Cyclic Stretch Research Articles

Real-Time Quantification of Nanoplastics-Induced Oxidative Stress in Stretching Alveolar Cells.

Nanoplastics from air pollutants can be directly inhaled into the alveoli in the lungs and further enter blood circulation, and numerous studies have revealed the close relation between internalized nanoplastics with many physiological disorders via intracellular oxidative stress. However, the dynamic process of nanoplastics-induced oxidative stress in lung cells under breath-mimicked conditions is still unclear, due to the lack of methods that can reproduce the mechanical stretching of the alveolar and simultaneously monitor the oxidative stress response. Here, we describe a biomimetic platform by culturing alveoli epithelial cells on a stretchable electrochemical sensor and integrating them into a microfluidic device. This allows reproducing the respiration of alveoli by cyclic stretching of the alveoli epithelial cells and monitoring the nanoplastics-induced oxidative stress by the built-in sensor. By this device, we prove that cyclic stretches can greatly enhance the cellular uptake of nanoplastics with the dependencies of strain amplitude. Importantly, oxidative stress evoked by internalized nanoplastics can be quantitatively monitored in real time. This work will promote the deep understanding about the cytotoxicity of inhaled nanoplastics in the pulmonary mechanical microenvironment.

Mechanical factors influence β-catenin localization and barrier properties.

Mechanical forces are of major importance in regulating vascular homeostasis by influencing endothelial cell behavior and functions. Adherens junctions are critical sites for mechanotransduction in endothelial cells. β-catenin, a component of adherens junctions and the canonical Wnt signaling pathway, plays a role in mechanoactivation. Evidence suggests that β-catenin is involved in flow sensing and responds to tensional forces, impacting junction dynamics. The mechanoregulation of β-catenin signaling is context-dependent, influenced by the type and duration of mechanical loads. In endothelial cells, β-catenin's nuclear translocation and signaling are influenced by shear stress and strain, affecting endothelial permeability. The study investigates how shear stress, strain, and surface topography impact adherens junction dynamics, regulate β-catenin localization, and influence endothelial barrier properties. Insight box Mechanical loads are potent regulators of endothelial functions through not completely elucidated mechanisms. Surface topography, wall shear stress and cyclic wall deformation contribute overlapping mechanical stimuli to which endothelial monolayer respond to adapt and maintain barrier functions. The use of custom developed flow chamber and bioreactor allows quantifying the response of mature human endothelial to well-defined wall shear stress and gradients of strain. Here, the mechanoregulation of β-catenin by substrate topography, wall shear stress, and cyclic stretch is analyzed and linked to the monolayer control of endothelial permeability.

YAP mechanotransduction under cyclic mechanical stretch loading for mesenchymal stem cell osteogenesis is regulated by ROCK.

While yes-associated protein (YAP) is now recognized as a potent mechanosensitive transcriptional regulator to affect cell growth and differentiation including the osteogenic transcription of mesenchymal stem cells (MSCs), most studies have reported the YAP mechanosensing of static mechanophysical cues such as substrate stiffness. We tested MSC response to dynamic loading, i.e., cyclic mechanical stretching, and assessed YAP mechanosensing and resultant MSC osteogenesis. We showed that cyclic stretching at 10% strain and 1Hz frequency triggered YAP nuclear import in MSCs. YAP phosphorylation at S127 and S397, which is required for YAP cytoplasmic retention, was suppressed by cyclic stretch. We also observed that anti-YAP-regulatory Hippo pathway, LATS phosphorylation, was significantly decreased by stretch. We confirmed the stretch induction of MSC osteogenic transcription and differentiation, and this was impaired under YAP siRNA suggesting a key role of YAP dynamic mechanosensing in MSC osteogenesis. As an underlying mechanism, we showed that the YAP nuclear transport by cyclic stretch was abrogated by ROCK inhibitor, Y27632. ROCK inhibitor also impaired the stretch induction of F-actin formation and MSC osteogenesis, thus implicating the role of the ROCK-F-actin cascade in stretch-YAP dynamic mechanosensing-MSC osteogenesis. Our results provide insight into bone tissue engineering and skeletal regenerative capacity of MSCs especially as regards the role of dynamic mechanical loading control of YAP-mediated MSC osteogenic transcription.

Cyclic Stretching Enhances Angiocrine Signals at Liver Bud Stage from Human Pluripotent Stem Cells in Two-Dimensional Culture.

Angiocrine signals during the development and growth of organs, including the liver, intestine, lung, and bone, are essential components of intercellular communication. The signals elicited during the liver bud stage are critical for vascularization and enhanced during the intercellular communication between the cells negative for kinase insert domain receptor (KDR) (KDR- cells) and the cells positive for KDR (KDR+ cells), which constitute the liver bud. However, the use of a human pluripotent stem cell (hPSC)-derived system has not facilitated the generation of a perfusable vascularized liver organoid that allows elucidation of liver development and has great potential for liver transplantation. This is largely owing to the lack of fundamental understanding to induce angiocrine signals in KDR- and KDR+ cells during the liver bud stage. We hypothesized that mechanical stimuli of cyclic stretching/pushing by the fetal heart adjacent to the liver bud could be the main contributor to promoting angiocrine signals in KDR- and KDR+ cells during the liver bud stage. In this study, we show that an organ-on-a-chip platform allows the emulation of an in vivo-like mechanical environment for the liver bud stage in vitro and investigate the role of cyclic mechanical stretching (cMS) to angiocrine signals in KDR- and KDR+ cells derived from hPSCs. RNA sequencing revealed that the expression of genes associated with epithelial-to-mesenchymal transition, including angiocrine signals, such as hepatocyte growth factor (HGF) and matrix metallopeptidase 9 (MMP9), were increased by cMS in cocultured KDR- and KDR+ cells. The expression and secretions of HGF and MMP9 were increased by 1.98- and 1.69-fold and 3.23- and 3.72-fold with cMS in the cocultured KDR- and KDR+ cells but were not increased by cMS in the monocultured KDR- and KDR+ cells, respectively. Finally, cMS during the liver bud stage did not lead to the dedifferentiation of hepatocytes, as the cells with cMS showed hepatic maker expression (CYP3A4, CYP3A7, ALB, and AAT) and 1.71-fold higher CYP3A activity than the cells without cMS, during 12 day-hepatocyte maturation after halting cMS. Our findings provide new insights into the mechanical factors during the liver bud stage and directions for future improvements in the engineered liver tissue.

The ion channel Trpc6a regulates the cardiomyocyte regenerative response to mechanical stretch.

Myocardial damage caused, for example, by cardiac ischemia leads to ventricular volume overload resulting in increased stretch of the remaining myocardium. In adult mammals, these changes trigger an adaptive cardiomyocyte hypertrophic response which, if the damage is extensive, will ultimately lead to pathological hypertrophy and heart failure. Conversely, in response to extensive myocardial damage, cardiomyocytes in the adult zebrafish heart and neonatal mice proliferate and completely regenerate the damaged myocardium. We therefore hypothesized that in adult zebrafish, changes in mechanical loading due to myocardial damage may act as a trigger to induce cardiac regeneration. Based on this notion we sought to identify mechanosensors which could be involved in detecting changes in mechanical loading and triggering regeneration. Here we show using a combination of knockout animals, RNAseq and in vitro assays that the mechanosensitive ion channel Trpc6a is required by cardiomyocytes for successful cardiac regeneration in adult zebrafish. Furthermore, using a cyclic cell stretch assay, we have determined that Trpc6a induces the expression of components of the AP1 transcription complex in response to mechanical stretch. Our data highlights how changes in mechanical forces due to myocardial damage can be detected by mechanosensors which in turn can trigger cardiac regeneration.

A novel modular opto-biomechatronics bioreactor for simultaneous isotropic mechanical stretch application and fluorescence microscopy under cell and tissue culture conditions

Mechanical stresses are an environmental challenge virtually all tissues in the body are exposed to and thus, are of fundamental interest to study cell reactions in mechanobiology. Yet, unlike acute short-term mechanical cell stimulations, long-term or cyclic mechano-stimulation as experienced in the body is difficult to reproduce. Bioreactors are designed to control cell culture conditions, but still, there are yet no technical solutions available to merge bioreactor and opto-biomechatronics technologies for cyclic stretch-applications and simultaneous live cell imaging. To close this gap, we have engineered an opto-biomechatronics module, consisting of our in-house developed IsoStretcher technology and customised epifluorescence optics, into an automated bioreactor platform. For this, redesigned polydimethylsiloxane (PDMS) chambers with closed geometry (∽700μL internal volume) to warrant sterile operation were developed. Those chambers could be flushed with cell solution for cell seeding in a sterile manner. The epifluorescence imaging module was engineered into the reactor underneath the IsoStretcher to allow for continuous image acquisition during long-term stretch cycles (hours to days). The system was validated on human fibroblast BJ foreskin cells, and Cal-520 Ca2＋ fluorescence was stably imaged using our in-built autofocus functionality. Cultures for 24h within the IsoStretcher-bioreactor preserved a normal cell morphology as compared to external incubator control cultures. Isotropic stretch was reliably transferred to the cell membranes. Our system with in-built bioreactor and opto-biomechatronics functionality provides a holistic technology platform for the growing field of mechanobiology to allow long-term observations of cultured single cells and confluent cell layers that are subjected to cyclic long-term isotropic stretch protocols.

Mechanical Unloading Promotes Osteoclastic Differentiation and Bone Resorption by Modulating the MSC Secretome to Favor Inflammation.

Aging, space flight, and prolonged bed rest have all been linked to bone loss, and no effective treatments are clinically available at present. Here, with the rodent hindlimb unloading (HU) model, we report that the bone marrow (BM) microenvironment was significantly altered, with an increased number of myeloid cells and elevated inflammatory cytokines. In such inflammatory BM, the osteoclast-mediated bone resorption was greatly enhanced, leading to a shifted bone remodeling balance that ultimately ends up with disuse-induced osteoporosis. Using Piezo1 conditional knockout (KO) mice (Piezo1fl/fl;LepRCre), we proved that lack of mechanical stimuli on LepR+ mesenchymal stem cells (MSCs) is the main reason for the pathological BM inflammation. Mechanically, the secretome of MSCs was regulated by mechanical stimuli. Inadequate mechanical load leads to increased production of inflammatory cytokines, such as interleukin (IL)-1α, IL-6, macrophage colony-stimulating factor 1 (M-CSF-1), and so on, which promotes monocyte proliferation and osteoclastic differentiation. Interestingly, transplantation of 10% cyclic mechanical stretch (CMS)-treated MSCs into HU animals significantly alleviated the BM microenvironment and rebalanced bone remodeling. In summary, our research revealed a new mechanism underlying mechanical unloading-induced bone loss and suggested a novel stem cell-based therapy to potentially prevent disuse-induced osteoporosis.

Mechanical strategies to promote vascularization for tissue engineering and regenerative medicine.

Vascularization is a major challenge in the field of tissue engineering and regenerative medicine. Mechanical factors have been demonstrated to play a fundamental role in vasculogenesis and angiogenesis and can affect the architecture of the generated vascular network. Through the regulation of mechanical factors in engineered tissues, various mechanical strategies can be used to optimize the preformed vascular network and promote its rapid integration with host vessels. Optimization of the mechanical properties of scaffolds, including controlling scaffold stiffness, increasing surface roughness and anisotropic structure, and designing interconnected, hierarchical pore structures, is beneficial for the in vitro formation of vascular networks and the ingrowth of host blood vessels. The incorporation of hollow channels into scaffolds promotes the formation of patterned vascular networks. Dynamic stretching and perfusion can facilitate the formation and maturation of preformed vascular networks in vitro. Several indirect mechanical strategies provide sustained mechanical stimulation to engineered tissues in vivo, which further promotes the vascularization of implants within the body. Additionally, stiffness gradients, anisotropic substrates and hollow channels in scaffolds, as well as external cyclic stretch, boundary constraints and dynamic flow culture, can effectively regulate the alignment of vascular networks, thereby promoting better integration of prevascularized engineered tissues with host blood vessels. This review summarizes the influence and contribution of both scaffold-based and external stimulus-based mechanical strategies for vascularization in tissue engineering and elucidates the underlying mechanisms involved.

Physiological cyclic stretching potentiates the cell–cell junctions in vascular endothelial layer formed on aligned fiber substrate

In vascular tissue engineering, formation of stable endothelial cell–cell and cell–substrate adhesions is essential for maintaining long-term patency of the tissue-engineered vascular grafts (TEVGs). In this study, sheet-like aligned fibrous substrates of poly(l-lactide-co-caprolactone) (PLCL) were prepared by electrospinning to provide basement membrane-resembling structural support to endothelial cells (ECs). Cyclic stretching at physiological and pathological levels was then applied to human umbilical vein endothelial cells (HUVECs) cultured on chosen fibrous substrate using a force-loading device, from which effects of the cyclic stretching on cell–cell and cell–substrate adhesions were examined. It was found that applying uniaxial 1 Hz cyclic stretch at physiological levels (5 % and 10 % elongation) strengthened the cell–cell junctions, thus leading to improved structural integrity, functional expression and resistance to thrombin-induced damaging impacts in the formed endothelial layer. The cell–cell junctions were disrupted at pathological level (15 % elongation) cyclic stretching, which however facilitated the formation of focal adhesions (FAs) at cell–substrate interface. Mechanistically, the effects of cyclic stretching on endothelial cell–cell and cell–substrate adhesions were identified to be correlated with the RhoA/ROCK signaling pathway. Results from this study highlight the relevance between applying dynamic mechanical stimulation and maintaining the structural integrity of the formed endothelial layer, and implicate a necessity to implement appropriate dynamic mechanical training (i.e., preconditioning) to obtain tissue-engineered blood vessels with long-term patency post-implantation.

4-octyl itaconate ameliorates ventilator-induced lung injury

Ventilator-induced lung injury (VILI) disturbs the disordered immune system and causes persistent inflammatory damage. 4-octyl itaconate (OI) is a synthetic cell-permeable itaconate derivative with antioxidant and anti-inflammatory effects. In this study, we assessed whether OI protects against VILI. OI was intraperitoneally injected for three days before mechanical ventilation (MV; 20 ml/kg at 70 breaths/min) for 2 h. Mouse lung vascular endothelial cells (MLVECs) were pretreated with OI (62.5, 125, and 250 μM) prior to cyclic stretch for 4 h. We found that OI attenuated VILI and inflammatory response. OI also increased superoxide dismutase, nuclear factor E2-related factor 2, and heme oxygenase-1 levels, and decreased reactive oxygen species and malondialdehyde levels. Furthermore, OI inhibited the expression of NLR family pyrin domain-containing 3 (NLRP3), caspase-1 p20, apoptosis-associated speck-like protein containing a CARD, and N-terminal fragment of gasdermin D. Therefore, OI attenuates VILI, potentially by suppressing oxidative stress and NLRP3 activation.

Gene expression of Postn and FGF7 in canine chordae tendineae and their effects on flexor tenocyte biology.

Chordae tendineae, referred to as heart tendinous cords, act as tendons connecting the papillary muscles to the valves in the heart. Their role is analogous to tendons in the musculoskeletal system. Despite being exposed to millions of cyclic tensile stretches over a human's lifetime, chordae tendineae rarely suffer from overuse injuries. On the other hand, musculoskeletal tendinopathy is very common and remains challenging in clinical treatment. The objective of this study was to investigate the mechanism behind the remarkable durability and resistance to overuse injuries of chordae tendineae, as well as to explore their effects on flexor tenocyte biology. The messenger RNAexpression profiles of chordae tendineae were analyzed using RNA sequencing and verified by quantitative reverse transcription polymerase chain reaction and immunohistochemistry. Interestingly, we found that periostin (Postn) and fibroblast growth factor 7 (FGF7) were expressed at significantly higher levels in chordae tendineae, compared to flexor tendons. We further treated flexor tenocytes in vitro with periostin and FGF7 to examine their effects on the proliferation, migration, apoptosis, and tendon-related gene expression of flexor tenocytes. The results displayed enhanced cell proliferation ability at an early stage and an antiapoptotic effect on tenocytes, while treated with periostin and/or FGF7 proteins. Furthermore, there was a trend of promoted tenocyte migration capability. These findings indicated that Postn and FGF7 may represent novel cytokines to target flexor tendon healing. Clinical significance: The preliminary discovery leads to a novel idea for treating tendinopathy in the musculoskeletal system using specific molecules identified from chordae tendineae.

Empagliflozin prevents oxidative stress in human coronary artery endothelial cells via the NHE/PKC/NOX axis

BackgroundEmpagliflozin (EMPA) ameliorates reactive oxygen species (ROS) generation in human endothelial cells (ECs) exposed to 10 % stretch, but the underlying mechanisms are still unclear. Pathological stretch is supposed to stimulate protein kinase C (PKC) by increasing intracellular calcium (Ca2+), therefore activating nicotinamide adenine dinucleotide phosphate oxidase (NOX) and promoting ROS production in human ECs. We hypothesized that EMPA inhibits stretch-induced NOX activation and ROS generation through preventing PKC activation. MethodsHuman coronary artery endothelial cells (HCAECs) were pre-incubated for 2 h before exposure to cyclic stretch (5 % or 10 %) with either vehicle, EMPA or the PKC inhibitor LY-333531 or PKC siRNA. PKC activity, NOX activity and ROS production were detected after 24 h. Furthermore, the Ca2+ chelator BAPTA-AM, NCX inhibitor ORM-10962 or NCX siRNA, sodium/potassium pump inhibitor ouabain and sodium hydrogen exchanger (NHE) inhibitor cariporide were applied to explore the involvement of the NHE/Na+/NCX/Ca2+ in the ROS inhibitory capacity of EMPA. ResultsCompared to 5 % stretch, 10 % significantly increased PKC activity, which was reduced by EMPA and PKC inhibitor LY-333531. EMPA and LY-333531 showed a similar inhibitory capacity on NOX activity and ROS generation induced by 10 % stretch, which was not augmented by combined treatment with both drugs. PKC-β knockdown inhibits the NOX activation induced by Ca2+ and 10 % stretch. BAPTA, pharmacologic or genetic NCX inhibition and cariporide reduced Ca2+ in static HCAECs and prevented the activation of PKC and NOX in 10%-stretched cells. Ouabain increased ROS generation in cells exposed to 5 % stretch. ConclusionEMPA reduced NOX activity via attenuation of the NHE/Na+/NCX/Ca2+/PKC axis, leading to less ROS generation in HCAECs exposed to 10 % stretch.

High hydrostatic pressure participates in atrial fibrosis through the p300/p53/Smad3 pathway.

As an independent risk factor of atrial fibrillation (AF), hypertension (HTN) can induce atrial fibrosis through cyclic stretch and hydrostatic pressure. The mechanism by which high hydrostatic pressure promotes atrial fibrosis is unclear yet. p300 and p53/Smad3 play important roles in the process of atrial fibrosis. This study investigated whether high hydrostatic pressure promotes atrial fibrosis by activating the p300/p53/Smad3 pathway. Biochemical experiments were used to study the expression of p300/p53/Smad3 pathway in left atrial appendage (LAA) tissues of patients with sinus rhythm (SR), AF, AF + HTN, and C57/BL6 mice, hypertensive C57/BL6 mice and atrial fibroblasts of mice. To investigate the roles of p300 and p53 in the process of atrial fibrosis, p300 and p53 in mice atrial fibroblasts were knocked in or knocked down, respectively. The expression of p300/p53/Smad3 and fibrotic factors was higher in patients with AF and AF + HTN than those with SR only. The expressions of p300/p53/Smad3 and fibrotic factors increased in hypertensive mice. Curcumin (Cur) and knocking down of p300 reversed the expressions of these factors. 40 mmHg hydrostatic pressure/overexpression of p300 upregulated the expressions of p300/p53/Smad3 and fibrotic factors in mice LAA fibroblasts. While Cur or knocking down p300 reversed these changes. Knocking down/overexpression of p53, the expressions of p53/Smad3 and fibrotic factors also decreased/increased, correspondingly. High hydrostatic pressure promotes atrial fibrosis by activating the p300/p53/Smad3 pathway, which further increases the susceptibility to AF.

Blood Flow Directs Yap/Taz-Mediated Transcriptional Regulation of Self-Renewal Programs to Control Developmental HSPC Expansion By Mechanical Stimulation of Piezo1

Hematopoietic stem and progenitor cells (HSPCs) emerge from artery-derived hemogenic endothelium (HE) in the vertebrate embryo during development. This process entails a cellular reprogramming from endothelial to hematopoietic gene signatures driven by the Runx1 transcription factor (TF), which is referred to as the endothelial-to-hematopoietic transition (EHT). A major goal of cellular therapeutics is to derive patient-specific HSPCs from iPSCs for clinical use, yet current differentiation protocols largely fail to recapitulate EHT to produce or expand long-lived multi-potent HSPCs in vitro suggesting an incomplete understanding of the in vivo regulatory cues. Physical forces of wall shear stress (WSS) and cyclic stretch (CS) produced by hemodynamic blood flow in embryos are one such cue required during EHT to generate HSPCs from HE, however the mechanisms by which these forces are sensed and converted into a “stemness” regulatory module remain undefined. Our previous work implicated the Hippo pathway TF YAP as essential for the maintenance, but not initiation, of the hematopoietic program in newly specified HE. Here, using scRNA-sequencing of sorted zebrafish trunk endothelial cells to capture all stages of EHT progression, we find that overexpression of YAP drives lipid metabolism, cell cycling, and propagation of a hematopoietic gene regulatory network (GRN) in the earliest specified HE cells. By employing a heat shock-inducible dominant negative YAP zebrafish line, we unmask a latent role for the YAP paralogue TAZ in hematopoiesis, which can promote CD41 + and Flk +/Myb + HSPC production upon reduced YAP function. YAP and TAZ initiate transcriptional responses downstream of mechanical stimuli and require DNA binding cofactors to regulate target genes. Surprisingly, luciferase assays in HEK293 cells demonstrated a potent synergistic effect of TAZ/RUNX1, but not YAP/RUNX1, in transcriptional regulation at RUNX enhancers. Finally, by pharmacologic and genetic manipulation, we identify the stretch-gated membrane ion channel Piezo1 as a potent regulator of CS-induced YAP/TAZ mechanotransduction in HE. Stimulation of zebrafish embryos with the Piezo1 small molecule agonist Yoda1 significantly increases HSPC number and YAP target gene expression in a YAP-dependent fashion. A similar modulation of blood and YAP target genes in human iPSC-derived CD34+ HE cells is seen with Yoda1, suggesting that this stretch-Piezo1-YAP axis can be chemically tuned in vitro to enhance HSPC differentiation. These results illuminate molecular details of how YAP/TAZ directs the production of HSPCs from HE in response to mechanical cues, and have broader implications for alternate regulatory effects of mechanically-stimulated Hippo TFs depending on the transcriptional milieu in cell-type specific contexts.

A Flexible PDMS-Based Optical Biosensor for Stretch Monitoring in Cardiac Tissue Samples

Cardiotoxicity, characterized by adverse impacts on normal heart function due to drug exposure, is a significant concern due to the potentially serious side effects associated with various pharmaceuticals. It is essential to detect the cardiotoxicity of a drug as early as possible in the testing phase of a medical composite. Therefore, there is a pressing need for more reliable in vitro models that accurately mimic the in vivo conditions of cardiac biopsies. In a functional beating heart, cardiac muscle cells are under the effect of static and cyclic stretches. It has been demonstrated that cultured cardiac biopsies can benefit from external mechanical loads that resemble the in vivo condition, increasing the probability of cardiotoxicity detection in the early testing stages. In this work, a biosensor is designed and fabricated to allow for stretch monitoring in biopsies and tissue cultures using an innovative sensing mechanism. The detection setup is based on a biocompatible, thin, flexible membrane—where the samples are attached—which is used as an optical waveguide to detect pressure-caused shape changes and stretches. Various prototypes have been fabricated with a cost-effective process, and different measurements have been carried out to experimentally validate the proposed measurement technique. From these evaluations, stretches of up to 1.5% have been measured, but the performed simulations point towards the possibility of expanding the considered technique up to 10–30% stretches.

Physical biology of cell-substrate interactions under cyclic stretch.

Mechanosensitive focal adhesion (FA) complexes mediate dynamic interactions between cells and substrates and regulate cellular function. Integrins in FA complexes link substrate ligands to stress fibers (SFs) and aid load transfer and traction generation. We developed a one-dimensional, multi-scale, stochastic finite element model of a fibroblast on a substrate that includes calcium signaling, SF remodeling, and FA dynamics. We linked stochastic dynamics, describing the formation and clustering of integrins to substrate ligands via motor-clutches, to a continuum level SF contractility model at various locations along the cell length. We quantified changes in cellular responses with substrate stiffness, ligand density, and cyclic stretch. Results show that tractions and integrin recruitments varied along the cell length; tractions were maximum at lamellar regions and reduced to zero at the cell center. Optimal substrate stiffness, based on maximum tractions exerted by the cell, shifted toward stiffer substrates at high ligand densities. Mean tractions varied biphasically with substrate stiffness and peaked at the optimal substrate stiffness. Cytosolic calcium increased monotonically with substrate stiffness and accumulated near lamellipodial regions. Cyclic stretch increased the cytosolic calcium, integrin concentrations, and tractions at lamellipodial and intermediate regions on compliant substrates. The optimal substrate stiffness under stretch shifted toward compliant substrates for a given ligand density. Stretch also caused cell deadhesions beyond a critical substrate stiffness. FA's destabilized on stiff substrates under cyclic stretch. An increase in substrate stiffness and cyclic stretch resulted in higher fibroblast contractility. These results show that chemomechanical coupling is essential in mechanosensing responses underlying cell-substrate interactions.

Increasing pulse pressure ex vivo, mimicking acute physical exercise, induces smooth muscle cell-mediated de-stiffening of murine aortic segments

The mechanisms by which physical activity affects cardiovascular function and physiology are complex and multifactorial. In the present study, cardiac output during rest or acute physical activity was simulated in isolated aortic segments of healthy C57BL/6J wild-type mice. This was performed using the Rodent Oscillatory Tension Set-up to study Arterial Compliance (ROTSAC) by applying cyclic stretch of different amplitude, duration and frequency in well-controlled and manageable experimental conditions. Our data show that vascular smooth muscle cells (VSMCs) of the aorta have the intrinsic ability to “de-stiffen” or “relax” after periods of high cyclic stretch and to “re-stiffen” slowly thereafter upon return to normal distension pressures. Thereby, certain conditions have to be fulfilled: 1) VSMC contraction and repetitive stretching (loading/unloading cycles) are a prerequisite to induce post-exercise de-stiffening; 2) one bout of high cyclic stretch is enough to induce de- and re-stiffening. Aortic de-stiffening was highly dependent on cyclic stretch amplitude and on the manner and timing of contraction with probable involvement of focal adhesion phosphorylation/activation. Results of this study may have implications for the therapeutic potential of regular and acute physical activity and its role in the prevention and/or treatment of cardiovascular disease.

Abstract 16846: Three-Dimensional Assessment of Ascending Aortic Motion by Dynamic CT in Sporadic and Marfan Thoracic Aortic Aneurysm

Background: The ascending aorta exhibits a complex, 3D motion related to cyclic pressure changes and downward traction forces related to left ventricular contraction. However, most metrics of disease severity are static (i.e., diameter) and among prior studies that have investigated ascending aortic motion, 2D imaging is typically employed. Vascular deformation mapping (VDM) is an image analysis technique that accurately quantifies 3D displacement of the aortic wall. Aims We aimed to quantify 3D metrics of cyclic ascending aortic motion and distensibility in patients with aTAA, and evaluate associations between motion metrics and patient characteristics in sporadic TAA (SaTAA) and Marfan syndrome (MFS). Methods: We included adult patients with aTAA, either SaTAA or MFS, and multi-phasic CTA of the thoracic aorta. VDM analysis of CTA data was performed with metrics including: 1) Axial, in-plane and rotational displacements at the root, 2) distensibility assessed by cyclic 2D cross-sectional area change (Dist-2D) at the root [(ΔA/A diastole х PP)], and 3) distensibility assessed by VDM-derived cyclic surface area stretch [(Area ratio-1)*1/PP] at the root and tubular ascending (Dist-3D). Aortic growth rate (GR) was measured from follow-up CTA. Results: We analyzed 51 with SaTAA (33% female, 62 ± 11 years old), and 14 with MFS (50% female, 42 ± 13 years old). In SaTAA, Dist-2D was associated (β=0.59, p=0.047) with axial displacement and AI was strongly associated (β=1.03 p=0.001) with rotation. In MFS, BAV and diameter were associated with in-plane displacement (β=5.50, p=0.004; β=-0.026, p=0.001). Among MFS patients with follow-up CTAs (n=10), ascending distensibility (Dist-3D) was marginally negatively associated with ascending GR (β= -0.27, p=0.058) Conclusion: 3D analysis of aortic motion in aTAA showed differences in root motion, namely related to valve disease and age, as well as differences in aortic distensibility that may predict ascending aortic GR in MFS.

Abstract 16995: Distensibility and Aortic Growth Rate Predict Type B Aortic Dissection in Marfan Syndrome

Introduction: Marfan syndrome (MFS) is associated with high aortic growth rate (GR) and incidence of aortic dissection (AD), including delayed type B AD (TBAD) after prophylactic root repair. Elevated aortic stiffness has been investigated as a biomarker in MFS, but it remains uncertain how stiffness relates to GR and risk of AD. Vascular deformation mapping (VDM) is a technique that uses clinical CT angiography (CTA) to yield accurate, 3D maps of aortic growth over surveillance (VDM-G). This technique can also be used to assess aortic stiffness via quantification of cyclic wall stretch between systole and diastole (VDM-D) using multi-phasic dynamic CTA. Research Question: Is distensibility of the descending thoracic aorta associated with aortic GR or development of TBAD in MFS patients? Goal: The aim of this study was to analyze stiffness of the descending thoracic aorta in MFS and its association with aortic GR and occurrence of TBAD. Methods: We identified 36 adult MFS patients with dynamic CTA studies (>1 year interval) between 2004-2020. Growth was quantified by VDM-G, and linearized descending aortic stiffness by VDM-D - via calculation of distensibility - as the ratio of cyclic aortic surface area change (AR) over pulse pressure [(AR-1)/PP]. Results: Patients were 38.9 ± 12.1 years old, 58% were male and 25 (69%) had prior ascending repair. Mean CT follow-up interval was 5.7 ± 3.4 years and 8 developed TBAD during follow-up with pre-dissection diameter of 31.12 ± 6.01 mm at tear location. Age (β -0.4, 95%CI: -0.07, -0.01, p=0.015) and baseline diameter (β 0.10, 95%CI: 0.02, 0.18, p=0.017 ) independently predicted GR on multivariable analysis, but distensibility did not (p=0.38). Both distensibility (OR 0.02, 95% CI: 0.00, 0.68, p=0.03) and GR (OR 8687.05, 95% CI: 1.43, 5x10^7, p=0.04) were independent predictors of TBAD during follow-up, but diameter was not (p=0.49). Conclusion: Aortic stiffness and growth rate are likely predictors of type B aortic dissection in MFS.

Cyclic stretch-induced exosomes from periodontal ligament cells promote osteoblasts osteogenic differentiation via the miR-181d-5p/TNF signaling pathway

ObjectiveTo investigate the effects of cyclic stretch-induced periodontal ligament cell (PDLC) exosomes on osteoblast differentiation, and to explore their regulatory role in mechanical force-related periodontal tissue remodeling. DesignAfter applying 20 % cyclic stretch loading to PDLCs, exosomes were extracted from the supernatant and co-cultured with osteoblasts to detect their effects on osteogenic differentiation. Meanwhile, the exosomes were sequenced by high-throughput microRNA sequencing for bioinformatic analysis and validation to explore exosome signaling pathways through miRNAs. ResultsCyclic stretch-induced PDLC exosomes could be taken up by osteoblasts and promoted osteogenic differentiation of osteoblasts, as demonstrated by the increased expression levels of osteogenesis-related factors and enhanced alkaline phosphatase (Alp) staining. Among the differentially expressed miRNAs between exosomes from cyclic stretch group and control group, miRNA-181d-5p was up-regulated significantly. The expression levels of osteogenesis-related factors and Alp staining were also increased in osteoblasts transfected with miR-181d-5p, and this effect might be related to the inhibitory role of exosomal miR-181d-5p on tumor necrosis factor (TNF). ConclusionsCyclic stretch-induced PDLC exosomes exhibited a promoting effect on osteogenic differentiation, which might result from the inhibition of TNF via exosomal miR-181d-5p.