Fetal membrane (amniochorion), the innermost lining of the intrauterine cavity, surround the fetus and enclose amniotic fluid. Unlike unidirectional blood flow, amniotic fluid subtly rocks back and forth, and thus, the innermost amnion epithelial cells are continuously exposed to low levels of shear stress from fluid undulation. Here, we tested the impact of fluid motion on amnion epithelial cells (AECs) as a bearer of force impact and their potential vulnerability to cytopathologic changes that can destabilize fetal membrane functions. A previously developed amnion membrane (AM) organ-on-chip (OOC) was utilized but with dynamic flow to culture human fetal amnion membrane cells. The applied flow was modulated to perfuse culture media back and forth for 48 h to mimic fluid motion. A static culture condition was used as a negative control, and oxidative stress (OS) condition was used as a positive control representing pathophysiological changes. The impacts of fluidic motion were evaluated by measuring cell viability, cellular transition, and inflammation. Additionally, scanning electron microscopy (SEM) imaging was performed to observe microvilli formation. The results show that regardless of the applied flow rate, AECs and AMCs maintained their viability, morphology, innate meta-state, and low production of pro-inflammatory cytokines. E-cadherin expression and microvilli formation in the AECs were upregulated in a flow rate-dependent fashion; however, this did not impact cellular morphology or cellular transition or inflammation. OS treatment induced a mesenchymal morphology, significantly higher vimentin to cytokeratin 18 (CK-18) ratio, and pro-inflammatory cytokine production in AECs, whereas AMCs did not respond in any significant manner. Fluid motion and shear stress, if any, did not impact AEC cell function and did not cause inflammation. Thus, when using an amnion membrane OOC model, the inclusion of a dynamic flow environment is not necessary to mimic in utero physiologic cellular conditions of an amnion membrane.

BACKGROUND: 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).
CONCLUSIONS: In conclusion, a small reduction in pulmonary inflammation after 24 h of support with OLEV suggests potential to reduce preterm lung injury.

Proximal tubule (PT) cells maintain a high-capacity apical endocytic pathway to recover essentially all proteins that escape the glomerular filtration barrier. The multi ligand receptors megalin and cubilin play pivotal roles in the endocytic uptake of normally filtered proteins in PT cells but also contribute to the uptake of nephrotoxic drugs, including aminoglycosides. We previously demonstrated that opossum kidney (OK) cells cultured under continuous fluid shear stress (FSS) are superior to cells cultured under static conditions in recapitulating essential functional properties of PT cells in vivo. To identify drivers of the high-capacity, efficient endocytic pathway in the PT, we compared FSS-cultured OK cells with less endocytically active static-cultured OK cells. Megalin and cubilin expression are increased, and endocytic uptake of albumin in FSS-cultured cells is > 5-fold higher compared with cells cultured under static conditions. To understand how differences in receptor expression, distribution, and trafficking rates contribute to increased uptake, we used biochemical, morphological, and mathematical modeling approaches to compare megalin traffic in FSS- versus static-cultured OK cells. Our model predicts that culturing cells under FSS increases the rates of all steps in megalin trafficking. Importantly, the model explains why, despite seemingly counterintuitive observations (a reduced fraction of megalin at the cell surface, higher colocalization with lysosomes, and a shorter half-life of surface-tagged megalin in FSS-cultured cells), uptake of albumin is dramatically increased compared with static-grown cells. We also show that FSS-cultured OK cells more accurately exhibit the mechanisms that mediate uptake of nephrotoxic drugs in vivo compared with static-grown cells. This culture model thus provides a useful platform to understand drug uptake mechanisms, with implications for developing interventions in nephrotoxic injury prevention.

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.

Mesenchymal Stem Cells, mesodermal origin and multipotent stem cells, have ability to differentiate into vascular endothelial cells. The cells are squamous in morphology, inlining, and protecting blood vessel tissue, as well as maintaining homeostatic conditions. ECs are essential in vascularization and blood vessels formation. The differentiation process, generally carried out in 2D culture systems, were relied on growth factors induction. Therefore, an artificial extracellular matrix with relevant mechanical properties is essential to build 3D culture models. Various 3D fabrication techniques, such as hydrogel-based and fibrous scaffolds, scaffold-free, and co-culture to endothelial cells were reviewed and summarized to gain insights. The obtained MSCs-derived ECs are shown by the expression of endothelial gene markers and tubule-like structure. In order to mimicking relevant vascular tissue, 3D-bioprinting facilitates to form more complex microstructures. In addition, a microfluidic chip with adequate flow rate allows medium perfusion, providing mechanical cues like shear stress to the artificial vascular vessels.

UNASSIGNED: Pulmonary hypertension (PH) is a refractory disease characterized by elevated pulmonary artery pressure and resistance. Drag-reducing polymers (DRPs) are blood-soluble macromolecules that reduce vascular resistance by altering the blood dynamics and rheology. Our previous work indicated that polyethylene oxide (PEO) can significantly reduce the medial wall thickness and vascular resistance of the pulmonary arteries, but the specific mechanism is still unclear.
UNASSIGNED: This study was designed to investigate the role and mechanism of PEO on intracellular calcium [Ca2 +] i and cytoskeletal proteins of endothelial cells (ECs) induced by low shear stress (LSS) in PH. Primary Pulmonary Artery Endothelial Cells (PAECs) were subjected to steady LSS (1 dyn/cm2) or physiological shear stress (SS) (10 dyn/cm2) for 20 h in a BioFlux 200 flow system. Calcium influx assays were conducted to evaluate the mechanisms of PEO on [Ca2 +] i. Subsequently, taking the key protein that induces cytoskeletal remodeling, the regulatory light chain (RLC) phosphorylation, as the breakthrough point, this study focused on the two key pathways of PEO that regulate phosphorylation of RLC: Myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK) pathways.
UNASSIGNED: Our current research revealed that PEO at LSS (1 dyn/cm2) significantly suppressed LSS-induced [Ca2 +] i and the expression level of transient receptor potential channel 1(TRPC1). In addition, ECs convert LSS stimuli into the upregulation of cytoskeletal proteins, including filamentous actin (F-actin), MLCK, ROCK, p-RLC, and pp-RLC. Further experiments using pharmacological inhibitors demonstrated that PEO at the LSS downregulated cytoskeleton-related proteins mainly through the ROCK and MLCK pathways.
UNASSIGNED: This study considered intracellular calcium and cytoskeleton rearrangement as entry points to study the application of PEO in the biomedical field, which has important theoretical significance and practical application value for the treatment of PH.

Background: Shear stress-induced Dickkopf-1 (DKK1) secretion by endothelial cells (ECs) promotes EC dysfunction and accelerates atherosclerosis (AS). However, the paracrine role of endothelial DKK1 in modulating adjacent smooth muscle cells (SMCs) in atherosclerosis remains unclear. This study investigated the role of EC-secreted DKK1 in SMC-derived foam cell formation under shear stress, in vitro and in vivo. Methods: Parallel-plate co-culture flow system was used to explore the cellular communication between ECs and SMCs under shear stress in vitro. Endothelium-specific knockout of DKK1 (DKK1ECKO/APOE-/-) and endothelium-specific overexpression of DKK1 (DKK1ECTg) mice were constructed to investigate the role of endothelial DKK1 in atherosclerosis and SMC-derived foam cell formation in vivo. RNA sequencing (RNA-seq) was used to identify the downstream targets of DKK1. Reverse transcription quantitative polymerase chain reaction (RT-qPCR), western blot, coimmunoprecipitation (Co-IP) assays and chromatin immunoprecipitation (ChIP) experiments were conducted to explore the underlying regulatory mechanisms. Results: DKK1 is transcriptionally upregulated in ECs under conditions of low shear stress, but not in co-cultured SMCs. However, DKK1 protein in co-cultured SMCs is increased via uptake of low shear stress-induced endothelial DKK1, thereby promoting lipid uptake and foam cell formation in co-cultured SMCs via the post-translational upregulation of scavenger receptor-A (SR-A) verified in parallel-plate co-culture flow system, DKK1ECKO and DKK1ECTg mice. RNA sequencing revealed that DKK1-induced SR-A upregulation in SMCs is dependent on Ubiquitin-specific Protease 53 (USP53), which bound to SR-A via its USP domain and cysteine at position 41, exerting deubiquitination to maintain the stability of the SR-A protein by removing the K48 ubiquitin chain and preventing proteasomal pathway degradation, thereby mediating the effect of DKK1 on lipid uptake in SMCs. Moreover, DKK1 regulates the transcription of USP53 by facilitating the binding of transcription factor CREB to the USP53 promoter. SMC-specific overexpression of USP53 via adeno-associated virus serotype 2 vectors in DKK1ECKO/APOE-/- mice reversed the alleviation of atherosclerotic plaque burden, SR-A expression and lipid accumulation in SMCs within plaques resulting from DKK1 deficiency. Conclusions: Our findings demonstrate that, endothelial DKK1, induced by pathological low shear stress, acts as an intercellular mediator, promoted the foam cell formation of SMCs. These results suggest that targeted intervention with endothelial DKK1 may confer beneficial effects on atherosclerosis.

BACKGROUND: Ependymal cilia play a major role in the circulation of cerebrospinal fluid. Although isolation of cilia is an essential technique for investigating ciliary structure, to the best of our knowledge, no report on the isolation and structural analysis of ependymal cilia from mouse brain is available.
METHODS: We developed a novel method for isolating ependymal cilia from mouse brain ventricles. We isolated ependymal cilia by partially opening the lateral ventricles and gently applying shear stress, followed by pipetting and ultracentrifugation.
RESULTS: Using this new method, we were able to observe cilia separately. The results demonstrated that our method successfully isolated intact ependymal cilia with preserved morphology and ultrastructure. In this procedure, the ventricular ependymal cell layer was partially detached.
METHODS: Compared to existing methods for isolating cilia from other tissues, our method is meticulously tailored for extracting ependymal cilia from the mouse brain. Designed with a keen understanding of the fragility of the ventricular ependyma, our method prioritizes minimizing tissue damage during the isolation procedure.
CONCLUSIONS: We isolated ependymal cilia from mouse brain by applying shear stress selectively to the ventricles. Our method can be used to conduct more detailed studies on the structure of ependymal cilia.

The efficacy of a sanitizer in biofilm removal may be influenced by a combination of factors such as sanitizer exposure time and concentration, bacterial species, surface topography, and shear stresses. We employed an inline biofilm reactor to investigate the interactions of these variables on biofilm removal with chlorine. The CDC bioreactor was used to grow E. coli O157:H7 and L. monocytogenes biofilms as a single species or with Ralstonia insidiosa as a dual-species biofilm on stainless steel, PTFE, and EPDM coupons at shear stresses 0.368 and 2.462 N/m2 for 48 hours. Coupons were retrieved from a CDC bioreactor and placed in an inline biofilm reactor and 100, 200, or 500 ppm of chlorine was supplied for 1- and 4 min. Bacterial populations in the biofilms were quantified pre- and posttreatment by plating on selective media. After chlorine treatment, reduction (Log CFU/cm2) in pathogen populations obtained from three replicates was analyzed for statistical significance. A 1-min chlorine treatment (500 ppm), on dual-species E. coli O157:H7 biofilms grown at high shear stress of 2.462 N/m2 resulted in significant E. coli O157:H7 reductions on SS 316L (2.79 log CFU/cm2) and PTFE (1.76 log CFU/cm2). Similar trend was also observed for biofilm removal after a 4-min chlorine treatment. Single species E. coli O157:H7 biofilms exhibited higher resistance to chlorine when biofilms were developed at high shear stress. The effect of chlorine in L. monocytogenes removal from dual-species biofilms was dependent primarily on the shear stress at which they were formed rather than the surface topography of materials. Besides surface topography, shear stresses at which biofilms were formed also influenced the effect of sanitizer. The removal of E. coli O157:H7 biofilms from EPDM material may require critical interventions due to difficulty in removing this pathogen. The inline biofilm reactor is a novel tool to evaluate the efficacy of a sanitizer in bacterial biofilm removal.

BACKGROUND: The endothelial glycocalyx (EG), covering the luminal side of endothelial cells, regulates vascular permeability and senses wall shear stress. In sepsis, EG undergoes degradation leading to increased permeability and edema formation. We hypothesized that restoring EG integrity using liposomal nanocarriers of preassembled glycocalyx (LNPG) will restore normal venular permeability in a lipopolysaccharide (LPS)-induced sepsis model of mice.
METHODS: To test this hypothesis, we designed a unique perfusion microchamber in which permeability of isolated venules could be assessed by measuring the concentration of Evans blue dye (EBD) in microliter-samples of extravascular solution (ES).
RESULTS: Histamine-induced time- and dose-dependent increases in EBD in the ES could be measured, confirming the sensitivity of the microchamber system. Notably, the histamine-induced increase in permeability was significantly attenuated by histamine receptor (H1) antagonist, triprolidine hydrochloride. Subsequently, mice were treated with LPS, or LPS + LNPG. Compared to control mice, venules from LPS-treated mice showed a significant increased permeability, which was significantly reduced by LNPG administration. Moreover, in the presence of wall shear stress, intraluminal administration of LNPG significantly reduced the permeability in isolated venules from LPS-treated mice. We have found no sex differences.
CONCLUSIONS: Our newly developed microchamber system allows us to quantitatively measure the permeability of isolated mesenteric venules. LPS-induced sepsis increases permeability of venules that is attenuated by in vivo LNPG administration, which is also reestablished endothelial responses to shear stress. Thus, LNPG presents a promising therapeutic potential for restoring EG function and thereby mitigating vasogenic edema due to increased permeability in sepsis.