controlled shear stress regimes were applied to EECs to replicate the conditions observed in DSS patients. We found that elevated shear stress triggered EEC alignment as well as endothelial-to-mesenchymal transformation (EndMT) signaling pathways driven by upregulation of SNAI1 gene expression. The EECs were then treated with a small molecule inhibitor of Snail1 protein, CYD19, to attempt to attenuate EndMT signaling, and subsequently subjected to pathological shear stress. The Snail1 inhibitor did downregulate selected markers of EndMT signaling, although only transiently. Interestingly, the application of shear stress had a greater effect on the EEC gene and protein expression than did the Snail1 inhibition. This investigation of EEC response to shear stress reveals the pronounced and complex effect of this mechanical stimulation on the EEC phenotype. Further study should reveal the mechanisms that drive fibrosis and the formation of the DSS membrane.

bioengineering↗

bioRxiv2022

Isolation and Characterization of Endocardial Endothelial Cells from the Left Ventricular Wall and Outflow Tract of the Porcine Heart

The heart contains six different types of endothelial cells, each with a unique function. We sought to characterize the endocardial endothelial cells (EECs), which line the chambers of the heart. EECs are relatively understudied, yet their dysregulation can lead to various cardiac pathologies. Due to the lack of commercial availability of this cell line, we developed a protocol for isolating EECs from porcine hearts and detailed our methodology for establishing populations of EECs through cell sorting. Additionally, we compared the EEC phenotype and fundamental behaviors to a well-studied endothelial cell line, human umbilical vein endothelial cells (HUVECs). The EECs were slightly smaller than HUVECs, and they stained positively for classic endothelial phenotypic markers such as CD31, von Willebrand Factor, and vascular endothelial (VE) cadherin. The EECs proliferated more quickly than HUVECs, yet migrated more slowly to cover a scratch wound assay. Finally, the EECs maintained their robust endothelial phenotype (expression of CD31) through more than a dozen passages. In contrast, the HUVECs showed significantly reduced CD31 expression in later passages. These important phenotypic differences between EECs and HUVECs highlight the need for researchers to utilize the most relevant cell lines when studying or modeling a disease of interest. Impact statementMany researchers model cardiovascular disease via tissue engineering to determine disease etiology on the cellular and molecular level. However, researchers usually rely on commercially-available cell lines, which can result in utilizing cells that are not specific to the region of the heart being modeled and could lead to incorrect conclusions. By providing a detailed protocol for isolating and purifying endocardial endothelial cells, we are enabling other researchers to access this cell line and model cardiovascular disease states more accurately.

pathology↗

bioRxiv2021

Effect of Substrate Stiffness on Human Intestinal Enteroids Infectivity by Enteroaggregative Escherichia coli

Human intestinal enteroids (HIE) models have contributed significantly to our understanding of diarrheal diseases and other intestinal infections, but their routine culture conditions fail to mimic the mechanical environment of the native intestinal wall. Because the mechanical characteristics of the intestine significantly alter how pathogens interact with the intestinal epithelium, we used different concentrations of polyethylene glycol (PEG) to generate soft ([~]2 kPa), medium ([~]10 kPa), and stiff ([~]100 kPa) hydrogel biomaterial scaffolds. The height of HIEs cultured in monolayers atop these hydrogels was 18 {micro}m whereas HIEs grown on rigid tissue culture surfaces (with stiffness in the GPa range) were 10 {micro}m. Substrate stiffness also influenced the amount of enteroaggregative E. coli (EAEC strain 042) adhered to the HIEs. We quantified a striking difference in adherence pattern; on the medium and soft gels, the bacteria formed clusters of >100 and even >1000 on both duodenal and jejunal HIEs (such as would be found in biofilms), but did not on glass slides and stiff hydrogels. All hydrogel cultured HIEs showed significant enrichment for gene and signaling pathways related to epithelial differentiation, cell junctions and adhesions, extracellular matrix, mucins, and cell signaling compared to the HIEs cultured on rigid tissue culture surfaces. Collectively, these results indicate that the HIE monolayers cultured on the hydrogels are primed for a robust engagement with their mechanical environment, and that the soft hydrogels promote the formation of larger EAEC aggregates, likely through an indirect differential effect on mucus. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/448533v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@f9c7b3org.highwire.dtl.DTLVardef@a5d5feorg.highwire.dtl.DTLVardef@184224forg.highwire.dtl.DTLVardef@1e8a28d_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

bioengineering↗

bioRxiv2019

A novel human enteroid-anaerobe co-culture system to study microbial-host interaction under physiological hypoxia

Mechanistic investigations of host-microbe interactions in the human gut are limited by current co-culture model systems. The intestinal epithelium requires oxygen for viability, while gut bacteria are facultative or obligate anaerobes. The ability to model host-commensal interactions under dynamic oxygen conditions is critical to understanding host-pathogen interactions in the human gut. Here, we demonstrate a simple, cost-effective method for co-culturing obligate anaerobic bacteria with human intestinal enteroid monolayers under variable oxygen conditions. The Enteroid-Anaerobe Co-Culture (EACC) system is able to recapitulate the steep oxygen gradient seen in vivo and induce expression of hypoxia-associated phenotypes such as increased barrier integrity and expression of antimicrobial peptide genes. Using clinical strains of the commensal anaerobes Bacteroides thetaiotaomicron and Blautia sp. on established patient-derived intestinal enteroid cell lines under physiological hypoxia, the EACC system can sustain host-anaerobe interactions for at least 24 hours. Following co-culture with anaerobic bacteria, we demonstrate patient-specific differences in epithelial response, reinforcing the potential to develop a personalized medicine approach to bacteriotherapy and host-microbe interaction investigations. Our innovative EACC system provides a robust model for investigating host-microbe interactions in complex, patient-derived intestinal tissues, that facilitates study of mechanisms underlying the role of the microbiome in health and disease.

bioengineering↗