DNA-functionalized artificial mechanoreceptor for de novo force-responsive signaling.

Synthetic signaling receptors enable programmable cellular responses coupling with customized inputs. However, engineering a designer force-sensing receptor to rewire mechanotransduction remains largely unexplored. Herein, we introduce nongenetically engineered artificial mechanoreceptors (AMRs) capable of reprogramming non-mechanoresponsive receptor tyrosine kinases (RTKs) to sense user-defined force cues, enabling de novo-designed mechanotransduction. AMR is a modular DNA-protein chimera comprising a mechanosensing-and-transmitting DNA nanodevice grafted on natural RTKs via aptameric anchors. AMR senses intercellular tensile force via an allosteric DNA mechano-switch with tunable piconewton-sensitive force tolerance, actuating a force-triggered dynamic DNA assembly to manipulate RTK dimerization and activate intracellular signaling. By swapping the force-reception ligands, we demonstrate the AMR-mediated activation of c-Met, a representative RTK, in response to the cellular tensile forces mediated by cell-adhesion proteins (integrin, E-cadherin) or membrane protein endocytosis (CI-M6PR). Moreover, AMR also allows the reprogramming of FGFR1, another RTK, to customize mechanobiological function, for example, adhesion-mediated neural stem cell maintenance.

Biomechanical stress regulates mammalian tooth replacement via the integrin ß1-RUNX2-Wnt pathway.

Renewal of integumentary organs occurs cyclically throughout an organism's lifetime, but the mechanism that initiates each cycle remains largely unknown. In a miniature pig model of tooth development that resembles tooth development in humans, the permanent tooth did not begin transitioning from the resting to the initiation stage until the deciduous tooth began to erupt. This eruption released the accumulated mechanical stress inside the mandible. Mechanical stress prevented permanent tooth development by regulating expression and activity of the integrin ß1-ERK1-RUNX2 axis in the surrounding mesenchyme. We observed similar molecular expression patterns in human tooth germs. Importantly, the release of biomechanical stress induced downregulation of RUNX2-wingless/integrated (Wnt) signaling in the mesenchyme between the deciduous and permanent tooth and upregulation of Wnt signaling in the epithelium of the permanent tooth, triggering initiation of its development. Consequently, our findings identified biomechanical stress-associated Wnt modulation as a critical initiator of organ renewal, possibly shedding light on the mechanisms of integumentary organ regeneration.

Potential Roles of YAP/TAZ Mechanotransduction in Spaceflight-Induced Liver Dysfunction.

Microgravity exposure during spaceflight causes the disordered regulation of liver function, presenting a specialized mechano-biological coupling process. While YAP/TAZ serves as a typical mechanosensitive pathway involved in hepatocyte metabolism, it remains unclear whether and how it is correlated with microgravity-induced liver dysfunction. Here, we discussed liver function alterations induced by spaceflight or simulated effects of microgravity on Earth. The roles of YAP/TAZ serving as a potential bridge in connecting liver metabolism with microgravity were specifically summarized. Existing evidence indicated that YAP/TAZ target gene expressions were affected by mechanotransductive pathways and phase separation, reasonably speculating that microgravity might regulate YAP/TAZ activation by disrupting these pathways via cytoskeletal remodeling or nuclear deformation, or disturbing condensates formation via diffusion limit, and then breaking liver homeostasis.

Numerical simulation of flow characteristics in a permeable liver sinusoid with leukocytes.

Double-layered channels of sinusoid lumen and Disse space separated by fenestrated liver sinusoidal endothelial cells (LSECs) endow the unique mechanical environment of the liver sinusoid network, which further guarantees its biological function. It is also known that this mechanical environment changes dramatically under liver fibrosis and cirrhosis, including the reduced plasma penetration and metabolite exchange between the two flow channels and the reduced Disse space deformability. The squeezing of leukocytes through narrow sinusoid lumen also affects the mechanical environment of liver sinusoid. To date, the detailed flow-field profile of liver sinusoid is still far from clear due to experimental limitations. It also remains elusive whether and how the varied physical properties of the pathological liver sinusoid regulate the fluid flow characteristics. Here a numerical model based on the immersed boundary method was established, and the effects of Disse space and leukocyte elasticities, endothelium permeability, and sinusoidal stenosis degree on fluid flow as well as leukocyte trafficking were specified upon a mimic liver sinusoid structure. Results showed that endothelium permeability dominantly controlled the plasma penetration velocity across the endothelium, whereas leukocyte squeezing promoted local penetration and significantly regulated wall shear stress on hepatocytes, which was strongly related to the Disse space and leukocyte deformability. Permeability and elasticity cooperatively regulated the process of leukocytes trafficking through the liver sinusoid, especially for stiffer leukocytes. This study will offer new insights into deeper understanding of the elaborate mechanical features of liver sinusoid and corresponding biological function.

E-selectin negatively regulates polymorphonuclear neutrophil transmigration through altered endothelial junction integrity.

Transendothelial migration (TEM) of neutrophils under blood flow is critical in the inflammatory cascade. However, the role of endothelial plasticity in this process is not fully understood. Therefore, we used an in vitro model to test the dynamics of human polymorphonuclear neutrophil (PMN) TEM across lipopolysaccharide-treated human umbilical vein endothelial cell (HUVEC) monolayers. Interestingly, shRNA-E-selectin knockdown in HUVECs destabilized endothelial junctional integrity by reducing actin branching and increasing stress fiber at cell-cell junctions. This process is accomplished by downregulating the activation of cortactin and Arp2/3, which in turn alters the adhesive function of VE-cadherin, enhancing PMN transmigration. Meanwhile, redundant P-selectins possess overlapping functions in E-selectin-mediated neutrophil adhesion, and transmigration. These results demonstrate, to our knowledge, for the first time, that E-selectins negatively regulate neutrophil transmigration through alterations in endothelial plasticity. Furthermore, it improves our understanding of the mechanisms underlying actin remodeling, and junctional integrity, in endothelial cells mediating leukocyte TEM.

Influence of microflow on hepatic sinusoid blood flow and red blood cell deformation.

Hepatic sinusoids present complex anatomical structures such as the endothelial sieve pores and the Disse space, which govern the microscopic blood flow in the sinusoids and are associated with structural variations in liver fibrosis and cirrhosis. However, the contributions of the permeability of endothelial and collagen layers and the roughness of hepatocyte microvilli to the features of this microflow remain largely unknown. Here, an immersed boundary method coupled with a lattice Boltzmann method was adopted in an in vitro hepatic sinusoidal model, and flow field and erythrocyte deformation analyses were conducted by introducing three new source terms including permeability of the endothelial layer, resistance of hepatocyte microvilli and collagen layers, and deformation of red blood cells (RBCs). Numerical calculations indicated that alterations in endothelial permeability could significantly affect the flow velocity and flow rate distributions in hepatic sinusoids. Interestingly, a biphasic regulating pattern of shear stress occurred simultaneously on the surface of hepatocytes and the lower side of endothelium, i.e., the shear stress increased with increased thickness of hepatocyte microvilli and collagen layer when the endothelial permeability was high but decreased with the increase of the thickness at low endothelial permeability. Additionally, this specified microflow manipulates typical RBC deformation inside the sinusoid, yielding one-third of the variation of deformable index with varied endothelial permeability. These simulations not only are consistent with experimental measurements using in vitro liver sinusoidal chip but also elaborate the contributions of endothelial and collagen layer permeability and wall roughness. Thus, our results provide a basis for further characterizing this microflow and understanding its effects on cellular migration and deformation in the hepatic sinusoids.

Matrix stiffness and shear stresses modulate hepatocyte functions in a fibrotic liver sinusoidal model.

Extracellular matrix (ECM) rigidity has important effects on cell behaviors and increases sharply in liver fibrosis and cirrhosis. Hepatic blood flow is essential in maintaining hepatocytes' (HCs) functions. However, it is still unclear how matrix stiffness and shear stresses orchestrate HC phenotype in concert. A fibrotic three-dimensional (3-D) liver sinusoidal model is constructed using a porous membrane sandwiched between two polydimethylsiloxane (PDMS) layers with respective flow channels. The HCs are cultured in collagen gels of various stiffnesses in the lower channel, whereas the upper channel is pre-seeded with liver sinusoidal endothelial cells (LSECs) and accessible to shear flow. The results reveal that HCs cultured within stiffer matrices exhibit reduced albumin production and cytochrome P450 (CYP450) reductase expression. Low shear stresses enhance synthetic and metabolic functions of HC, whereas high shear stresses lead to the loss of HC phenotype. Furthermore, both mechanical factors regulate HC functions by complementing each other. These observations are likely attributed to mechanically induced mass transport or key signaling molecule of hepatocyte nuclear factor 4α (HNF4α). The present study results provide an insight into understanding the mechanisms of HC dysfunction in liver fibrosis and cirrhosis, especially from the viewpoint of matrix stiffness and blood flow.NEW & NOTEWORTHY A fibrotic three-dimensional (3-D) liver sinusoidal model was constructed to mimic different stages of liver fibrosis in vivo and to explore the cooperative effects of matrix stiffness and shear stresses on hepatocyte (HC) functions. Mechanically induced alterations of mass transport mainly contributed to HC functions via typical mechanosensitive signaling.

Mechanical strength determines Ca2+ transients triggered by the engagement of ß2 integrins to their ligands.

Lymphocyte function-associated antigen-1 (LFA-1) and macrophage-1 antigen (Mac-1) are key adhesion receptors to mediate neutrophil (PMN) recruitment and intracellular calcium (Ca2+) signaling. Binding of LFA-1 and Mac-1 to their ligands is essential in triggering Ca2+ transients and activating Ca2+-dependent kinases involved in cytoskeletal remodeling and migratory function. While mechanical forces are critical in regulating integrin-mediated Ca2+ transients, it is still unclear how the bond strength of ß2-integrin-ligand pair affects Ca2+ responses. Here three typical ligands with known mechanical features with LFA-1 and Mac-1 in our previous work were adopted to quantify their capabilities in inducing Ca2+ transients in adherent PMNs under shear flow. Data indicated that LFA-1 dominates Ca2+ transients in PMNs on intercellular adhesive molecule 1 (ICAM-1) and junctional adhesion molecule-A (JAM-A), while Mac-1 mediates Ca2+ transients induced by receptor for advanced glycation end products (RAGE), consistent with their corresponding bond strengths. These results link ß2 integrin-ligand bond strength with Ca2+ transients in PMNs, suggesting high bond strength gives rise to strong Ca2+ response especially under physiological-like shear flow. The outcomes provide a new insight in understanding the mechanical regulatory mechanisms of PMN recruitment.

Mechanically Regulated Outside-In Activation of an I-Domain-Containing Integrin.

Integrins are heterodimeric transmembrane proteins that mediate cellular adhesion and bidirectional mechanotransductions through their conformational allostery. The allosteric pathway of an I-domain-containing integrin remains unclear because of its complexity and lack of effective experiments. For a typical I-domain-containing integrin αXß2, molecular dynamics simulations were employed here to investigate the conformational dynamics in the first two steps of outside-in activation, the bindings of both the external and internal ligands. Results showed that the internal ligand binding is a prerequisite to the allosteric transmission from the α- to ß-subunits and the exertion of external force to integrin-ligand complex. The opening state of αI domain with downward movement and lower half unfolding of α7-helix ensures the stable intersubunit conformational transmission through external ligand binding first and internal ligand binding later. Reverse binding order induces a, to our knowledge, novel but unstable swingout of ß-subunit Hybrid domain with the retained close states of both αI and ßI domains. Prebinding of external ligand greatly facilitates the following internal ligand binding and vice versa. These simulations furthered the understanding in the outside-in activation of I-domain-containing integrins from the viewpoint of internal allosteric pathways.

Microgravity-induced hepatogenic differentiation of rBMSCs on board the SJ-10 satellite.

Bone marrow-derived mesenchymal stem cells (BMSCs) are able to differentiate into functional hepatocytelike cells, which are expected to serve as a potential cell source in regenerative medicine, tissue engineering, and clinical treatment of liver injury. Little is known about whether and how space microgravity is able to direct the hepatogenic differentiation of BMSCs in the actual space microenvironment. In this study, we examined the effects of space microgravity on BMSC hepatogenic differentiation on board the SJ-10 Recoverable Scientific Satellite. Rat BMSCs were cultured and induced in hepatogenic induction medium for 3 and 10 d in custom-made space cell culture hardware. Cell growth was monitored periodically in orbit, and the fixed cells and collected supernatants were retrieved back to the Earth for further analyses. Data indicated that space microgravity improves the differentiating capability of the cells by up-regulating hepatocyte-specific albumin and cytokeratin 18. The resulting cells tended to be maturated, with an in-orbit period of up to 10 d. In space, mechanosensitive molecules of ß1-integrin, ß-actin, α-tubulin, and Ras homolog gene family member A presented enhanced expression, whereas those of cell-surface glycoprotein CD44, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, vinculin, cell division control protein 42 homolog, and Rho-associated coiled-coil kinase yielded reduced expression. Also observed in space were the depolymerization of actin filaments and the accumulation of microtubules and vimentin through the altered expression and location of focal adhesion complexes, Rho guanosine 5'-triphosphatases, as well as the enhanced exosome-mediated mRNA transfer. This work furthers the understanding of the underlying mechanisms of space microgravity in directing hepatogenic differentiation of BMSCs.-Lü, D., Sun, S., Zhang, F., Luo, C., Zheng, L., Wu, Y., Li, N., Zhang, C., Wang, C., Chen, Q., Long, M. Microgravity-induced hepatogenic differentiation of rBMSCs on board the SJ-10 satellite.

Epidermal restriction confers robustness to organ shapes.

The shape of comparable tissues and organs is consistent among individuals of a given species, but how this consistency or robustness is achieved remains an open question. The interaction between morphogenetic factors determines organ formation and subsequent shaping, which is ultimately a mechanical process. Using a computational approach, we show that the epidermal layer is essential for the robustness of organ geometry control. Specifically, proper epidermal restriction allows organ asymmetry maintenance, and the tensile epidermal layer is sufficient to suppress local variability in growth, leading to shape robustness. The model explains the enhanced organ shape variations in epidermal mutant plants. In addition, differences in the patterns of epidermal restriction may underlie the initial establishment of organ asymmetry. Our results show that epidermal restriction can answer the longstanding question of how cellular growth noise is averaged to produce precise organ shapes, and the findings also shed light on organ asymmetry establishment.

Mechanical Point Loading Induces Cortex Stiffening and Actin Reorganization.

Global cytoskeleton reorganization is well-recognized when cells are exposed to distinct mechanical stimuli, but the localized responses at a specified region of a cell are still unclear. In this work, we mapped the cell-surface mechanical property of single cells in situ before and after static point loading these cells using atomic force microscopy in PeakForce-Quantitative Nano Mechanics mode. Cell-surface stiffness was elevated at a maximum of 1.35-fold at the vicinity of loading site, indicating an enhanced structural protection of the cortex to the cell. Mechanical modeling also elucidated the structural protection from the stiffened cell cortex, in which 9-15% and 10-19% decrease of maximum stress and strain of the nucleus were obtained. Furthermore, the flat-ended atomic force microscopy probes were used to capture cytoskeleton reorganization after point loading quantitatively, revealing that the larger the applied force and the longer the loading time are, the more pronounced cytoskeleton reorganization is. Also, point loading using a microneedle combined with real-time confocal microscopy uncovered the fast dynamics of actin cytoskeleton reorganization for actin-stained live cells after point loading (<10 s). These results furthered the understandings in the transmission of localized mechanical forces into an adherent cell.

Impact of real-time shedding on binding kinetics of membrane-remaining L-selectin to PSGL-1.

L-selectin shedding induced by various cytokines is crucial in activating neutrophils (PMNs) in inflammatory cascade. While the real-time shedding in vivo lasts ~10 min after PMN activation, the impact of time-dependent shedding on binding kinetics of membrane-remaining L-selectins to its ligands is poorly understood at transient or steady state. Here, we developed an in vitro L-selectin shedding dynamics approach, together with competitive assays of cell adhesion, and proposed a theoretical model for quantifying the impact of real-time shedding on the binding kinetics of membrane-remaining L-selectins to P-selectin glycoprotein ligand-1 (PSGL-1). Our data indicated that the extent of L-selectin shedding on PMA activation is higher, but the terminating time is longer for Jurkat cells than those for human PMNs. Meanwhile, fMLF or IL-8 stimulation yields the longer terminating time than that on PMA stimulation but results in a similar shedding extent for PMNs. L-selectin shedding reduces L-selectin-PSGL-1-mediated cell adhesion in three ways: decreasing membrane-anchored L-selectins, increasing soluble L-selectins competitively binding to ligands, and presenting conformational alteration of membrane-remaining L-selectins themselves. Compared with those on intact cells, the binding affinities of membrane-remaining L-selectin-PSGL-1 pairs were all enhanced at initial and lowered at the late shedding phase for both PMN and Jurkat cells even with varied transition time points. The rolling velocities of both PMNs and Jurkat cells were increased following mechanically or biochemically induced shedding of L-selectin under shear flow. These findings help to further our understanding of the function of time-dependent L-selectin shedding during the inflammation cascade.

Binding of intercellular adhesion molecule 1 to ß2-integrin regulates distinct cell adhesion processes on hepatic and cerebral endothelium.

Flowing polymorphonuclear neutrophils (PMNs) are forced to recruit toward inflamed tissue and adhere to vascular endothelial cells, which is primarily mediated by the binding of ß2-integrins to ICAM-1. This process is distinct among different organs such as liver and brain; however, the underlying kinetic and mechanical mechanisms regulating tissue-specific recruitment of PMNs remain unclear. Here, binding kinetics measurement showed that ICAM-1 on murine hepatic sinusoidal endothelial cells (LSECs) bound to lymphocyte function-associated antigen-1 (LFA-1) with higher on- and off-rates but lower effective affinity compared with macrophage-1 antigen (Mac-1), whereas ICAM-1 on cerebral endothelial cells (BMECs or bEnd.3 cells) bound to LFA-1 with higher on-rates, similar off-rates, and higher effective affinity compared with Mac-1. Physiologically, free crawling tests of PMN onto LSEC, BMEC, or bEnd.3 monolayers were consistent with those kinetics differences between two ß2-integrins interacting with hepatic sinusoid or cerebral endothelium. Numerical calculations and Monte Carlo simulations validated tissue-specific contributions of ß2-integrin-ICAM-1 kinetics to PMN crawling on hepatic sinusoid or cerebral endothelium. Thus, this work first quantified the biophysical regulation of PMN adhesion in hepatic sinusoids compared with cerebral endothelium.

Dynamic contributions of P- and E-selectins to ß2-integrin-induced neutrophil transmigration.

Leukocyte transendothelial migration is a key step in their recruitment to sites of inflammation. However, synergic regulation of endothelium-expressed selectins on leukocyte transmigration remains unclear. In this study, an in vitro model was developed to investigate the dynamic contributions of P- and E-selectin to polymorphonuclear neutrophil (PMN) transmigration under static conditions. Human umbilical vein endothelial cells (HUVECs) were treated with LPS for 4 or 12 h to induce different expression of selectins and intercellular adhesion molecule (ICAM)-1. PMN transmigration was increased significantly by LPS stimulation, which was higher on 4-h than on 12-h LPS-treated HUVECs. Blocking and competitive tests indicated that P-selectin engages PSGL-1 to activate ß2-integrin and initiate PMN transmigration within the first 15 min, whereas E-selectin engages CD44 to influence PMN transmigration after 15 min. P- and E-selectin-induced ß2-integrin activation is likely conducted through the spleen tyrosine kinase signaling pathway. Complicated complementary and competitive mechanisms are involved in the interaction of P-/E-selectins and their ligands to promote PMN transmigration. These results provide direct evidence of the distinct and dynamic contribution of P- and E-selectins in mediating PMN transmigration and give new insight into PMN interaction with the vessel wall.-Gong, Y., Zhang, Y., Feng, S., Liu, X., Lü, S., Long, M. Dynamic contributions of P- and E-selectins to ß2-integrin-induced neutrophil transmigration.

Mechanical remodeling of normally sized mammalian cells under a gravity vector.

Translocation of the dense nucleus along a gravity vector initiates mechanical remodeling of a cell, but the underlying mechanisms of cytoskeletal network and focal adhesion complex (FAC) reorganization in a mammalian cell remain unclear. We quantified the remodeling of an MC3T3-E1 cell placed in upward-, downward-, or edge-on-orientated substrate. Nucleus longitudinal translocation presents a high value in downward orientation at 24 h or in edge-on orientation at 72 h, which is consistent with orientation-dependent distribution of perinuclear actin stress fibers and vimentin cords. Redistribution of total FAC area and fractionized super mature adhesion number coordinates this dependence at short duration. This orientation-dependent remodeling is associated with nucleus flattering and lamin A/C phosphorylation. Actin depolymerization or Rho-associated protein kinase signaling inhibition abolishes the orientation dependence of nucleus translocation, whereas tubulin polymerization inhibition or vimentin disruption reserves the dependence. A biomechanical model is therefore proposed for integrating the mechanosensing of nucleus translocation with cytoskeletal remodeling and FAC reorganization induced by a gravity vector.-Zhang, C., Zhou, L., Zhang, F., Lü, D., Li, N., Zheng, L., Xu, Y., Li, Z., Sun, S., Long, M. Mechanical remodeling of normally sized mammalian cells under a gravity vector.

Neutrophil adhesion and crawling dynamics on liver sinusoidal endothelial cells under shear flow.

Neutrophil (polymorphonuclear leukocyte, PMN) recruitment in the liver sinusoid takes place in almost all liver diseases and contributes to pathogen clearance or tissue damage. While PMN rolling unlikely appears in liver sinusoids and Mac-1 or CD44 is assumed to play respective roles during in vivo local or systematic inflammatory stimulation, the regulating mechanisms of PMN adhesion and crawling dynamics are still unclear from those in vivo studies. Here we developed a two-dimensional in vitro sinusoidal model with primary liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs) to investigate TNF-α-induced PMN recruitment under shear flow. Our data demonstrated that LFA-1 dominates the static or shear resistant adhesion of PMNs while Mac-1 decelerates PMN crawling on LSEC monolayer. Any one of LFA-1, Mac-1, and CD44 molecules is not able to work effectively for mediating PMN transmigration across LSEC monolayer. The presence of KCs only affects the randomness of PMN crawling. These findings further the understandings of PMN recruitment under shear flow in liver sinusoids.

ß1 integrin signaling in asymmetric migration of keratinocytes under mechanical stretch in a co-cultured wound repair model.

BACKGROUND: Keratinocyte (KC) migration in re-epithelization is crucial in repairing injured skin. But the mechanisms of how mechanical stimuli regulate the migration of keratinocytes have been poorly understood. METHODS: Human immortalized keratinocyte HaCaT cells were co-cultured with skin fibroblasts on PDMS membranes and transferred to the static stretch device developed in-house for additional 6 day culture under mechanical stretch to mimic surface tension in skin. To detect the expression of proteins on different position at different time points and the effect of ß1 integrin mechanotransduction on HaCaT migration, Immunofluorescence, Reverse transcription-polymerase chain reaction, Flow cytometry, Western blotting assays were applied. RESULTS: Mechanical receptor of ß1 integrin that recognizes its ligand of collagen I was found to be strongly associated with migration of HaCaT cells since the knockdown of ß1 integrin via RNA silence eliminated the key protein expression dynamically. Here the expression of vinculin was lower but that of Cdc42 was higher for the cells at outward edge than those at inward edge, respectively, supporting that the migration capability of keratinocytes is inversely correlated with the formation of focal adhesion complexes but positively related to the lamellipodia formation. This asymmetric expression feature was further confirmed by high or low expression of PI3K for outward- or inward-migrating cells. And ERK1/2 phosphorylation was up-regulated by mechanical stretch. CONCLUSION: We reported here, a novel mechanotransduction signaling pathways were ß1 integrin-dependent pattern of keratinocytes migration under static stretch in an in vitro co-culture model. These results provided an insight into underlying molecular mechanisms of keratinocyte migration under mechanical stimuli.

Distinct binding affinities of Mac-1 and LFA-1 in neutrophil activation.

Macrophage-1 Ag (Mac-1) and lymphocyte function-associated Ag-1 (LFA-1), two ß2 integrins expressed on neutrophils (PMNs), mediate PMN recruitment cascade by binding to intercellular adhesive molecule 1. Distinct functions of LFA-1-initiating PMN slow rolling and firm adhesion but Mac-1-mediating cell crawling are assumed to be governed by the differences in their binding affinities and kinetic rates. In this study, we applied an adhesion frequency approach to compare their kinetics in the quiescent and activated states using three molecular systems, constitutively expressed receptors on PMNs, wild-type and high-affinity (HA) full-length constructs transfected on 293T cells, and wild-type and HA recombinant extracellular constructs. Data indicate that the difference in binding affinity between Mac-1 and LFA-1 is on-rate dominated with slightly or moderately varied off-rate. This finding was further confirmed when both ß2 integrins were activated by chemokines (fMLF or IL-8), divalent cations (Mg(2+) or Mn(2+)), or disulfide bond lockage on an HA state. Structural analyses reveal that such the kinetics difference is likely attributed to the distinct conformations at the interface of Mac-1 or LFA-1 and intercellular adhesive molecule 1. This work furthers the understandings in the kinetic differences between Mac-1 and LFA-1 and in their biological correlations with molecular activation and structural bases.

FAK-p38 signaling serves as a potential target for reverting matrix stiffness-modulated liver sinusoidal endothelial cell defenestration.

Liver sinusoidal endothelial cells (LSECs) are highly specific endothelial cells which play an essential role in the maintenance of liver homeostasis. During the progression of liver fibrosis, matrix stiffening promotes LSEC defenestration, however, the underlying mechanotransduction mechanism remains poorly understood. Here, we applied stiffness-tunable hydrogels to assess the matrix stiffening-induced phenotypic changes in primary mouse LSECs. Results indicated that increased stiffness promoted LSEC defenestration through cytoskeletal reorganization. LSECs sensed the increased matrix stiffness via focal adhesion kinase (FAK), leading to the activation of p38-mitogen activated protein kinase activated protein kinase 2 (MK2) pathway, thereby inducing actin remodeling via LIM Kinase 1 (LIMK1) and Cofilin. Interestingly, inhibition of FAK or p38-MK2 pathway was able to effectively restore the fenestrae to a certain degree in LSECs isolated from early to late stages of liver fibrosis mice. Thus, this study highlights the impact of mechanotransduction in LSEC defenestration, and provides novel insights for potential therapeutic interventions for liver fibrosis.