Here we report a proof-of-concept for development of pancreatic islet-targeting nanoparticles for immunomodulatory therapy of autoimmune Type 1 Diabetes. Modified with a unique islet-homing peptide, these polymeric nanomaterials exhibit 3-fold greater binding to islet endothelial cells and a 200-fold greater anti-inflammatory effect through targeted islet endothelial cell delivery of an immunosuppressant drug. Our findings also underscore the need to carefully tailor drug loading and nanoparticle dosage to achieve maximal vascular targeting and immunosuppression.
Diabetes; pancreatic islet; nanoparticle; drug delivery; vascular endothelium; inflammation; tissue targeting
This paper focuses on the development of magnetic cellular switches to enable magnetic control of intracellular functions in living mammalian cells, including receptor signal transduction and gene transcription. Our approach takes advantage of the mechanosensitivity of adenosine 3′,5′-monophosphate (cAMP) induction and downstream transcription controlled by the cAMP regulatory element (CRE) to engineer gene constructs that optically report gene expression in living cells. We activate transcription of these gene reporters by applying magnetic (mechanical) stress to magnetic microbeads bound to cell surface integrin receptors. In these gene reporter constructs, CRE motifs drive the expression of fluorescent proteins or enzymes that produce fluorescent products, such as DsRed and β-lactamase (BLA), respectively. We demonstrate that a chemical inducer of cAMP (forskolin) increases expression of CRE-DsRed in living cells. More importantly, a threefold increase in CRE-BLA expression is induced by application of mechanical stress to magnetic microbeads (4.5 µm) bound to cell surface integrin receptors. Induction of cAMP could be detected within 5 min using a protein fragment complementation assay involving interactions between the KID and KIX domains of the CRE binding protein linked to complementary halves of the BLA enzyme. These studies confirm that application of magnetic stress to integrins induces gene transcription by activating the cAMP-dependent transcription factor CREB. Ongoing studies focus on optimizing sensitivity and reducing signal-to-noise by establishing stable cell lines that express these gene reporters. These studies collectively demonstrate the feasibility of using magnetic technologies to control function in living mammalian cells and, hence, support the possibility of developing magnetically-actuated cellular components for use in future micro- and nanotechnologies.
Biological cells; biological control systems; biological signal transduction; biomagnetics
Mesenchymal condensation is critical for organogenesis, yet little is known about how this process is controlled. Here we show that Fgf8 and Sema3f produced by early dental epithelium respectively attract and repulse mesenchymal cells, which causes them to pack tightly together during mouse tooth development. Resulting mechanical compaction-induced changes in cell shape induce odontogenic transcription factors (Pax9, Msx1) and chemical cue (BMP4), and mechanical compression of mesenchyme is sufficient to induce tooth-specific cell fate switching. The inductive effects of cell compaction are mediated by suppression of the mechanical signaling molecule RhoA, and its over-expression prevents odontogenic induction. Thus, the mesenchymal condensation that drives tooth formation is induced by antagonistic epithelial morphogens that manifest their pattern-generating actions mechanically via changes in mesenchymal cell shape and altered mechanotransduction.
Mesenchymal condensation; organogenesis; epithelial-mesenchymal interactions; mechanical forces; cell shape; Fgf8; Sema3f; Nrp2; Pax9; RhoA; tooth
HLA-B27 is associated with spontaneous viral clearance in hepatitis C virus (HCV) infection. Viral escape within the immunodominant HLA-B27 restricted HCV-specific CD8+ T cell epitope NS5B2841-2849 (ARMILMTHF) has been shown to be limited by viral fitness costs as well as broad T cell cross-recognition, suggesting a potential mechanism of protection by HLA-B27. Here, we studied the subdominant HLA-B27 restricted epitope NS5B2936-2944 (GRAAICGKY) in order to further define the mechanisms of protection by HLA-B27. We identified a unique pattern of escape mutations within this epitope in a large cohort of HCV genotype 1a infected patients. The predominant escape mutations represented conservative substitutions at the main HLA-B27 anchor residue or a T cell receptor contact site, neither of which impaired viral replication capacity as assessed in a subgenomic HCV replicon system. In contrast, however, in a subset of HLA-B27+ subjects rare escape mutations arose at the HLA-B27 anchor residue R2937, which nearly abolished viral replication. Notably, these rare mutations only occurred in conjunction with the selection of two equally rare, and structurally proximal, upstream mutations. Co-expression of these upstream mutations with the rare escape mutations dramatically restored viral replication capacity from <5% to ≥70% of wild-type levels.
The selection of rare CTL escape mutations in this HLA-B27 restricted epitope dramatically impairs viral replicative fitness unless properly compensated. These data support a role for the targeting of highly-constrained regions by HLA-B27 in its ability to assert immune control of HCV and other highly variable pathogens.
Hepatitis C virus; T cell response; viral escape; HLA-B27; viral fitness
Angiogenesis is crucial for lung development. Although there has been considerable exploration, the mechanism by which lung vascular and alveolar formation is controlled is still not completely understood. Here we show that low-density lipoprotein receptor-related protein 5 (LRP5), a component of the Wnt ligand-receptor complex, regulates angiogenesis and alveolar formation in the lung by modulating expression of the angiopoietin (Ang) receptor, Tie2, in vascular endothelial cells (ECs). Vascular development in whole mouse lungs and in cultured ECs is controlled by LRP5 signaling, which is, in turn, governed by a balance between the activities of the antagonistic Tie2 ligands, Ang1 and Ang2. Under physiological conditions when Ang1 is dominant, LRP5 knockdown decreases Tie2 expression and thereby, inhibits vascular and alveolar development in the lung. Conversely, when Ang2 dominates under hyperoxia treatment in neonatal mice, high LRP5 and Tie2 expression suppress angiogenesis and lung development. These findings suggest that the LRP5-Tie2-Ang signaling axis plays a central role in control of both angiogenesis and alveolarization during postnatal lung development, and that deregulation of this signaling mechanism might lead to developmental abnormalities of the lung, such as are observed in bronchopulmonary dysplasia (BPD).
Epoxyeicosatrienoic acids (EETs) are small molecules produced by cytochrome P450 epoxygenases. They are lipid mediators that act as autocrine or paracrine factors to regulate inflammation and vascular tone. As a result, drugs that raise EET levels are in clinical trials for the treatment of hypertension and many other diseases. However, despite their pleiotropic effects on cells, little is known about the role of these epoxyeicosanoids in cancer. Here, using genetic and pharmacological manipulation of endogenous EET levels, we demonstrate that EETs are critical for primary tumor growth and metastasis in a variety of mouse models of cancer. Remarkably, we found that EETs stimulated extensive multiorgan metastasis and escape from tumor dormancy in several tumor models. This systemic metastasis was not caused by excessive primary tumor growth but depended on endothelium-derived EETs at the site of metastasis. Administration of synthetic EETs recapitulated these results, while EET antagonists suppressed tumor growth and metastasis, demonstrating in vivo that pharmacological modulation of EETs can affect cancer growth. Furthermore, inhibitors of soluble epoxide hydrolase (sEH), the enzyme that metabolizes EETs, elevated endogenous EET levels and promoted primary tumor growth and metastasis. Thus, our data indicate a central role for EETs in tumorigenesis, offering a mechanistic link between lipid signaling and cancer and emphasizing the critical importance of considering possible effects of EET-modulating drugs on cancer.
Physical interactions between cells and the extracellular matrix (ECM) guide directional migration by spatially controlling where cells form focal adhesions (FAs), which in turn regulate the extension of motile processes. Here we show that physical control of directional migration requires the FA scaffold protein paxillin. Using single-cell sized ECM islands to constrain cell shape, we found that fibroblasts cultured on square islands preferentially activated Rac and extended lamellipodia from corner, rather than side regions after 30 min stimulation with PDGF, but that cells lacking paxillin failed to restrict Rac activity to corners and formed small lamellipodia along their entire peripheries. This spatial preference was preceded by non-spatially constrained formation of both dorsal and lateral membrane ruffles from 5–10 min. Expression of paxillin N-terminal (paxN) or C-terminal (paxC) truncation mutants produced opposite, but complementary, effects on lamellipodia formation. Surprisingly, pax−/− and paxN cells also formed more circular dorsal ruffles (CDRs) than pax+ cells, while paxC cells formed fewer CDRs and extended larger lamellipodia even in the absence of PDGF. In a two-dimensional (2D) wound assay, pax−/− cells migrated at similar speeds to controls but lost directional persistence. Directional motility was rescued by expressing full-length paxillin or the N-terminus alone, but paxN cells migrated more slowly. In contrast, pax−/− and paxN cells exhibited increased migration in a three-dimensional (3D) invasion assay, with paxN cells invading Matrigel even in the absence of PDGF. These studies indicate that paxillin integrates physical and chemical motility signals by spatially constraining where cells will form motile processes, and thereby regulates directional migration both in 2D and 3D. These findings also suggest that CDRs may correspond to invasive protrusions that drive cell migration through 3D extracellular matrices.
Integrins are ubiquitous transmembrane mechanoreceptors that elicit changes in intracellular biochemistry in response to mechanical force application, but these alterations generally proceed over seconds to minutes. Stress-sensitive ion channels represent another class of mechanoreceptors that are activated much more rapidly (within msec), and recent findings suggest that calcium influx through Transient Receptor Potential Vanilloid-4 (TRPV4) channels expressed in the plasma membrane of bovine capillary endothelial cells is required for mechanical strain-induced changes in focal adhesion assembly, cell orientation and directional migration. However, whether mechanically stretching a cell’s extracellular matrix (ECM) adhesions might directly activate cell surface ion channels remains unknown. Here we show that forces applied to β1 integrins result in ultra-rapid (within 4 msec) activation of calcium influx through TRPV4 channels. The TRPV4 channels were specifically activated by mechanical strain in the cytoskeletal backbone of the focal adhesion, and not by deformation of the lipid bilayer or submembranous cortical cytoskeleton alone. This early-immediate calcium signaling response required the distal region of the β1 integrin cytoplasmic tail that contains a binding site for the integrin-associated transmembrane CD98 protein, and external force application to CD98 within focal adhesions activated the same ultra-rapid calcium signaling response. Local direct strain-dependent activation of TRPV4 channels mediated by force transfer from integrins and CD98 may therefore enable compartmentalization of calcium signaling within focal adhesions that is critical for mechanical control of many cell behaviors that underlie cell and tissue development.
The advent of the mid-urethral sling (MUS) 15 years ago has drastically changed the surgical management of stress urinary incontinence (SUI). Both retropubic and transobturator MUS can be placed in the ambulatory setting with excellent results. The tension-free vaginal tape (TVT) sling has the most robust and long-term data, but more recent literature suggests that the transobturator tape sling may offer comparable efficacy in appropriately selected patients. Single incision sling (SIS) is the newest addition to the MUS group and was developed in an attempt to minimize morbidity and create an anti-incontinence procedure that could be performed in the office. The efficacy of SIS remains unknown as the current literature regarding SIS lacks long-term results and comparative trials. The suprapubic arc sling appears to have equally effective outcomes in at least the short-term when compared with TVT. Although evolution of the SIS has led to a less invasive procedure with decreased post-op pain and reduced recovery time, durability of efficacy could be the endpoint we are sacrificing. Until longer-term data and more quality comparison trials are available, tailoring one's choice of MUS to the individual patient and her unique clinical parameters remains the best option.
Incontinence surgery; mid-urethral sling; stress urinary incontinence; tension-free vaginal tape; transobturator tape
In vitro 3D culture is an important model for tissues in
vivo. Cells in different locations of 3D tissues are
physiologically different, because they are exposed to different concentrations
of oxygen, nutrients, and signaling molecules, and to other environmental
factors (temperature, mechanical stress, etc). The majority of high-throughput
assays based on 3D cultures, however, can only detect the
average behavior of cells in the whole 3D construct.
Isolation of cells from specific regions of 3D cultures is possible, but relies
on low-throughput techniques such as tissue sectioning and micromanipulation.
Based on a procedure reported previously (“cells-in-gels-in-paper”
or CiGiP), this paper describes a simple method for culture of arrays of thin
planar sections of tissues, either alone or stacked to create more complex 3D
tissue structures. This procedure starts with sheets of paper patterned with
hydrophobic regions that form 96 hydrophilic zones. Serial spotting of cells
suspended in extracellular matrix (ECM) gel onto the patterned paper creates an
array of 200 micron-thick slabs of ECM gel (supported mechanically by cellulose
fibers) containing cells. Stacking the sheets with zones aligned on top of one
another assembles 96 3D multilayer constructs. De-stacking the layers of the 3D
culture, by peeling apart the sheets of paper, “sections” all 96
cultures at once. It is, thus, simple to isolate 200-micron-thick
cell-containing slabs from each 3D culture in the 96-zone array. Because the 3D
cultures are assembled from multiple layers, the number of cells plated
initially in each layer determines the spatial distribution of cells in the
stacked 3D cultures. This capability made it possible to compare the growth of
3D tumor models of different spatial composition, and to examine the migration
of cells in these structures.
The actin cross-linking protein filamin A reduces migration, invasion, and metastasis of breast cancer cells.
The actin cross-linking protein filamin A (FLNa) functions as a scaffolding protein and couples cell cytoskeleton to extracellular matrix and integrin receptor signaling. In this study, we report that FLNa suppresses invasion of breast cancer cells and regulates focal adhesion (FA) turnover. Two large progression tissue microarrays from breast cancer patients revealed a significant decrease of FLNa levels in tissues from invasive breast cancer compared with benign disease and in lymph node–positive compared with lymph node–negative breast cancer. In breast cancer cells and orthotopic mouse breast cancer models, down-regulation of FLNa stimulated cancer cell migration, invasion, and metastasis formation. Time-lapse microscopy and biochemical assays after FLNa silencing and rescue with wild-type or mutant protein resistant to calpain cleavage revealed that FLNa regulates FA disassembly at the leading edge of motile cells. Moreover, FLNa down-regulation enhanced calpain activity through the mitogen-activated protein kinase–extracellular signal-regulated kinase cascade and stimulated the cleavage of FA proteins. These results document a regulation of FA dynamics by FLNa in breast cancer cells.
When challenged with extracellular fluid shear stress, vascular endothelial cells are known to release nitric oxide, an important vasodilator. Here, we show that the ability of cultured endothelial cells to sense a low range of fluid shear depends on apical membrane organelles, called cilia, and that cilia are compartments required for proper localization and function of the mechanosensitive polycystin-1 molecule.
Methods and Results
Cells with the Pkd1null/null or Tg737orpk/orpk mutation encoded for polycystin-1 or polaris, respectively, are unable to transmit extracellular shear stress into intracellular calcium signaling and biochemical nitric oxide synthesis. Cytosolic calcium and nitric oxide recordings further show that fluid shear sensing is a cilia-specific mechanism because other mechanical or pharmacological stimulation does not abolish calcium and nitric oxide signaling in polycystin-1 and polaris mutant endothelial cells. Polycystin-1 localized in the basal body of Tg737orpk/orpk endothelial cells is insufficient for a fluid shear stress response. Furthermore, the optimal shear stress to which the cells respond best does not alter the apical cilia structure but modifies the responsiveness of cells to higher shear stresses through proteolytic modification of polycystin-1.
We demonstrate for the first time that polycystin-1 (required for cilia function) and polaris (required for cilia structure) are crucial mechanosensitive molecules in endothelial cells. We propose that a distinctive communication with the extracellular microenvironment depends on the proper localization and function of polycystin-1 in cilia.
blood flow; blood pressure; endothelium; endothelium-derived factors; physiology; polycystic kidney diseases
This article is based on a lecture I presented as the recipient of the 2009 Pritzker Distinguished Lecturer Award at the Biomedical Engineering Society annual meeting in October 2009. Here, I review more than thirty years of research from my laboratory, beginning with studies designed to test the theory that cells use tensegrity (tensional integrity) architecture to stabilize their shape and sense mechanical signals, which I believed to be critical for control of cell function and tissue development. Although I was trained as a cell biologist, I found that the tools I had at my disposal were insufficient to experimentally test these theories, and thus I ventured into engineering to find critical solutions. This path has been extremely fruitful as it has led to confirmation of the critical role that physical forces play in developmental control, as well as how cells sense and respond to mechanical signals at the molecular level through a process known as cellular mechanotransduction. Many of the predictions of the cellular tensegrity model relating to cell mechanical behaviors have been shown to be valid, and this vision of cell structure led to discovery of the central role that transmembrane adhesion receptors, such as integrins, and the cytoskeleton play in mechanosensing and mechanochemical conversion. In addition, these fundamental studies have led to significant unexpected technology fallout, including development of micromagnetic actuators for non-invasive control of cellular signaling, microfluidic systems as therapeutic extracorporeal devices for sepsis therapy, and new DNA-based nanobiotechnology approaches that permit construction of artificial tensegrities that mimic properties of living materials for applications in tissue engineering and regenerative medicine.
Mechanotransduction; Tensegrity; Cell mechanics; Prestress; Cytoskeleton; Integrin; Biomimetic
Tensegrity or tensional integrity is a property of a structure that relies on a balance between components that are either in pure compression or in pure tension for its stability [1,2]. Tensegrity structures exhibit extremely high strength-to-weight ratios and great resilience, and are therefore widely used in engineering, robotics and architecture [3,4]. Here we report nanoscale, prestressed, three-dimensional tensegrity structures in which rigid bundles of DNA double helices resist compressive forces exerted by segments of single-stranded DNA that act as tension-bearing cables. Our DNA tensegrity structures can self-assemble against forces up to 14 pN, which is twice the stall force of powerful molecular motors such as kinesin or myosin [5,6]. The forces generated by this molecular prestressing mechanism can be employed to bend the DNA bundles or to actuate the entire structure through enzymatic cleavage at specific sites. In addition to being building blocks for nanostructures, tensile structural elements made of single-stranded DNA could be used to study molecular forces, cellular mechanotransduction, and other fundamental biological processes.
The role of nutrients and metabolism in cellular differentiation is poorly understood. Using RNAi screening, metabolic profiling and small-molecule probes, we discovered three metabolic enzymes whose knockdown induces differentiation of mouse C2C12 myoblasts even in the presence mitogens: phosphoglycerate kinase (Pgk1), hexose-6-phosphate dehydrogenase (H6pd) and ATP citrate lyase (Acl). These enzymes and the pathways they regulate provide novel targets for the control of myogenic differentiation in myoblasts and rhabdomyosarcoma cells.
taurodeoxycholic acid (TUDCA); glycochenodeoxycholic acid; 3-phosphoglycerate; phosphoenol pyruvate (PEP); cyclosporin A (CsA); trichostatin A (TSA); pravastatin; atorvastatin; fluvastatin
Pityriasis rosea is an acute, self-limited papulosquamous dermatosis of the trunk and extremities. Many atypical forms of the disease have been reported in the literature [Ahmed et al.: Clin Exp Dermatol 2000;25:624–626; Imamura et al.: Dermatologica 1985;171:474–477]. It is rare to find pityriasis rosea in multiple family members (within a household) at the same time. There have been only 4 reported cases where a couple has contracted pityriasis rosea simultaneously [Miller et al.: Arch Derm Syphilol 1941;44:66-68; Niles et al.: Arch Derm Syphilol 1940;41:264].
Pityriasis rosea; Familial; Human herpes virus; Herald patch
With the recent revolution in Molecular Biology and the deciphering of the Human Genome, our understanding of the building blocks that comprise living systems has advanced rapidly. We have yet to understand, however, how the physical forces that animate life affect the synthesis, folding, assembly, and function of these molecular building blocks. We are equally uncertain as to how these building blocks interact dynamically to create coupled regulatory networks from which integrative biological behaviors emerge. Here we review several recent advances in the field of biomechanics at the cellular and molecular levels, and we set forth some of the challenges confronting the field. Living systems work and move as multi-molecular collectives, and in order to understand key aspects of health and disease we must first be able to explain how physical forces and mechanical structures contribute to the ‘active’ material properties of living cells and tissues, as well as how these forces impact information processing and cellular decision making. Such insights will no doubt inform basic biology and rational engineering of effective new approaches to clinical therapy.
Virtual reality immersion (VRI), an advanced computer-generated technique, decreased subjective reports of pain in experimental and procedural medical therapies. Furthermore, VRI significantly reduced pain-related brain activity as measured by functional magnetic resonance imaging. Resemblance between anatomical and neuroendocrine pathways of pain and pruritus may prove VRI to be a suitable adjunct for basic and clinical studies of the complex aspects of pruritus.
To compare effects of VRI with audiovisual distraction (AVD) techniques for attenuation of pruritus in patients with atopic dermatitis and psoriasis vulgaris.
Twenty-four patients suffering from chronic pruritus – 16 due to atopic dermatitis and eight due to psoriasis vulgaris – were randomly assigned to play an interactive computer game using a special visor or a computer screen. Pruritus intensity was self-rated before, during and 10 min after exposure using a visual analogue scale ranging from 0 to 10. The interviewer rated observed scratching on a three-point scale during each distraction program.
Student’s t tests were significant for reduction of pruritus intensity before and during VRI and AVD (P=0.0002 and P=0.01, respectively) and were significant only between ratings before and after VRI (P=0.017). Scratching was mostly absent or mild during both programs.
VRI and AVD techniques demonstrated the ability to diminish itching sensations temporarily. Further studies on the immediate and late effects of interactive computer distraction techniques to interrupt itching episodes will open potential paths for future pruritus research.
Atopic dermatitis; Audiovisual distraction; Pruritus; Psoriasis; Virtual reality
Mechanical stresses that are preferentially transmitted across the cell surface via transmembrane integrin receptors activate gene transcription by triggering production of intracellular chemical second messengers, such as cAMP. Here we show that the sensitivity of the cAMP signaling pathway to mechanical stresses transferred across β1 integrins is mediated by force-dependent activation of the heterotrimeric G protein subunit Gαs within focal adhesions at the site of stress application. Gαs is recruited to focal adhesions that form within minutes following clustering of β1 integrins induced by cell binding to magnetic microbeads coated with activating integrin ligands, and β1 integrin and Gαs co-precipitate when analyzed biochemically. Stress application to activated β1 integrins using magnetic twisting cytometry increases Gαs recruitment and activates these large G proteins within focal adhesions, as measured by binding of biotinylated azido-anilido-GTP, whereas application of similar stresses to inactivated, integrins or control histocompatibility antigens has little effect. This response is relevant physiologically as application of mechanical strain to cells bound to flexible extracellular matrix-coated substrates induce translocation of phospho-CREB to the nucleus, which can be attenuated by inhibiting Gαs activity, either using the inhibitor melittin or suppressing its expression using siRNA. Although integrins are not typical G protein-coupled receptors, these results show that integrins focus mechanical stresses locally on heterotrimeric G proteins within focal adhesions at the site of force application, and transduce mechanical stimuli into an intracellular cAMP signaling response by activating Gαs at these membrane signaling sites.
mechanotransduction; G protein; integrin; focal adhesion; shear stress; mechanical strain; magnetic
To advance cancer research in a transformative way, we must redefine the problem. Although epithelial cancers, such as breast cancer, may be caused by random somatic gene mutations, the reality is that this is only one of many ways to induce tumor formation. Cancers also can be produced in experimental systems in vitro and in vivo, for example, by inducing sustained alterations of extracellular matrix (ECM) structure. Moreover, certain epithelial cancers can be induced to ‘reboot’ and regenerate normal tissue morphology when combined with embryonic mesenchyme or exogenous ECM scaffolds that are produced through epithelial-stromal interactions. At the same time, work in the field of Mechanical Biology has revealed that many cell behaviors critical for cancer formation (e.g., growth, differentiation, motility, apoptosis) can be controlled by physical interactions between cells and their ECM adhesions that alter the mechanical force balance in the ECM, cell and cytoskeleton. Epithelial tumor progression also can be induced in vitro by changing ECM mechanics or altering cytoskeletal tension generation through manipulation of the Rho GTPase signaling pathway. Mechanical interactions between capillary cells and ECM that are mediated by Rho signaling similarly mediate control of capillary cell growth and angiogenesis, which are equally critical for cancer progression and metastasis. These findings question basic assumptions in the cancer field, and raise the intriguing possibility that cancer may be a reversible disease that results from progressive deregulation of tissue architecture, which leads to physical changes in cells and altered mechanical signaling. This perspective raises the possibility of developing a tissue engineering approach to cancer therapy in which biologically-inspired materials that mimic the embryonic microenvironment are used to induce cancers to revert into normal tissues.
mechanical; extracellular matrix; stroma; cell traction; cytoskeleton; cancer therapy
Cyclic mechanical strain produced by pulsatile blood flow regulates the orientation of endothelial cells lining blood vessels, and influences critical processes such as angiogenesis. Mechanical stimulation of stretch-activated calcium channels is known to mediate this reorientation response, however, the molecular basis remains unknown. Here we show that cyclically stretching capillary endothelial cells adherent to flexible extracellular matrix substrates activates mechanosensitive TRPV4 ion channels that, in turn, stimulate phosphatidyl inositol-3-kinase-dependent activation and binding of additional ·1 integrin receptors, which promotes cytoskeletal remodeling and cell reorientation. Inhibition of integrin activation using blocking antibodies and knockdown of TRPV4 channels using specific siRNA suppress strain-induced capillary cell reorientation. Thus, mechanical forces that physically deform extracellular matrix may guide capillary cell reorientation through a strain-dependent ‘integrin to integrin’ signaling mechanism mediated by force-induced activation of mechanically-gated TRPV4 ion channels on the cell surface.
mechanical strain; integrin; TRPV4; endothelial cell; reorientation; cytoskeleton
Angiogenesis is controlled by physical interactions between cells and extracellular matrix as well as soluble angiogenic factors, such as VEGF. However, the mechanism by which mechanical signals integrate with other microenvironmental cues to regulate neovascularization remains unknown. Here we show that the Rho inhibitor, p190RhoGAP, controls capillary network formation in vitro and retinal angiogenesis in vivo by modulating the balance of activities between two antagonistic transcription factors – TFII-I and GATA2 – that govern gene expression of the VEGF receptor, VEGFR2. Moreover, this novel angiogenesis signaling pathway is sensitive to extracellular matrix elasticity as well as soluble VEGF. This is the first known functional cross-antagonism between transcription factors that controls tissue morphogenesis, and that responds to both mechanical and chemical cues.
VEGFR2; p190RhoGAP; TFII-I; GATA2; mechanotransduction; angiogenesis; capillary endothelial cell
The formation of focal adhesions governs cell shape and function; however, there are few measurements of the binding kinetics of focal adhesion proteins in living cells. Here, we used the fluorescence recovery after photobleaching (FRAP) technique, combined with mathematical modeling and scaling analysis to quantify dissociation kinetics of focal adhesion proteins in capillary endothelial cells. Novel experimental protocols based on mathematical analysis were developed to discern the rate-limiting step during FRAP. Values for the dissociation rate constant kOFF ranged over an order of magnitude from 0.009 ± 0.001/s for talin to 0.102 ± 0.010/s for FAK, indicating that talin is bound more strongly than other proteins in focal adhesions. Comparisons with in vitro measurements reveal that multiple focal adhesion proteins form a network of bonds, rather than binding in a pair-wise manner in these anchoring structures in living cells.
Stress fibers are contractile bundles in the cytoskeleton that stabilize cell structure by exerting traction forces on extracellular matrix. Individual stress fibers are molecular bundles composed of parallel actin and myosin filaments linked by various actin-binding proteins, which are organized end-on-end in a sarcomere-like pattern within an elongated three-dimensional network. While measurements of single stress fibers in living cells show that they behave like tensed viscoelastic fibers, precisely how this mechanical behavior arises from this complex supramolecular arrangement of protein components remains unclear. Here we show that computationally modeling a stress fiber as a multi-modular tensegrity network can predict several key behaviors of stress fibers measured in living cells, including viscoelastic retraction, fiber splaying after severing, non-uniform contraction, and elliptical strain of a puncture wound within the fiber. The tensegrity model also can explain how they simultaneously experience passive tension and generate active contraction forces; in contrast, a tensed cable net model predicts some, but not all, of these properties. Thus, tensegrity models may provide a useful link between molecular and cellular scale mechanical behaviors, and represent a new handle on multi-scale modeling of living materials.
Cell mechanics; cell structure; computer model; contractility; cytoskeleton; tensegrity
Anyone who is skilled in the art of physical therapy knows that the mechanical properties, behavior and movement of our bodies are as important for human health as chemicals and genes. However, only recently have scientists and physicians begun to appreciate the key role that mechanical forces play in biological control at the molecular and cellular levels. This article provides a brief overview of a lecture presented at the 1st International Fascia Research Congress that convened at Harvard Medical School in Boston, MA on October 4, 2007. (see figure 1) In this lecture, I described what we have learned over the past thirty years as a result of our research focused on the molecular mechanisms by which cells sense mechanical forces and convert them into changes in intracellular biochemistry and gene expression – a process called “mechanotransduction”. This work has revealed that molecules, cells, tissues, organs, and our entire bodies use “tensegrity” architecture to mechanically stabilize their shape, and to seamlessly integrate structure and function at all size scales. Through use of this tension-dependent building system, mechanical forces applied at the macroscale produce changes in biochemistry and gene expression within individual living cells. This structure-based system provides a mechanistic basis to explain how application of physical therapies might influence cell and tissue physiology.
tensegrity; mechanotransduction; cytoskeleton; integrins; cell tension; physical therapy