Biology subjects

Keswani, S.

Publications and source records attributed to Keswani, S..

5 recordsLinked to original sources

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↗

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↗

bioRxiv2020

Filamentous Bacteriophage Delay Healing of Pseudomonas-Infected Wounds

We have identified a novel role for filamentous bacteriophage in the delayed healing associated with chronic Pseudomonas aeruginosa (Pa) wound infections. Pf phage delays wound re-epithelialization in the absence of live Pa, indicative that Pf effects on wound healing are independent of Pa pathogenesis. Pf phage directly inhibits autocrine signaling of CXCL1 (KC) to impede keratinocyte migration and wound re-epithelization. In agreement with these studies, a prospective cohort study of 36 human patients with chronic Pa wound infections revealed that wounds infected with Pf positive strains of Pa took longer to heal and were more likely to increase in size compared to wounds infected with Pf negative strains. Together, these data implicate Pf phage in the delayed wound healing associated with Pa infection through direct manipulation of mammalian target cells. We propose that Pf phage may have potential as a biomarker and therapeutic target in delayed wound healing.

microbiology↗