A nasal airway-on-chip model to evaluate airflow pre-conditioning during epithelial cell maturation at the air-liquid interface.

The function of a well-differentiated nasal epithelium is largely affected by airflow-induced wall shear stress, yet few in vitro models recapitulate this dynamic condition. Models which do expose cells to airflow exclusively initiate flow after the differentiation process has occurred. In vivo, basal cells are constantly replenishing the epithelium under airflow conditions, indicating that airflow may affect the development and function of the differentiated epithelium. To address this gap in the field, we developed a physiologically relevant microphysiological model of the human nasal epithelium and investigated the effects of exposing cells to airflow during epithelial maturation at the air-liquid interface. The nasal airway-on-chip platform was engineered to mimic bi-directional physiological airflow during normal breathing. Primary human nasal epithelial cells were seeded on chips and subjected to either: 1) no flow, 2) single flow (0.5 dyne/cm2flow on Day 21 of ALI only), or 3) pre-conditioning flow (0.05 dyne/cm2on Days 14-20 and 0.5 dyne/cm2flow on Day 21) treatments. Cells exposed to pre-conditioning showed decreased morphological changes and mucus secretions, as well as a decreased inflammation, compared to unconditioned cells. Our results indicate that flow exposure only post-differentiation may impose acute stress on cells, while pre-conditioning may potentiate a properly functioning epithelium in vitro. .

ECMO PRODUCES VERY RAPID CHANGES IN PRIMARY HEMOSTASIS DETECTED BY PFA- 200 DURING LUNG TRANSPLANTATION: AN OBSERVATIONAL STUDY.

BACKGROUND: The pathology of primary hemostasis is a common complication of extracorporeal membrane oxygenation (ECMO) support. Scientific data describing its changes in patients on short-term ECMO support and the ability and speed of the restoration of its functions are limited. AIMS: The aim of this study was to describe the pathology of primary hemostasis induced by short-term ECMO support and its development over time using PFA-200®, ROTEM® platelet and von Willebrand factor (vWF) analyses. METHODS: In patients undergoing lung transplantation surgery using intra-operative veno-arterial ECMO support, blood samples were analyzed using the following tests: PFA-200®, ROTEM® platelet tests, vWF antigen, ristocetin cofactor (RCo) and collagen binding protein (CB) before, during and after ECMO support. RESULTS: Blood samples from 32 patients were analyzed. All three PFA-200® tests (COL/EPI, COL/ADP and COL/P2Y) showed significant deterioration during ECMO support with rapid restoration after ECMO cessation (p<0.05), suggesting an ECMO-induced primary hemostasis disorder. A significant increase of vWF antigen after ECMO cessation (p<0.05) was found with an increase of ristocetin cofactor and collagen binding protein levels, although it was not significant (p>0.05). CONCLUSIONS: Short-term ECMO support induces primary hemostasis pathology. It occurs immediately after initiation but is rapidly restored after ECMO cessation, which is detectable by PFA-200®. Despite there being persistent platelet dysfunction after ECMO cessation as seen with the ROTEM® platelet results, the increased levels of vWF antigen might explain the normal results of primary hemostasis detected by PFA-200®.

Hydrogel-Integrated Millifluidic Systems: Advancing the Fabrication of Mucus-Producing Human Intestinal Models.

The luminal surface of the intestinal epithelium is protected by a vital mucus layer, which is essential for lubrication, hydration, and fostering symbiotic bacterial relationships. Replicating and studying this complex mucus structure in vitro presents considerable challenges. To address this, we developed a hydrogel-integrated millifluidic tissue chamber capable of applying precise apical shear stress to intestinal models cultured on flat or 3D structured hydrogel scaffolds with adjustable stiffness. The chamber is designed to accommodate nine hydrogel scaffolds, 3D-printed as flat disks with a storage modulus matching the physiological range of intestinal tissue stiffness (~3.7 kPa) from bioactive decellularized and methacrylated small intestinal submucosa (dSIS-MA). Computational fluid dynamics simulations were conducted to confirm a laminar flow profile for both flat and 3D villi-comprising scaffolds in the physiologically relevant regime. The system was initially validated with HT29-MTX seeded hydrogel scaffolds, demonstrating accelerated differentiation, increased mucus production, and enhanced 3D organization under shear stress. These characteristic intestinal tissue features are essential for advanced in vitro models as they critically contribute to a functional barrier. Subsequently, the chamber was challenged with human intestinal stem cells (ISCs) from the terminal ileum. Our findings indicate that biomimicking hydrogel scaffolds, in combination with physiological shear stress, promote multi-lineage differentiation, as evidenced by a gene and protein expression analysis of basic markers and the 3D structural organization of ISCs in the absence of chemical differentiation triggers. The quantitative analysis of the alkaline phosphatase (ALP) activity and secreted mucus demonstrates the functional differentiation of the cells into enterocyte and goblet cell lineages. The millifluidic system, which has been developed and optimized for performance and cost efficiency, enables the creation and modulation of advanced intestinal models under biomimicking conditions, including tunable matrix stiffness and varying fluid shear stresses. Moreover, the readily accessible and scalable mucus-producing cellular tissue models permit comprehensive mucus analysis and the investigation of pathogen interactions and penetration, thereby offering the potential to advance our understanding of intestinal mucus in health and disease.

In silico model of stent performance in multi-layered artery using 2-way fluid-structure interaction: Influence of boundary conditions and vessel length.

BACKGROUND AND OBJECTIVE: Atherosclerotic lesions of coronary arteries (stenosis) are caused by the buildup of lipids and blood-borne substances within the artery wall. Their qualitative and rapid assessment is still a challenging task. The primary therapy for this pathology involves implanting coronary stents, which help to restore the blood flow in atherosclerosis-prone arteries. In-stent restenosis is a stenting-procedure complication detected in about 10-40% of patients. A numerical study using 2-way fluid-structure interaction (FSI) assesses the stenting procedure quality and can decrease the number of negative post-operative results. Nevertheless, boundary conditions (BCs) used in simulation play a crucial role in implementation of an adequate computational analysis. METHODS: Three CoCr stents designs were modelled with the suggested approach. A multi-layer structure describing the artery and plaque with anisotropic hyperelastic mechanical properties was adopted in this study. Two kinds of boundary conditions for a solid domain were examined - fixed support (FS) and remote displacement (RD) - to assess their impact on the hemodynamic parameters to predict restenosis. Additionally, the influence of artery elongation (short-artery model vs. long-artery model) on numerical results with the FS boundary condition was analyzed. RESULTS: The comparison of FS and RD boundary conditions demonstrated that the variation of hemodynamic parameters values did not exceed 2%. The analysis of short-artery and long-artery models revealed that the difference in hemodynamic parameters was less than 5.1%, and in most cases, it did not exceed 2.5%. The RD boundary conditions were found to reduce the computation time by up to 1.7-2.0 times compared to FS. Simple stent model was shown to be susceptible to restenosis development, with maximum WSS values equal to 183 Pa, compared to much lower values for other two stents. CONCLUSIONS: The study revealed that the stent design significantly affected the hemodynamic parameters as restenosis predictors. Moreover, the stress-strain state of the system artery-plaque-stent also depends on a proper choice of boundary conditions.

Effect of cortical bone thickness on shear stress and force in orthodontic miniscrew-bone interface - A finite element analysis.

Miniscrews are widely used in orthodontics as an anchorage device while aligning teeth. Shear stress in the miniscrew-bone interface is an important factor when the miniscrew makes contact with the bone. The objective of this study was to analyze the shear stress and force in the screw-bone interface for varying Cortical Bone Thickness (CBT) using Finite Element Analysis (FEA). Varying CBT of 1.09 mm (1.09CBT) and 2.66 mm (2.66CBT) with miniscrews of Ø1.2 mm, 10 mm length (T1), Ø1.2 mm, 6 mm length (T2) and Ø1.6 mm, 8 mm length (T3) were analyzed. Six Finite Element (FE) models were developed with cortical, cancellous bone, miniscrews and gingiva as a prism. A deflection of 0.1 mm was applied on the neck of the miniscrews at 0°, +30° and -30° angles. The shear stress and force in the screw-bone interface were assessed. The results showed that the CBT affects the shear stress and force in the screw-bone interface region in addition to the screw dimensions and deflection angulations. T1 screw generated lesser shear stress in 1.09CBTand 2.66CBTcompared to T2 and T3 screws. Higher CBT is preferred for better primary stability in shear aspect. Clinically applied forces of 200 gms to 300 gms to an anchorage device induces shear stress in the miniscrew-bone interface region might cause stress shielding. Thus, clinicians need to consider the effect of varying CBT and the size of the miniscrews for the stability, reduced stress shielding and better anchorage during orthodontic treatment.

Optimized lung expansion ventilation modulates ventilation-induced lung injury in preterm lambs.

INTRODUCTION: Preterm infants close to viability commonly require mechanical ventilation (MV) for respiratory distress syndrome. Despite commonly used lung-sparing ventilation techniques, rapid lung expansion during MV induces lung injury, a risk factor for bronchopulmonary dysplasia. This study investigates whether ventilation with optimized lung expansion is feasible and whether it can further minimize lung injury. Therefore, optimized lung expansion ventilation (OLEV) was compared to conventional volume targeted ventilation. METHODS: Twenty preterm lambs were surgically delivered after 132 days of gestation. Nine animals were randomized to receive OLEV for 24 h, and seven received standard MV. Four unventilated animals served as controls (NV). Lungs were sampled for histological analysis at the end of the experimental period. RESULTS: Ventilation with OLEV was feasible, resulting in a significantly higher mean ventilation pressure (0.7-1.3 mbar). Temporary differences in oxygenation between OLEV and MV did not reach clinically relevant levels. Ventilation in general tended to result in higher lung injury scores compared to NV, without differences between OLEV and MV. While pro-inflammatory tumor necrosis factor-α messenger RNA (mRNA) levels increased in both ventilation groups compared to NV, only animals in the MV group showed a higher number of CD45-positive cells in the lung. In contrast, mean (standard deviations) surfactant protein-B mRNA levels were significantly lower in OLEV, 0.63 (0.38) compared to NV 1.03 (0.32) (p = .023, one-way analysis of variance). CONCLUSION: In conclusion, a small reduction in pulmonary inflammation after 24 h of support with OLEV suggests potential to reduce preterm lung injury.

Shear-Sensitive circRNA-LONP2 Promotes Endothelial Inflammation and Atherosclerosis by Targeting NRF2/HO1 Signaling.

Hemodynamic shear stress is a frictional force that acts on vascular endothelial cells and is essential for endothelial homeostasis. Physiological laminar shear stress (LSS) suppresses endothelial inflammation and protects arteries from atherosclerosis. Herein, we screened differentially expressed circular RNAs (circRNAs) that were significantly altered in LSS-stimulated endothelial cells and found that circRNA-LONP2 was involved in modulating the flow-dependent inflammatory response. Furthermore, endothelial circRNA-LONP2 overexpression promoted endothelial inflammation and atherosclerosis in vitro and in vivo. Mechanistically, circRNA-LONP2 competitively sponged miR-200a-3p and subsequently promoted Kelch-like ECH-associated protein 1, Yes-associated protein 1, and enhancer of zeste homolog 2 expression, thereby inactivating nuclear factor erythroid 2-related factor 2/heme oxygenase-1 signaling, promoting oxidative stress and endothelial inflammation, and accelerating atherosclerosis. LSS-induced down-regulation of circRNA-LONP2 suppresses endothelial inflammation, at least in part, by activating the miR-200a-3p-mediated nuclear factor erythroid 2-related factor 2/heme oxygenase-1 signaling pathway. CircRNA-LONP2 may serve as a new therapeutic target for atherosclerosis.

Decoupling shear stress and pressure effects in the biomechanics of autosomal dominant polycystic kidney disease using a perfused kidney-on-chip.

Kidney tubular cells are submitted to two distinct mechanical forces generated by the urine flow: shear stress and hydrostatic pressure. In addition, the mechanical properties of the surrounding extracellular matrix modulate tubule deformation under constraints. These mechanical factors likely play a role in the pathophysiology of kidney diseases as exemplified by autosomal dominant polycystic kidney disease, in which pressure, flow and matrix stiffness have been proposed to modulate the cystic dilation of tubules with PKD1 mutations. The lack of in vitro systems recapitulating the mechanical environment of kidney tubules impedes our ability to dissect the role of these mechanical factors. Here we describe a perfused kidney-on-chip with tunable extracellular matrix mechanical properties and hydrodynamic constraints, that allows a decoupling of shear stress and flow. We used this system to dissect how these mechanical cues affect Pkd1 -/- tubule dilation. Our results show two distinct mechanisms leading to tubular dilation. For PCT cells (proximal tubule), overproliferation mechanically leads to tubular dilation, regardless of the mechanical context. For mIMCD-3 cells (collecting duct), tube dilation is associated with a squamous cell morphology but not with overproliferation and is highly sensitive to extracellular matrix properties and hydrodynamic constraints. Surprisingly, flow alone suppressed Pkd1 -/- mIMCD-3 tubule dilation observed in static conditions, while the addition of luminal pressure restored it. Our in vitro model emulating nephron geometrical and mechanical organization sheds light on the roles of mechanical constraints in ADPKD and demonstrates the importance of controlling intraluminal pressure in kidney tubule models.

Krüppel-like Factor 2 is an endo-protective transcription factor in diabetic kidney disease.

Diabetic kidney disease (DKD) is a microvascular complication of diabetes, and glomerular endothelial cell (GEC) injury is a key driver of DKD pathogenesis. Krüppel-like factor 2 (KLF2), a shear stress-induced transcription factor, was identified among the genes that are highly upregulated in early DKD. In the kidney, KLF2 expression is mostly restricted to endothelial cells, but its expression is also found in immune cell subsets. KLF2 expression is upregulated in response to increased shear stress by the activation of mechanosensory receptors, but suppressed by inflammatory cytokines, both of which characterize the early diabetic kidney milieu. KLF2 expression is reduced in progressive DKD and hypertensive nephropathy in humans and mice, likely due to high glucose and inflammatory cytokines such as TNF-α. However, KLF2 expression is increased in settings of glomerular hyperfiltration-induced shear stress without metabolic dysregulation, such as in settings of unilateral nephrectomy. Lower KLF2 expression is associated with CKD progression in patients with unilateral nephrectomy, consistent with its endoprotective role. KLF2 confers endoprotection by inhibition of inflammation, thrombotic activation, and angiogenesis, and thus KLF2 is considered a protective factor for cardiovascular disease (CVD). Based on similar mechanisms, KLF2 also exhibits renoprotection, and its reduced expression in endothelial cells worsens glomerular injury and albuminuria in settings of diabetes or unilateral nephrectomy. Thus, KLF2 confers endo-protective effects in both CVD and DKD, and its agonists could be potentially developed as a novel class of drugs for cardio-renal protection in diabetic patients.

The relation between aortic morphology and transcatheter aortic heart valve thrombosis: Particle tracing and platelet activation in larger aortic roots with and without neo-sinus.

Transcatheter aortic heart valve thrombosis (THVT) affects long-term valve durability, transvalvular pressure gradient and leaflet mobility. In this study, we conduct high-fidelity fluid-structure interaction simulations to perform Lagrangian particle tracing in a generic model with larger aortic diameters (THVT model) with and without neo-sinus which is compared to a model of unaffected TAVI patients (control model). Platelet activation indices are computed for each particle to assess the risk of thrombus formation induced by high shear stresses followed by flow stagnation. Particle tracing indicates that fewer particles contribute to sinus washout of the THVT model with and without neo-sinus compared to the control model (-34.9%/-34.1%). Stagnating particles in the native sinus of the THVT model show higher platelet activation indices than for the control model (+39.6% without neo-sinus, +45.3% with neo-sinus). Highest activation indices are present for particles stagnating in the neo-sinus of the larger aorta representing THVT patients (+80.2% compared to control). This fluid-structure interaction (FSI) study suggests that larger aortas lead to less efficient sinus washout in combination with higher risk of platelet activation among stagnating particles, especially within the neo-sinus. This could explain (a) a higher occurrence of thrombus formation in transcatheter valves compared to surgical valves without neo-sinus and (b) the neo-sinus as the prevalent region for thrombi in TAV. Pre-procedural identification of larger aortic roots could contribute to better risk assessment of patients and improved selection of a patient-specific anti-coagulation therapy.

A Microphysiological HHT-on-a-Chip Platform Recapitulates Patient Vascular Lesions.

Hereditary Hemorrhagic Telangiectasia (HHT) is a rare congenital disease in which fragile vascular malformations (VM) - including small telangiectasias and large arteriovenous malformations (AVMs) - focally develop in multiple organs. There are few treatment options and no cure for HHT. Most HHT patients are heterozygous for loss-of-function mutations affecting Endoglin (ENG) or Alk1 (ACVRL1); however, why loss of these genes manifests as VMs remains poorly understood. To complement ongoing work in animal models, we have developed a fully human, cell-based microphysiological model based on our Vascularized Micro-organ (VMO) platform (the HHT-VMO) that recapitulates HHT patient VMs. Using inducible ACVRL1 -knockdown, we control timing and extent of endogenous Alk1 expression in primary human endothelial cells (EC). Resulting HHT-VMO VMs develop over several days. Interestingly, in chimera experiments AVM-like lesions can be comprised of both Alk1-intact and Alk1-deficient EC, suggesting possible cell non-autonomous effects. Single cell RNA sequencing data are consistent with microvessel pruning/regression as contributing to AVM formation, while loss of PDGFB implicates mural cell recruitment. Finally, lesion formation is blocked by the VEGFR inhibitor pazopanib, mirroring positive effects of this drug in patients. In summary, we have developed a novel HHT-on-a-chip model that faithfully reproduces HHT patient lesions and that can be used to better understand HHT disease biology and identify potential new HHT drugs.

The Ramus Intermedius: A Bridge to Survival in the Setting of Triple-Vessel Total Occlusion.

Coronary artery disease continues to remain the leading cause of mortality worldwide. Coronary blood supply is provided through the right and left main coronary arteries. The left main coronary artery (LMCA) in turn gives rise to the left anterior descending (LAD) and left circumflex (LCX) arteries. In some cases, LMCA may trifurcate into the ramus intermedius (RI) in addition to the LAD and LCX arteries. Atherosclerotic plaque formation and rupture with subsequent clot formation and occlusion of coronary arteries are the underlying mechanisms of myocardial infarction. Though the clinical implications of the presence of ramus intermedius (RI) are controversial some data suggest that the RI is associated with an increased risk of atherosclerotic plaque formation in the LMCA and the proximal LAD. Conversely, it has been proposed that the RI provides an additional collateral source of blood supply to the myocardium and may potentially contribute to improved survival. Case reports tout the benefits of RI, specifically in the setting of multivessel coronary artery occlusions. Whether it increases the risk of atherosclerotic plaque formation or whether it is protective has yet to be determined. We present a case of a 58-year-old male who presented with acute coronary syndrome and cardiogenic shock due to total ostial occlusion of LAD. The patient had also chronic total occlusions of the right coronary artery and LCX but a patent RI, which was the only source of blood supply to the myocardium and practically determined the patient's survival. Additionally, we performed a literature review to identify similar cases, to support RI's potentially protective role in enhancing survival.

Temperature-responsive hydrogel-grafted vessel-on-a-chip: Exploring cold-induced endothelial injury.

Cold-induced vasoconstriction is a significant contributor that leads to chilblains and hypothermia in humans. However, current animal models have limitations in replicating cold-induced acral injury due to their low sensitivity to cold. Moreover, existing in vitro vascular chips composed of endothelial cells and perfusion systems lack temperature responsiveness, failing to simulate the vasoconstriction observed under cold stress. This study presents a novel approach where a microfluidic bioreactor of vessel-on-a-chip was developed by grafting the inner microchannel surface of polydimethylsiloxane with a thermosensitive hydrogel skin composed of N-isopropyl acrylamide and gelatin methacrylamide. With a lower critical solution temperature set at 30°C, the gel layer exhibited swelling at low temperatures, reducing the flow rate inside the channel by 10% when the temperature dropped from 37°C to 4°C. This well mimicked the blood stasis observed in capillary vessels in vivo. The vessel-on-a-chip was further constructed by culturing endothelial cells on the surface of the thermosensitive hydrogel layer, and a perfused medium was introduced to the cells to provide a physiological shear stress. Notably, cold stimulation of the vessel-on-a-chip led to cell necrosis, mitochondrial membrane potential (ΔΨm) collapse, cytoskeleton disaggregation, and increased levels of reactive oxygen species. In contrast, the static culture of endothelial cells showed limited response to cold exposure. By faithfully replicating cold-induced endothelial injury, this groundbreaking thermosensitive vessel-on-a-chip technology offers promising advancements in the study of cold-induced cardiovascular diseases, including pathogenesis and therapeutic drug screening.

Simulation and Modeling of the Adhesion of Staphylococcus aureus onto Inert Surfaces under Fluid Shear Stress.

Bacterial adhesion to biotic and abiotic surfaces under fluid shear stress plays a major role in the pathogenesis of infections linked to medical implants and tissues. This study employed an automated BioFlux 200 microfluidic system and video microscopy to conduct real-time adhesion assays, examining the influence of shear stress on adhesion kinetics and spatial distribution of Staphylococcus aureus on glass surfaces. The adhesion rate exhibited a non-linear relationship with shear stress, with notable variations at intermediate levels. Empirical adhesion events were simulated with COMSOL Multiphysics® and Python. Overall, COMSOL accurately predicted the experimental trend of higher rates of bacterial adhesion with decreasing shear stress but poorly characterized the plateauing phenomena observed over time. Python provided a robust mathematical representation of the non-linear relationship between cell concentration, shear stress, and time but its polynomial regression approach was not grounded on theoretical physical concepts. These insights, combined with advancements in AI and machine learning, underscore the potential for synergistic computational techniques to enhance our understanding of bacterial adhesion to surfaces, offering a promising avenue for developing novel therapeutic strategies.

A new method for scaling inlet flow waveform in hemodynamic analysis of aortic dissection.

Computational fluid dynamics (CFD) simulations have shown great potentials in cardiovascular disease diagnosis and postoperative assessment. Patient-specific and well-tuned boundary conditions are key to obtaining accurate and reliable hemodynamic results. However, CFD simulations are usually performed under non-patient-specific flow conditions due to the absence of in vivo flow and pressure measurements. This study proposes a new method to overcome this challenge by tuning inlet boundary conditions using data extracted from electrocardiogram (ECG). Five patient-specific geometric models of type B aortic dissection were reconstructed from computed tomography (CT) images. Other available data included stoke volume (SV), ECG, and 4D-flow magnetic resonance imaging (MRI). ECG waveforms were processed to extract patient-specific systole to diastole ratio (SDR). Inlet boundary conditions were defined based on a generic aortic flow waveform tuned using (1) SV only, and (2) with ECG and SV (ECG + SV). 4D-flow MRI derived inlet boundary conditions were also used in patient-specific simulations to provide the gold standard for comparison and validation. Simulations using inlet flow waveform tuned with ECG + SV not only successfully reproduced flow distributions in the descending aorta but also provided accurate prediction of time-averaged wall shear stress (TAWSS) in the primary entry tear (PET) and abdominal regions, as well as maximum pressure difference, ∆Pmax, from the aortic root to the distal false lumen. Compared with simulations with inlet waveform tuned with SV alone, using ECG + SV in the tuning method significantly reduced the error in false lumen ejection fraction at the PET (from 149.1% to 6.2%), reduced errors in TAWSS at the PET (from 54.1% to 5.7%) and in the abdominal region (from 61.3% to 11.1%), and improved ∆Pmax prediction (from 283.1% to 18.8%) However, neither of these inlet waveforms could be used for accurate prediction of TAWSS in the ascending aorta. This study demonstrates the importance of SDR in tailoring inlet flow waveforms for patient-specific hemodynamic simulations. A well-tuned flow waveform is essential for ensuring that the simulation results are patient-specific, thereby enhancing the confidence and fidelity of computational tools in future clinical applications.

A cross sectional pilot study utilising STrain Analysis and Mapping of the Plantar Surface (STAMPS) to measure plantar load characteristics within a healthy population.

BACKGROUND: No in-shoe systems, measuring both components of plantar load (plantar pressure and shear stress) are available for use in patients with diabetes. The STAMPS (STrain Analysis and Mapping of the Plantar Surface) system utilises digital image correlation (DIC) to determine the strain sustained by a deformable insole, providing a more complete understanding of plantar shear load at the foot-surface interface. RESEARCH QUESTIONS: What is the normal range and pattern of strain at the foot-surface interface within a healthy population as measured by the STAMPS system? Is STAMPS a valid tool to measure the effects of plantar load? METHODS: A cross-sectional study of healthy participants was undertaken. Healthy adults without foot pathology or diabetes were included. Participants walked 20 steps with the STAMPS insole in a standardised shoe. Participants also walked 10 m with the Novel Pedar® plantar pressure measurement insole within the standardised shoe. Both measurements were repeated three times. Outcomes of interest were global and regional values for peak resultant strain (SMAG) and peak plantar pressure (PPP). RESULTS: In 18 participants, median peak SMAG and PPP were 35.01 % and 410.6kPa respectively. The regions of the hallux and heel sustained the highest SMAG (29.31 % (IQR 24.56-31.39) and 20.50 % (IQR 15.59-24.12) respectively) and PPP (344.8kPa (IQR 268.3 - 452.5) and 279.3kPa (IQR 231.3-302.1) respectively). SMAG was moderately correlated with PPP (r= 0.65, p < 0.001). Peak SMAG was located at the hallux in 55.6 % of participants, at the 1st metatarsal head (MTH) in 16.7 %, the heel in 16.7 %, toes 3-5 in 11.1 % and the MTH2 in 5.6 %. SIGNIFICANCE: The results demonstrate the STAMPS system is a valid tool to measure plantar strain. Further studies are required to investigate the effects of elevated strain and the relationship with diabetic foot ulcer formation.

Mechanosensitive FHL2 tunes endothelial function.

Endothelial tissues are essential mechanosensors in the vasculature and facilitate adaptation to various blood flow-induced mechanical cues. Defects in endothelial mechanoresponses can perturb tissue remodelling and functions leading to cardiovascular disease progression. In this context, the precise mechanisms of endothelial mechanoresponses contributing to normal and diseased tissue functioning remain elusive. Here, we sought to uncover how flow-mediated transcriptional regulation drives endothelial mechanoresponses in healthy and atherosclerotic-prone tissues. Using bulk RNA sequencing, we identify novel mechanosensitive genes in response to healthy unidirectional flow (UF) and athero-prone disturbed flow (DF). We find that the transcription as well as protein expression of Four-and-a-half LIM protein 2 (FHL2) are enriched in athero-prone DF both in vitro and in vivo. We then demonstrate that the exogenous expression of FHL2 is necessary and sufficient to drive discontinuous adherens junction morphology and increased tissue permeability. This athero-prone phenotype requires the force-sensitive binding of FHL2 to actin. In turn, the force-dependent localisation of FHL2 to stress fibres promotes microtubule dynamics to release the RhoGEF, GEF-H1, and activate the Rho-ROCK pathway. Thus, we unravelled a novel mechanochemical feedback wherein force-dependent FHL2 localisation promotes hypercontractility. This misregulated mechanoresponse creates highly permeable tissues, depicting classic hallmarks of atherosclerosis progression. Overall, we highlight crucial functions for the FHL2 force-sensitivity in tuning multi-scale endothelial mechanoresponses.

Identification of a subpopulation of highly adherent endothelial cells for seeding synthetic vascular grafts.

OBJECTIVE: There is an unmet clinical need for alternatives to autologous vessel grafts. Small-diameter (<6mm) synthetic vascular grafts are not suitable because of unacceptable patency rates. This mainly occurs due to the lack of an endothelial cell (EC) monolayer to prevent platelet activation, thrombosis, and intimal hyperplasia. There are no reliable methods to endothelialize small-diameter grafts, as most seeded ECs are lost due to exposure to fluid shear stress (SS) after implantation. The goal of this work is to determine if EC loss is a random process or if it is possible to predict which cells are more likely to remain adherent. METHODS: In initial studies, we sorted ECs using fluid SS and identified a subpopulation of ECs that are more likely to resist detachment. We use RNA-sequencing (RNA-seq) to examine gene expression of adherent ECs compared to the whole population. Using fluorescence activated cell sorting (FACS), we sorted ECs based on the expression level of a candidate marker and studied their retention in small-diameter vascular grafts in vitro. RESULTS: Transcriptomic analysis revealed that fibronectin leucine rich transmembrane protein 2 (FLRT2), encoding protein FLRT2, is downregulated in the ECs that are more likely to resist detachment. When seeded onto vascular grafts and exposed to SS, ECs expressing low levels of FLRT2 exhibit 59.2±7.4% retention compared to 24.5±6.1% retention for the remainder of the EC population. CONCLUSIONS: For the first time, we show EC detachment is not an entirely random process. This provides validation for the concept that we can seed small-diameter vascular grafts only with highly adherent ECs to maintain a stable endothelium and improve graft patency rates.

Fluid shear stress-mediated Piezo1 alleviates osteocyte apoptosis by activating the PI3K/Akt pathway.

Glucocorticoid-induced osteoporosis serves as a primary cause for secondary osteoporosis and fragility fractures, representing the most prevalent adverse reaction associated with prolonged glucocorticoid use. In this study, to elucidate the impact and underlying mechanisms of fluid shear stress (FSS)-mediated Piezo1 on dexamethasone (Dex)-induced apoptosis, we respectively applied Dex treatment for 6 h, FSS at 9 dyne/cm2 for 30 min, Yoda1 treatment for 2 h, and Piezo1 siRNA transfection to intervene in MLO-Y4 osteocytes. Western blot analysis was used to assess the expression of Cleaved Caspase-3, Bax, Bcl-2, and proteins associated with the PI3K/Akt pathway. Additionally, qRT-PCR was utilized to quantify the mRNA expression levels of these molecules. Hoechst 33258 staining and flow cytometry were utilized to evaluate the apoptosis levels. The results indicate that FSS at 9 dyne/cm2 for 30 min significantly upregulates Piezo1 in osteocytes. Following Dex-induced apoptosis, the phosphorylation levels of PI3K and Akt are markedly suppressed. FSS-mediated Piezo1 exerts a protective effect against Dex-induced apoptosis by activating the PI3K/Akt pathway. Additionally, downregulating the expression of Piezo1 in osteocytes using siRNA exacerbates Dex-induced apoptosis. To further demonstrate the role of the PI3K/Akt signaling pathway, after intervention with the PI3K pathway inhibitor, the activation of the PI3K/Akt pathway by FSS-mediated Piezo1 in osteocytes was significantly inhibited, reversing the anti-apoptotic effect. This study indicates that under FSS, Piezo1 in MLO-Y4 osteocytes is significantly upregulated, providing protection against Dex-induced apoptosis through the activation of the PI3K/Akt pathway.