Cortical Stiffness Research Articles

Endothelial basement membrane laminin 511 is essential for shear stress response

Shear detection and mechanotransduction by arterial endothelium requires junctional complexes containing PECAM‐1 and VE‐cadherin, as well as firm anchorage to the underlying basement membrane. While considerable information is available for junctional complexes in these processes, gained largely from in vitro studies, little is known about the contribution of the endothelial basement membrane. Using resistance artery explants, we show that the integral endothelial basement membrane component, laminin 511 (laminin α5), is central to shear detection and mechanotransduction and its elimination at this site results in ablation of dilation in response to increased shear stress. Loss of endothelial laminin 511 correlates with reduced cortical stiffness of arterial endothelium in vivo, smaller integrin β1‐positive/vinculin‐positive focal adhesions, and reduced junctional association of actin–myosin II. In vitro assays reveal that β1 integrin‐mediated interaction with laminin 511 results in high strengths of adhesion, which promotes p120 catenin association with VE‐cadherin, stabilizing it at cell junctions and increasing cell–cell adhesion strength. This highlights the importance of endothelial laminin 511 in shear response in the physiologically relevant context of resistance arteries.

Lactation-Induced Changes in the Volume of Osteocyte Lacunar-Canalicular Space Alter Mechanical Properties in Cortical Bone Tissue.

Osteocytes can remove and remodel small amounts of their surrounding bone matrix through osteocytic osteolysis, which results in increased volume occupied by lacunar and canalicular space (LCS). It is well established that cortical bone stiffness and strength are strongly and inversely correlated with vascular porosity, but whether changes in LCS volume caused by osteocytic osteolysis are large enough to affect bone mechanical properties is not known. In the current studies we tested the hypotheses that (1) lactation and postlactation recovery in mice alter the elastic modulus of bone tissue, and (2) such local changes in mechanical properties are related predominantly to alterations in lacunar and canalicular volume rather than bone matrix composition. Mechanical testing was performed using microindentation to measure modulus in regions containing solely osteocytes and no vascular porosity. Lactation caused a significant (∼13%) reduction in bone tissue-level elastic modulus (p < 0.001). After 1 week postweaning (recovery), bone modulus levels returned to control levels and did not change further after 4 weeks of recovery. LCS porosity tracked inversely with changes in cortical bone modulus. Lacunar and canalicular void space increased 7% and 15% with lactation, respectively (p < 0.05), then returned to control levels at 1 week after weaning. Neither bone mineralization (assessed by high-resolution backscattered scanning electron microscopy) nor mineral/matrix ratio or crystallinity (assessed by Raman microspectroscopy) changed with lactation. Thus, changes in bone mechanical properties induced by lactation and recovery appear to depend predominantly on changes in osteocyte LCS dimensions. Moreover, this study demonstrates that tissue-level cortical bone mechanical properties are rapidly and reversibly modulated by osteocytes in response to physiological challenge. These data point to a hitherto unappreciated role for osteocytes in modulating and maintaining local bone mechanical properties. © 2016 American Society for Bone and Mineral Research.

Diets High in Fat or Fructose Differentially Modulate Bone Health and Lipid Metabolism.

Diets high in fat or carbohydrates can lead to obesity and diabetes, two interrelated conditions that have been associated with osteoporosis. Here, we contrasted the effects of a high fat (HF) versus fructose-enriched carbohydrate (CH) versus regular chow (SC) diet on bone morphology, fat content and metabolic balance in BALB/cByJ mice over a 15-week period. For 13weeks, there were no differences in body mass between groups with small differences in the last 2weeks. Even without the potentially confounding factor of altered body mass and levels of load bearing, HF consumption was detrimental to bone in the distal femur with lower trabecular bone volume fraction and thinner cortices than controls. These differences in bone were accompanied by twofold greater abdominal fat content and fourfold greater plasma leptin concentrations. High-fat feeding caused a decrease in de-novo lipid synthesis in the liver, kidney, white adipose and brown adipose tissue. In contrast to HF, the fructose diet did not significantly impact bone quantity or architecture. Fructose consumption also did not significantly alter leptin levels or de-novo lipid synthesis but reduced epididymal adipose tissue and increased brown adipose tissue. Cortical stiffness was lower in the CH than in HF mice. There were no differences in glucose or insulin levels between groups. Together, a diet high in fat had a negative influence on bone structure, adipose tissue deposition and lipid synthesis, changes that were largely avoided with a fructose-enriched diet.

Shear wave elastography imaging for assessing the chronic pathologic changes in advanced diabetic kidney disease.

ObjectiveThe assessment of the grade of renal fibrosis in diabetic kidney disease (DKD) requires renal biopsy, which may be associated with certain risks. To assess the severity of chronic pathologic changes in DKD, we performed a quantitative analysis of renal parenchymal stiffness in advanced DKD, using shear wave elastography (SWE) imaging.Patients and methodsTwenty-nine diabetic patients with chronic kidney disease (CKD) grades 3–4 due to DKD, and 23 healthy subjects were enrolled. Combined conventional ultrasound and SWE imaging were performed on all participants. The length, width, and cortical thickness and stiffness were recorded for each kidney.ResultsCortical thickness was lower in patients with DKD than in healthy subjects (13.8±2.2 vs 14.8±1.6 mm; P=0.002) and in DKD patients with CKD grade 4 than in those with grade 3 (13.0±3.5 vs 14.7±2.1 mm; P<0.001). Cortical stiffness was greater in patients with DKD than in healthy subjects (23.72±14.33 vs 9.02±2.42 kPa; P<0.001), in DKD patients with CKD grade 4 than in those with grade 3 (30.4±16.2 vs 14.6±8.1 kPa; P<0.001), and in DKD patients with CKD grade 3b, than in those with CKD grade 3a (15.7±6.7 vs 11.0±4.2 kPa; P=0.03). Daily proteinuria was higher in DKD patients with CKD grade 4 than in those with grade 3 (5.52±0.96 vs 1.13±0.72; P=0.001), and in DKD patients with CKD grade 3b, than in those with CKD grade 3a (1.59±0.59 vs 0.77±0.48; P<0.001). Cortical stiffness was inversely correlated with the estimated glomerular filtration rate (r=−0.65, P<0.001) and with cortical thickness (r=−0.43, P<0.001) in patients with DKD.ConclusionsIn patients with advanced DKD, SWE imaging may be utilized as a simple and practical method for quantitative evaluation of the chronic morphological changes and for the differentiation between CKD grades.

Efficient dispersion analysis of guided waves in cortical bone

Guided wave propagation is at the heart of the axial transmission techniques designed to assess bone health status. The method involves matching observed and predicted dispersion characteristics of guided waves. The strength of the technique is that it can infer more than one bone property from the measured ultrasonic data, such as cortical thickness, stiffness, or porosity. The suitability of the model chosen for the inversion has recently been debated and the question has been raised whether the physical model must take the soft tissue coating influence into account as well as perhaps other factors such as bone curvature. We present in this talk a series of experiments conducted on bone-mimicking phantoms (plates or tubes) with or without soft tissue-mimicking coating showing evidence 1/ that the experimental guided wave branches in the range of 0.4-1.6 MHz mainly exhibit sensitivity to the influence of the solid subsystem (bone) and 2/ that a simple non absorbing transverse isotropic free plate model provides an appropriate inverse model in all investigated cases, i.e., coated or non-coated plates and tubes. Finally, we demonstrate effectiveness of the inversion procedure in characterizing cortical bone using ex vivo and in vivo data.

Regular Exercise Reduces Endothelial Cortical Stiffness in Western Diet-Fed Female Mice.

We recently showed that Western diet-induced obesity and insulin resistance promotes endothelial cortical stiffness in young female mice. Herein, we tested the hypothesis that regular aerobic exercise would attenuate the development of endothelial and whole artery stiffness in female Western diet-fed mice. Four-week-old C57BL/6 mice were randomized into sedentary (ie, caged confined, n=6) or regular exercise (ie, access to running wheels, n=7) conditions for 16 weeks. Exercise training improved glucose tolerance in the absence of changes in body weight and body composition. Compared with sedentary mice, exercise-trained mice exhibited reduced endothelial cortical stiffness in aortic explants (sedentary 11.9±1.7 kPa versus exercise 5.5±1.0 kPa; P<0.05), as assessed by atomic force microscopy. This effect of exercise was not accompanied by changes in aortic pulse wave velocity (P>0.05), an in vivo measure of aortic stiffness. In comparison, exercise reduced femoral artery stiffness in isolated pressurized arteries and led to an increase in femoral internal artery diameter and wall cross-sectional area (P<0.05), indicative of outward hypertrophic remodeling. These effects of exercise were associated with an increase in femoral artery elastin content and increased number of fenestrae in the internal elastic lamina (P<0.05). Collectively, these data demonstrate for the first time that the aortic endothelium is highly plastic and, thus, amenable to reductions in stiffness with regular aerobic exercise in the absence of changes in in vivo whole aortic stiffness. Comparatively, the same level of exercise caused destiffening effects in peripheral muscular arteries, such as the femoral artery, that perfuse the working limbs.

In Vivo Characterization of Cortical Bone Using Guided Waves Measured by Axial Transmission.

Cortical bone loss is not fully assessed by the current X-ray methods, and there is an unmet need in identifying women at risk of osteoporotic fracture, who should receive a treatment. The last decade has seen the emergence of the ultrasound (US) axial transmission (AT) techniques to assess a cortical bone. Recent AT techniques exploit the multimode waveguide response of the long bones such as the radius. A recent ex vivo study by our group evidenced that a multimode AT approach can yield simultaneous estimates of cortical thickness (Ct.Th) and stiffness. The aim of this paper is to move one step forward to evaluate the feasibility of measuring multimode guided waves (GW) in vivo and to infer from it cortical thickness. Measurements were taken on the forearm of 14 healthy subjects with the goal to test the accuracy of the estimated thickness using the bidirectional AT method implemented on a dedicated 1-MHz linear US array. This setup allows determining in vivo the dispersion curves of GW transmitted in the cortical layer of the radius. An inverse procedure based on the comparison between the measured and modeled dispersion curves predicted by a 2-D transverse isotropic free plate waveguide model allowed an estimation of cortical thickness, despite the presence of soft tissue. The Ct.Th values were validated by comparison with the site-matched estimates derived from X-ray high-resolution peripheral quantitative computed tomography. Results showed a significant correlation between both measurements ( r2 = 0.7 , , and [Formula: see text] mm). This pilot study demonstrates the potential of bidirectional AT for the in vivo assessment of cortical thickness, a bone strength-related factor.

Chlorinated Lipids Induce Increased Cortical Stiffness in Microvascular Endothelial Cells

Elastic properties of the vascular endothelium are critical to its role in controlling blood flow, pressure, vascular tone, adhesion of platelets and leukocytes, permeability and other homeostatic functions. Dysregulation of these endothelial functions are hallmarks of the inflammation produced in response to ischemia‐reperfusion, but it is not clear to what extent altered endothelial elasticity plays a role in the endothelial response to specific pro‐inflammatory stimuli. Cellular and plasma lipids that have been chlorinated by reacting with hypochlorous acid (HOCl) produced by neutrophil myeloperoxidase (MPO) have been shown to increase several markers of endothelial inflammatory activation. However, their effect on endothelial elasticity is unknown. We have begun to explore this issue in cultured endothelial cells. Human microvascular endothelial cells (HMEC‐1) were cultured to varying levels of confluence, incubated with 10 μM of 2‐chlorohexadecanoic acid (Cl‐HA), hexadecanoic acid (HA), 2‐chlorohexadecanal (Cl‐HDA), or hexadecanal (HDA), or 10 ng/ml TNF‐α, and cortical stiffness was measured using atomic force microscopy (AFM). Treatment with chlorinated lipids showed confluence‐dependent increases in stiffness, while non‐chlorinated lipids tended to decrease stiffness when compared with EtOH diluent. While the effect of chlorinated lipid varied with confluence, TNF‐α (positive control) produced consistent results independent of culture confluence. These results show for the first time, that the chlorinated lipids known to be produced through reaction with neutrophil MPO‐derived HOCl, increase cortical stiffness in microvascular endothelial cells.Support or Funding InformationRO1‐GM115553 &amp; R01‐AA221081

Ex Vivo Assessment of Sentinel Lymph Nodes in Breast Cancer Using Shear Wave Elastography.

Axillary lymph node status is one of the important prognostic factors in early-stage breast cancer. Despite the combined use of sonography, fine-needle aspiration, and sentinel lymph node (SLN) dissection, there is a gap between the potential effectiveness of those techniques and current success to determine the axillary lymph node status. The main aim of this study was to evaluate the baseline accuracy of shear wave elastography for differentiation of benign versus malignant SLNs in an ex vivo artifact-free environment. Thirty patients with breast cancer scheduled for SLN dissection were enrolled prospectively after informed consent and Institutional Review Board approval were obtained. After dissection, lymph nodes were embedded in ultrasound gel and examined with grayscale sonography and shear wave elastography. Findings were compared to histopathologic results. A total of 64 SLNs obtained from the 30 patients were evaluated. Twelve of them (18.8%) were metastatic, and 52 (81.2%) were benign. The mean cortical thickness (benign versus metastatic, 1.6 versus 4.4 mm), short-axis length (4.63 versus 7.50 mm), cortical stiffness (10.7 versus 25.5 kPa), and hilar stiffness (7.5 versus 11.3 kPa) were statistically higher in metastatic lymph nodes (P ≤ .02). Area under the receiver operator characteristic curve values for these variables were 0.814, 0.768, 0.786, and 0.759, respectively. Cortical thickness was found to have the highest diagnostic performance, followed by cortical stiffness. Shear wave elastography can be used with grayscale sonography for evaluation of cases to decide on needle biopsy sampling. However, it cannot be used as a replacement for fine-needle aspiration or SLN dissection.

A Parallel-Plate Flow Chamber for Mechanical Characterization of Endothelial Cells Exposed to Laminar Shear Stress.

Shear stresses induced by laminar fluid flow are essential to properly recapitulate the physiological microenvironment experienced by endothelial cells (ECs). ECs respond to these stresses via mechanotransduction by modulating their phenotype and biomechanical characteristics, which can be characterized by Atomic Force Microscopy (AFM). Parallel Plate Flow Chambers (PPFCs) apply unidirectional laminar fluid flow to EC monolayers in vitro. Since ECs in sealed PPFCs are inaccessible to AFM probes, cone-and-plate viscometers (CPs) are commonly used to apply shear stress. This paper presents a comparison of the efficacies of both methods. Computational Fluid Dynamic simulation and validation testing using EC responses as a metric have indicated limitations in the use of CPs to apply laminar shear stress. Monolayers subjected to laminar fluid flow in a PPFC respond by increasing cortical stiffness, elongating, and aligning filamentous actin in the direction of fluid flow to a greater extent than CP devices. Limitations using CP devices to provide laminar flow across an EC monolayer suggest they are better suited when studying EC response for disturbed flow conditions. PPFC platforms allow for exposure of ECs to laminar fluid flow conditions, recapitulating cellular biomechanical behaviors, whereas CP platforms allow for mechanical characterization of ECs under secondary flow.

Balanced cortical stiffness is important for efficient migration of Dictyostelium cells in confined environments

Dictyostelium discoideum cells resemble in many aspects human leukocytes and serve as a model to study actin cytoskeleton dynamics and cell migration of highly motile cells. Dictyostelium cells deficient in the actin-binding protein filamin (ddFLN) showed a surprisingly subtle change in phenotype with no or only minor effects in single cell motility. These findings were in contrast to the strong actin-crosslinking activities measured for filamin in vitro. In the present study, we set out to revisit the role of ddFLN in cell migration. For this purpose, we examined migration of wild-type, ddFLN-null and ddFLN-overexpressing cells under different conditions. In addition to cyclic-AMP chemotaxis assays using micropipettes, we explored cell migration under more confined conditions: an under-agarose 2D assay and a 3D assay employing a collagen matrix that was adapted from assays for leukocytes. Using 3D migration conditions, cells deficient in ddFLN displayed only a minor impairment of motility, similar to the results obtained for migration in 2D. However, cells overexpressing ddFLN showed a remarkable decrease in the speed of migration in particular in 3D environments. We suggest that these results are in line with an increased stiffening of the cortex due to the crosslinking activity of overexpressed ddFLN. Our conclusion is that the absolute level of ddFLN is critical for efficient migration. Furthermore, our results show that under conditions of increased mechanical stress, Dictyostelium cells, like leukocytes, switch to a bleb-based mode of movement.

Aldosterone synthase knockout mouse as a model for sodium-induced endothelial sodium channel up-regulation in vascular endothelium.

Recently, a novel feedforward activation of the endothelial epithelial sodium channel (ENaC) [endothelial sodium channel (EnNaC)] by sodium was reported that counteracts ENaC function in kidney. In the absence of aldosterone, a rise in extracellular sodium (>145 mM) increases EnNaC surface abundance, thereby stiffening the cortex of vascular endothelial cells (ECs) in vitro. The latter reduces the release of NO-the hallmark of endothelial dysfunction. Here, we test whether high extracellular sodium per se increases EnNaC expression and cortical stiffness in an aldosterone synthase (Cyp11b2)-deficient (AS(-/-)) mouse model. Therefore, we employed in situ ECs of ex vivo aorta preparations from wild-type (WT) and AS(-/-). EnNaC surface expression (-16%) and cortical stiffness (-22%) were reduced in AS(-/-), compared with WT, whereas NO secretion was exclusively detectable in AS(-/-). EnNaC inhibition with benzamil decreased stiffness in both, while mineralocorticoid receptor antagonism diminished stiffness only in the WT. In the absence of aldosterone, high sodium (150 mM) increased EnNaC surface expression ex vivo (plus 19%) and cortical stiffness ex vivo (plus 41%) and in vivo (plus 44%). Application of aldosterone adjusted the stiffness of AS(-/-) to the WT level. We conclude that high sodium per se determines EnNaC expression and consequently endothelial cortical nanomechanics, thus likely contributing to endothelial dysfunction.

Cortical actin regulation modulates vascular contractility and compliance in veins.

Most cardiovascular research focuses on arterial mechanisms of disease, largely ignoring venous mechanisms. Here we examine ex vivo venous stiffness, spanning tissue to molecular levels, using biomechanics and magnetic microneedle technology, and show for the first time that venous stiffness is regulated by a molecular actin switch within the vascular smooth muscle cell in the wall of the vein. This switch connects the contractile apparatus within the cell to adhesion structures and facilitates stiffening of the vessel wall, regulating blood flow return to the heart. These studies also demonstrate that passive stiffness, the component of total stiffness not attributable to vascular smooth muscle activation, is severalfold lower in venous tissue than in arterial tissue. We show here that the activity of the smooth muscle cells plays a dominant role in determining total venous stiffness and regulating venous return. The literature on arterial mechanics is extensive, but far less is known about mechanisms controlling mechanical properties of veins. We use here a multi-scale approach to identify subcellular sources of venous stiffness. Portal vein tissue displays a severalfold decrease in passive stiffness compared to aortic tissues. The α-adrenergic agonist phenylephrine (PE) increased tissue stress and stiffness, both attenuated by cytochalasin D (CytoD) and PP2, inhibitors of actin polymerization and Src activity, respectively. We quantify, for the first time, cortical cellular stiffness in freshly isolated contractile vascular smooth muscle cells using magnetic microneedle technology. Cortical stiffness is significantly increased by PE and CytoD inhibits this increase but, surprisingly, PP2 does not. No detectable change in focal adhesion size, measured by immunofluorescence of FAK and zyxin, accompanies the PE-induced changes in cortical stiffness. Probing with phospho-specific antibodies confirmed activation of FAK/Src and ERK pathways and caldesmon phosphorylation. Thus, venous tissue stiffness is regulated both at the level of the smooth muscle cell cortex, via cortical actin polymerization, and by downstream smooth muscle effectors of Src/ERK signalling pathways. These findings identify novel potential molecular targets for the modulation of venous capacitance and venous return in health and disease.

Cystic fibrosis transmembrane conductance regulator is involved in polyphenol-induced swelling of the endothelial glycocalyx

Previous studies show that polyphenol-rich compounds can induce a swelling of the endothelial glycocalyx (eGC). Our goal was to reveal the mechanism behind the eGC-swelling. As polyphenols are potent modulators of fibrosis transmembrane conductance regulator (CFTR) Cl− channel, the hypothesis was tested whether polyphenol-induced increase in CFTR activity is responsible for the eGC-swelling. The impact of the polyphenols resveratrol, (−)-epicatechin, and quercetin on nanomechanics of living endothelial GM7373 cells was monitored by AFM-nanoindentation. The tested polyphenols lead to eGC-swelling with a simultaneous decrease in cortical stiffness. EGC-swelling, but not the change in cortical stiffness, was prevented by the inhibition of CFTR. Polyphenol-induced eGC-swelling could be mimicked by cytochalasin D, an actin-depolymerizing agent. Thus, in the vascular endothelium, polyphenols induce eGC-swelling by softening cortical actin and activating CFTR. Our findings imply that CFTR plays an important role in the maintenance of vascular homeostasis and may explain the vasoprotective properties of polyphenols. From the Clinical EditorMany vascular problems clinically can be attributed to a dysregulation of endothelial glycocalyx (eGC). The underlying mechanism however remains unclear. In this article, the authors used nanoindentation and showed that polyphenols could swell the endothelial glycocalyx and alter its function. This investigative method can lead to further mechanistic studies of other molecular pathways.

Emerging Brain Morphologies from Axonal Elongation.

Understanding the characteristic morphology of our brain remains a challenging, yet important task in human evolution, developmental biology, and neurosciences. Mathematical modeling shapes our understanding of cortical folding and provides functional relations between cortical wavelength, thickness, and stiffness. Yet, current mathematical models are phenomenologically isotropic and typically predict non-physiological, periodic folding patterns. Here we establish a mechanistic model for cortical folding, in which macroscopic changes in white matter volume are a natural consequence of microscopic axonal growth. To calibrate our model, we consult axon elongation experiments in chick sensory neurons. We demonstrate that a single parameter, the axonal growth rate, explains a wide variety of in vitro conditions including immediate axonal thinning and gradual thickness restoration. We embed our axonal growth model into a continuum model for brain development using axonal orientation distributions motivated by diffusion spectrum imaging. Our simulations suggest that white matter anisotropy-as an emergent property from directional axonal growth-intrinsically induces symmetry breaking, and predicts more physiological, less regular morphologies with regionally varying gyral wavelengths and sulcal depths. Mechanistic modeling of brain development could establish valuable relationships between brain connectivity, brain anatomy, and brain function.

Chorein addiction in VPS13A overexpressing rhabdomyosarcoma cells.

Chorein encoded by VPS13A (vacuolar protein sorting-associated protein 13A) is defective in chorea-acanthocytosis. Chorein fosters neuronal cell survival, cortical actin polymerization and cell stiffness. In view of its anti-apoptotic effect in neurons, we explored whether chorein is expressed in cancer cells and influences cancer cell survival. RT-PCR was employed to determine transcript levels, specific siRNA to silence chorein, FACS analysis to follow apoptosis and Western blotting to quantify protein abundance. Chorein transcripts were detected in various cancer cell types. The mRNA coding for chorein and chorein protein were most abundant in drug resistant, poorly differentiated human rhabdomyosarcoma cells. Chorein silencing significantly reduced the ratio of phosphorylated (and thus activated) to total phosphoinositide 3 kinase (PI-3K), pointing to inactivation of this crucial pro-survival signaling molecule. Moreover, chorein silencing diminished transcript levels and protein expression of anti-apoptotic BCL-2 and enhanced transcript levels of pro-apoptotic Bax. Silencing of chorein in rhabdomyosarcoma cells was followed by mitochondrial depolarization, caspase 3 activation and stimulation of early and late apoptosis. In conclusion, chorein is expressed in various cancer cells. In cells with high chorein expression levels chorein silencing promotes apoptotic cell death, an effect paralleled by down-regulation of PI-3K activity and BCL-2/Bax expression ratio.

Characterization of Biomechanical Properties of Primary Endothelial Cells Exposed to Shear Stress

Mechanotransduction, the process where cells convert biophysical signals into chemical signals, directs the behavior and function of endothelial cells (ECs) because of their unique position on the luminal surface of blood vessels. ECs are in direct contact with blood and can sense changes in shear stress caused by alterations in flow rate. To investigate the biophysical implications of this response we recapitulated physiological levels of shear stress in vitro using two systems, imaged and measured the consequent cytoskeletal changes with immunofluorescence, and quantified cortical stiffness distributions. We developed a sampling method for Atomic Force Microscopy (AFM) stiffness measurements by which force-indentation data is collected in a uniform grid (force map) over multiple cells within a monolayer. In this way, the sampling bias resulting from the selection of force-indentation measurements was reduced. Hertzian contact modeling for AFM was used to specifically isolate the cortical stiffness of the cell using a pyramidal indenter and small indentation depths (100 nm). Nonparametric statistics were used to compare the distributions of moduli from a given experiment, as the data distribution was not Gaussian. Actin filament alignment was quantified using Hough Transforms on immunofluorescence images and increased when cells were exposed to shear stress. The medians of stiffness distributions within sheared EC monolayers increased in magnitude when compared to static monolayers, yielding a method-dependent increase in cortical stiffness of up to 60%. This demonstrates that physiological levels of shear stress dramatically affect ECs morphology in a way that cannot be simulated under static conditions. We concluded that the biophysical milieu of a cell's physiological microenvironment must be recapitulated in order to represent characteristic cellular mechanical properties in vitro.

Biomechanical properties of bone in a mouse model of Rett syndrome

Rett syndrome (RTT) is an X-linked genetic disorder and a major cause of intellectual disability in girls. Mutations in the methyl-CpG binding protein 2 (MECP2) gene are the primary cause of the disorder. Despite the dominant neurological phenotypes, MECP2 is expressed ubiquitously throughout the body and a number of peripheral phenotypes such as scoliosis, reduced bone mineral density and skeletal fractures are also common and important clinical features of the disorder. In order to explore whether MeCP2 protein deficiency results in altered structural and functional properties of bone and to test the potential reversibility of any defects, we have conducted a series of histological, imaging and biomechanical tests of bone in a functional knockout mouse model of RTT. Both hemizygous Mecp2stop/y male mice in which Mecp2 is silenced in all cells and female Mecp2stop/+ mice in which Mecp2 is silenced in ~50% of cells as a consequence of random X-chromosome inactivation, revealed significant reductions in cortical bone stiffness, microhardness and tensile modulus. Microstructural analysis also revealed alterations in both cortical and cancellous femoral bone between wild-type and MeCP2-deficient mice. Furthermore, unsilencing of Mecp2 in adult mice cre-mediated stop cassette deletion resulted in a restoration of biomechanical properties (stiffness, microhardness) towards wild-type levels. These results show that MeCP2-deficiency results in overt, but potentially reversible, alterations in the biomechanical integrity of bone and highlights the importance of targeting skeletal phenotypes in considering the development of pharmacological and gene-based therapies.

Environmental particulate (PM2.5) augments stiffness-induced alveolar epithelial cell mechanoactivation of transforming growth factor beta.

Dysfunctional pulmonary homeostasis and repair, including diseases such as pulmonary fibrosis (PF), chronic obstructive pulmonary disease (COPD), and tumorigenesis have been increasing over the past decade, a fact that heavily implicates environmental influences. Several investigations have suggested that in response to increased transforming growth factor - beta (TGFβ) signaling, the alveolar type II (ATII) epithelial cell undergoes phenotypic changes that may contribute to the complex pathobiology of PF. We have previously demonstrated that increased tissue stiffness associated with PF is a potent extracellular matrix (ECM) signal for epithelial cell activation of TGFβ. The work reported here explores the relationship between tissue stiffness and exposure to environmental stimuli in the activation of TGFβ. We hypothesized that exposure of ATII cells to fine particulate matter (PM2.5) will result in enhanced cell contractility, TGFβ activation, and subsequent changes to ATII cell phenotype. ATII cells were cultured on increasingly stiff substrates with or without addition of PM2.5. Exposure to PM2.5 resulted in increased activation of TGFβ, increased cell contractility, and elongation of ATII cells. Most notably, on 8 kPa substrates, a stiffness greater than normal but less than established fibrotic lung, addition of PM2.5 resulted in increased cortical cell stiffness, enhanced actin staining and cell elongation; a result not seen in the absence of PM2.5. Our work suggests that PM2.5 exposure additionally enhances the existing interaction between ECM stiffness and TGFβ that has been previously reported. Furthermore, we show that this additional enhancement is likely a consequence of intracellular reactive oxygen species (ROS) leading to increased TGFβ signaling events. These results highlight the importance of both the micromechanical and biochemical environment in lung disease initiation and suggest that individuals in early stages of lung remodeling during fibrosis may be more susceptible than healthy individuals when exposed to environmental injury adjuvants.

Probing Cellular Mechanoadaptation Using Cell-Substrate De-Adhesion Dynamics: Experiments and Model

Physical properties of the extracellular matrix (ECM) are known to regulate cellular processes ranging from spreading to differentiation, with alterations in cell phenotype closely associated with changes in physical properties of cells themselves. When plated on substrates of varying stiffness, fibroblasts have been shown to exhibit stiffness matching property, wherein cell cortical stiffness increases in proportion to substrate stiffness up to 5 kPa, and subsequently saturates. Similar mechanoadaptation responses have also been observed in other cell types. Trypsin de-adhesion represents a simple experimental framework for probing the contractile mechanics of adherent cells, with de-adhesion timescales shown to scale inversely with cortical stiffness values. In this study, we combine experiments and computation in deciphering the influence of substrate properties in regulating de-adhesion dynamics of adherent cells. We first show that NIH 3T3 fibroblasts cultured on collagen-coated polyacrylamide hydrogels de-adhere faster on stiffer substrates. Using a simple computational model, we qualitatively show how substrate stiffness and cell-substrate bond breakage rate collectively influence de-adhesion timescales, and also obtain analytical expressions of de-adhesion timescales in certain regimes of the parameter space. Finally, by comparing stiffness-dependent experimental and computational de-adhesion responses, we show that faster de-adhesion on stiffer substrates arises due to force-dependent breakage of cell-matrix adhesions. In addition to illustrating the utility of employing trypsin de-adhesion as a biophysical tool for probing mechanoadaptation, our computational results highlight the collective interplay of substrate properties and bond breakage rate in setting de-adhesion timescales.