- Email: [email protected]
New technologies for dissecting the arteriolar myogenic response
Opinion
TRENDS in Pharmacological Sciences
Vol.28 No.7
New technologies for dissecting the arteriolar myogenic response Michael A. Hill, Zhe Sun, Luis Martinez-Lemus and Gerald A. Meininger Dalton Cardiovascular Research Center and Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, MO 65211, USA
The arteriolar myogenic response is crucial for the setting of vascular resistance and for providing a level of tone upon which vasodilators can act. Despite its physiological importance, questions remain regarding the underlying signaling mechanisms of the arteriolar myogenic response. Does an increase in pressure within an arteriole exert its effects via the extracellular matrix, an action on cell membranes and/or deformation of cytoskeletal structures? Recent advances in methodology, particularly involving sophisticated imaging approaches, are enabling the study of forces at single-cell and even subcellular levels. Atomic force microscopy (AFM) not only enables detection of cell morphology and stiffness but also allows discrete forces to be applied to single smooth muscle cells and subsequent responses to be observed. Importantly, the repertoire of approaches involving AFM can be expanded by using it in combination with other imaging approaches – including fluorescence imaging for cellular signals such as Ca2+, and total internal reflectance fluorescence, fluorescence resonance energy transfer and confocal microscopy for probing cellular contact function. Combinations of these advanced imaging and nanomechanical approaches will be instructive to studies of intact vessels and the circulatory system in general.
events has not been attained. With regard to arteriolar constriction resulting from an increase in intraluminal pressure (the ‘myogenic response’), the initial description of this vascular response is generally credited to Bayliss in 1902 [5]. Bayliss observed a link between reduced intravascular pressure and increased regional blood flow, which he attributed to the reduced distending pressure acting on the blood vessel wall. Since then, advances in understanding have come mainly from studies of whole animals, exteriorized organs, microvascular preparations and isolated arterioles (for review, see Refs [6–8]). These approaches have provided convincing evidence that pressure-induced vasoconstriction contributes to the setting of peripheral vascular resistance, the autoregulation of blood flow and the regulation of capillary pressure. Mechanistically and conceptually, in its simplest form, increased smooth muscle cell membrane tension (resulting from an increase in intraluminal pressure) is thought to lead to membrane depolarization, voltage-gated Ca2+ entry, activation of actomyosin interaction and contraction [7–10] (see Figure Ia in Box 1). Recently, the contributions of Ca2+ sensitization of the contractile proteins and cytoskeletal rearrangement have been proposed as additional Glossary
Introduction Vascular cells are continually exposed to mechanical forces, as typified by the transmural forces exerted by blood pressure and the shear stresses generated by the flow of blood (Figure 1). Importantly, cells of the vascular wall (both smooth muscle and endothelial) are able to respond to these stimuli by acutely activating signaling pathways to modulate vessel diameter while, in the longer term, altering the structure of the vessel wall, in part by modulation of biosynthetic pathways. Through the ability of these mechanically initiated signaling pathways, both short- and long-term alterations of vasomotor tone, local control of blood flow and vascular resistance can be affected. Although this article focuses predominately on pressure–stretch activation of vascular smooth muscle, the reader is referred to recent reviews of shear-stress-induced activation of endothelial cells [1–4]. Despite the importance of mechanically activated mechanisms of control of the microcirculation and vessel wall, a complete knowledge of the underlying cellular Corresponding author: Hill, M.A. ([email protected]). Available online 18 June 2007. www.sciencedirect.com
Atomic force microscopy (AFM): method enabling the application and measurement of forces in the pN–nN range by precisely controlling the deformation of a thin cantilever. The cantilever carries a tip that is either sharp or a surface such as a bead, and can be functionalized with biological molecules that act as binding partners for the cell or surface being studied. Fluorescence (Forster) resonance energy transfer (FRET): method using the non-radiative transfer of energy from a donor to an acceptor molecule. Energy transfer relies heavily on the distance separating the donor and acceptor molecules. Interference reflection microscopy (IRM): an optical technique for detecting the topography of the side of a cell in contact with a planar substrate. IRM uses the zero-order reflection interference pattern of the cell basal plane to measure the separation (<100 nm) and areas of contact (focal adhesions or close contacts) between the cell and a glass substrate. Laser tweezer: method that enables the application and measurement of forces in the pN range by manipulating a bead using laser beams (optical trap). Mechanical activity: force generated through (smooth muscle) cell contractility or relaxation. Paramagnetic bead: method enabling the application of force, through torque, on a pN–nN scale. Torque is provided by a magnetic field or magnetic needle. RGD peptides: synthetic amino acid sequences containing arginine-glycineaspartate, which are analogs of sequences found in ECM proteins. Binding of the RGD sequence to integrins initiates signaling cascades. Stiffness: Young’s modulus, a measure of the elasticity of a cell in response to the loading of a compression force. Total internal reflectance fluorescence (TIRF): a microscopy technique that excites the cell-labeling fluorophores with an evanescent wave (near-field standing wave exhibiting exponential decay), enabling quantitative measurement of fluorescent labeling within 200-nm distance from a glass substrate.
0165-6147/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2007.05.006
Opinion
TRENDS in Pharmacological Sciences
Vol.28 No.7
309
Figure 1. Mechanical forces and the vascular wall. The arteriolar wall is continually exposed to transmural and shearing forces as a result of the closed nature of the circulatory system. Complexity of the vessel wall occurs as a result of junctional contacts, both between vascular cells and with the ECM proteins. Gap junctional communication enables the vessel wall to act as a functional syncytium, facilitating the transfer of signals along – and perhaps through – the cells of the vessel wall. Contacts between cells and ECM proteins provide a mechanism for ‘sensing’ deformation caused by mechanical forces and, perhaps, initiating signal transduction mechanisms. Abbreviation: MEGJ, myoendothelial gap junction. Modified, with permission, from Ref. [24].
mechanisms that can modulate this pressure-dependent response [11–15]. Despite these advances in understanding, however, the exact molecular mechanisms through which a change in intraluminal pressure is sensed have remained elusive. In particular, the presence of a pressuresensitive mechanosensor, or mechanically activated receptor, is yet to be conclusively demonstrated. It is our opinion, however, that developments in novel and sophisticated imaging techniques [e.g. atomic force microscopy (AFM) (see Glossary), fluorescence resonance energy transfer
(FRET), total internal reflectance fluorescence (TIRF) microscopy, interference reflection microscopy (IRM) and multi-photon microscopy] – together with nanoscale- and picoscale-force measuring devices such as AFM, laser tweezers and paramagnetic microspheres, and approaches from disciplines such as nanomedicine and proteomics – are providing new tools for achieving this goal. These developments in imaging and nanoscale-force measurement are enabling, in particular, the study of candidate mechanosensors, including the axis formed by
Box 1. Signaling events underlying the arteriolar myogenic response Increased intraluminal pressure within an arteriole is thought to be detected by either cell stretch or an increase in wall tension [6,7]. This is conveyed through a direct effect on mechanically sensitive protein elements such as ion channels or the membrane lipid bilayer itself, or through interactions between extracellular elements (e.g. ECM proteins) and cell surface ‘receptors’ such as integrins. Activation of these pathways then leads to the opening of non-selective cation channels, membrane depolarization and the opening of voltage-gated Ca2+ entry [10,55]. Increased [Ca2+]i results in Ca2+/calmodulindependent activation of myosin light-chain kinase, phosphorylation of the 20-kDa myosin regulatory light chains and contractile interaction of actin and myosin [9,56] (Figure Ia). In addition to these events, it is evident that the mechanical stimulus drives other signaling Ca2+dependent and -independent events, including Ca2+ release, kinase activation and, perhaps, phosphatase inhibition. These pathways might contribute to modulation of the contractile response through alterations in Ca2+ sensitivity, Ca2+ handling, activation of ion channels such as BKCa and cytoskeletal rearrangements [11–14,57]. www.sciencedirect.com
Although Figure Ia presents these events in a linear fashion, culminating in contraction, it is equally – if not more – likely that the mechanical stimulation that occurs during an increase in intravascular pressure results in parallel activation of multiple, perhaps interacting, pathways (Figure Ib). Such a situation would be expected if mechanical deformation of the membrane, as would occur during cell stretch, simultaneously applies force to varying mechanosensors or proteins whose activity is modulated by nanoscale forces. Interaction between such pathways would invariably occur on multiple levels. For example, myogenic contraction might decrease tension and the resultant driving force for Ca2+ entry, leading to a biphasic global Ca2+ signal; decreased tension might also relieve a stimulus for adaptive remodeling and structural modification of the vessel wall; Ca2+ entry, in addition to contraction, might activate opposing hyperpolarizing events, as proposed for the BKCa channel. Importantly, the separation of these events requires the ability to apply nanoscale forces precisely to the membrane, perhaps through specific ligand–receptor interactions.
310
Opinion
TRENDS in Pharmacological Sciences
Vol.28 No.7
Figure I. Signaling events underlying the arteriolar myogenic response. Mechanosensors can include different ECM-protein–membrane interactions, mechanosensitive ion channels, mechanosensitive proteins and enzymes, and mechanically activated cytoskeletal rearrangements (denoted by * in the figure).
www.sciencedirect.com
Opinion
TRENDS in Pharmacological Sciences
extracellular matrix (ECM), cell membrane integrins and the cytoskeleton [16,17]. Importantly, this will enable our thinking about mechanotransduction to be further expanded from a purely linear set of events (see Figure Ia in Box 1) to consider more fully that physical forces can activate more than one pathway (see Figure Ib in Box 1). In this regard, it is clear from basic studies of mechanotransduction that cells of the vascular wall interact in a complex manner with their surrounding environment. Thus, it is conceivable that signals generated in response to physical forces within the vessel wall can be conducted in parallel through multiple cellular elements undergoing strain or deformation. Conceptually, these affected elements could include specific ECM proteins (e.g. fibronectin, collagen and laminin), cell surface adhesion receptor links, mechanosensitive ion channels and enzymes, and perturbation of membrane lipid or cytoskeletal components. Further benefits of some of these techniques include the ability to follow dynamic processes in (near) real-time and to consider important parameters such as the directionality of a given stimulus and cell polarity. Importantly, many of these processes cannot currently be studied using classical in vivo microcirculatory and isolated vessel techniques. The use of these imaging techniques is also enhanced by the ability to manipulate cells and vascular preparations genetically to enable the expression of fluorescently labeled reporter molecules or mutated proteins or to alter the expression of given signaling molecules. In this regard, many studies have shown successful transfection of cultured vascular cells and even intact vascular preparations. This has enabled the introduction of reporter molecules for fluorescence imaging, fluorescence-based measurements of ions such as Ca2+ and FRET partners for dynamically following candidate signaling molecules in mechanotransduction [18,19]. Transfection in intact arterioles has been used for regulating the activity of kinases, including Rho and sphingosine kinase, and the introduction of oligonucleotides and small interfering (si)RNA has been used to decrease the expression of candidate signaling molecules (e.g. for TrpC6 and TrpM4) [20–23]. Collectively, these imaging and molecular approaches provide investigators with novel platforms from which to complement pharmacological studies and, ultimately, to improve the understanding of cellular events associated with vascular cell mechanotransduction. Interactions between the ECM and vascular smooth muscle cells Recent attention has been given to the hypothesis that mechanical stimuli exert their effects on vascular cells via the interaction of ECM proteins with cell surface ‘receptors’ such as integrins [17,24]. Integrins represent a heterogeneous family of receptors that exist as heterodimers formed from the 14 a- and seven b-subunits expressed in vascular smooth muscle [24]. It has been shown that synthetic RGD peptides and anti-integrin function-blocking antibodies can inhibit myogenic vascular tone and pressure-induced myogenic signaling [24]. Specifically, in arterioles taken from skeletal muscle, synthetic RGD peptides lower intracellular Ca2+ concentrations ([Ca2+]i) in vascular smooth muscle, leading to vasodilatation via a mechanism www.sciencedirect.com
Vol.28 No.7
311
involving the avb3 integrin, whereas application of a non-vasodilatory concentration of RGD or anti-b3 antibody to the same arteriolar preparation inhibits acute pressureinduced vasoconstriction [24–26]. Such effects of integrinmediated signaling might result from interactions with smooth muscle cell ion channels that regulate vasomotor activity [27–29]. Complexity, however, exists because heterogeneity in integrin-mediated responses occurs between tissues (e.g. skeletal muscle compared with coronary and renal arterioles, and conduit compared with microvessels [24,30,31]). Additionally, the mechanisms by which binding to integrins elicits a particular vascular response can depend on cell-specific integrin signaling (e.g. specifically affecting either smooth muscle or endothelial cells) and which of the various integrin dimers are involved. Clearly, these wholevessel and pharmacological approaches, although providing data to support the hypothesis that an ECM–integrin axis is involved in myogenic signaling, do not enable the specificity of this interaction to be studied at the molecular level. This, therefore, currently limits the ability to understand precisely events such as integrin-mediated mechanotransduction in the arteriolar myogenic response. More than one way to apply a force: twisting, pulling and swelling Several nanoscale approaches have proved valuable in studying the effects of mechanical forces on cells, including those of the vasculature: for example, via the movement of cell-attached magnetic beads [32–34] and with laser tweezers [35–37]. In the former, movement and, hence, stretch can be applied by a magnetic field creating torque on matrixprotein-coated beads, or a directional stimulus can be applied using a magnetic needle. In the latter, proteincoated beads bound to cell surface receptors are trapped in an energy well formed by an infrared laser beam. Displacement of the laser beam can then be used to apply a controlled force to the cell. Stretching of single vascular smooth muscle cells has also been accomplished using modified patch pipettes [38,39]. In electrophysiological studies using this approach, cells were anchored at one site and the lateral movement of a second pipette was used to apply a stretch (100–130% of resting-cell length) stimulus. A third patch pipette was then used to record ion channel events using the whole-cell-recording mode. In these studies, membrane stretch was shown to activate non-selective cation currents, with subsequent opening of voltage-gated Ca2+ channels and large-conductance K+ channels. The effect of membrane stretch has also been studied under conditions of hypo-osmotic cell swelling [40,41], again linking mechanical stimulation to the opening of ion channels. These methods are typically used to study whole-cell stretch and do not currently enable the contribution of single molecules (e.g. integrin–ECM interactions) to be directly studied, although pharmacological approaches can be used (e.g. the addition of synthetic RGD peptides and function-blocking antibodies). Probing interactions between the ECM and vascular smooth muscle cells: AFM The advent of techniques such as biological AFM seems to provide a method for approaching the problem with
312
Opinion
TRENDS in Pharmacological Sciences
the required level of specificity: that is, where singlematrix-molecule–integrin interactions can be discerned. AFM is proving to be a flexible technique that, although first used for topographical studies, can provide information on the mechanical properties of cells, including stiffness, mechanical activity and ligand–receptor interactions, and can be used to measure and apply discrete nanoscale and picoscale forces [42–44]. The ability to measure and apply force, combined with the ability to ‘biofunctionalize’ the AFM tip with protein molecules [45–48], enables the study of mechanical activation via the ECM-protein–integrin axis (Box 2). The ability of AFM to apply forces on a nanoscale also lends itself to examining whether smooth muscle
Vol.28 No.7
mechanotransduction is equally effective when stimuli are applied to different regions of the cell membrane, raising issues such as whether the cell experiences stretch equally as well along its long and short axes. For smooth muscle cells resident in the arteriolar wall, an increase in intraluminal pressure passively stretches cells along their long axis because of their largely circumferential arrangement. However, in certain situations (e.g. during traumatic injury), vessels might be exposed to considerable longitudinal stretch that applies a mechanical force across the short axes of the smooth muscle cells. Interestingly, arterioles subjected to controlled in vitro stretch along these two directions show markedly different [Ca2+]i responses [49].
Box 2. Modes of AFM: the ability to apply controlled nanoscale and picoscale forces AFM (Figure I) provides a flexible approach for the topographical mapping of the cell surface, the measurement of forces associated with binding events, the estimation of biophysical properties of the cytoskeleton – including elasticity – and the application of mechanical stimuli [26,42–44]. These studies can be performed on intact cells, without the need for fixation, and in physiological solutions. AFM is
accomplished through the interaction of a fine tip attached to a cantilever of known spring constant that, for topographical imaging, can be raster-scanned across the surface of a given specimen (hence, AFM is a mode of scanning probe microscopy). Measured spring deflection–indentation at each point enables the reconstruction of images or force maps via digital techniques [42].
Figure I. Example modes of AFM operation, from simple scanning to situations in which the tip or probe is biofunctionalized to increase its specificity of interaction with the cell surface. (a)(i) A native, or non-functionalized, AFM probe enables the collection of detailed topographic information from live cells. (ii) Detail of the underlying cytoskeleton. (iii) A calculated topography map showing the relative height of the cell. Such images are usually achieved in the contact or tapping mode operation of AFM, where the probe is controlled to scan across the cell surface with a preset contact force (ii). (b)(i) AFM probes functionalized with ligand molecules to enable the detection of individual ligand–receptor interactions on the surface of a cell. In force mode operation, the probe is first controlled to contact the cell surface to enable the formation of ligand–receptor bonds. (ii) The probe is then lifted from the cell surface to break the ligand–receptor bonds physically. (iii) The data obtained can be used to resolve the characteristic binding force of a specific ligand–receptor interaction. Thus, for fibronectin–integrin interactions, we have calculated a binding force of 39 pN. (c)(i) Tips on AFM probes can be replaced by microspheres, which have a much larger surface area and can be coated with a variety of ligand molecules. (ii) Bringing the fibronectin-coated microspheres into contact with the vascular smooth muscle cell (VSMC) surface induces the formation of focal-adhesion-like structures, including the formation of integrin clusters and cytoskeletal connections. The AFM probe can then be controlled to apply either pulling or pushing forces to the VSMC through this molecular structure. The cellular response to the forces can be measured by using the AFM to monitor the up-and-down movement of the microsphere. (iii) As contact time continues, an increasing force is required to detach FN-coated microspheres from the VSMC surface, indicating that the molecular structures formed around the microspheres strengthen with time. Panels (a)(iii), (b)(iii) and (c)(iii) reproduced, with permission, from Ref. [46]. www.sciencedirect.com
Opinion
TRENDS in Pharmacological Sciences
As a further example of a relationship between cellular polarity and mechanical signals, endothelial cells align both their long axis and their cytoskeletal apparatus with the direction of flow-induced shear stress [50,51], whereas vascular smooth muscle cells organize themselves perpendicular to mechanical stress fields (Figure 1). It is not surprising that regions of the cell membrane might show differing responses to mechanical stimuli given the relatively recent appreciation of specialized signaling domains and complexes such as cholesterol-rich structures (e.g. caveolae and rafts), focal adhesions, adherens junctions and connexins. Thus, vascular cells might show a degree of polarity with respect to how they respond to mechanical forces: an issue that could be investigated further using nanomechanical approaches. Sun et al. [46] used AFM probes that were biofunctionalized with fibronectin to examine the characteristics of the binding of this ECM protein with cell surface integrins. In these studies, the fibronectin-coated probe was lowered onto cultured arteriolar smooth muscle cells and allowed to adhere for varying lengths of time, after which the probe was retracted from the cell surface and the rupture force was recorded (Box 2). The specificity of binding was shown by the use of anti-integrin antibodies and RGD peptides, with the authors concluding that the fibronectin binding was mediated largely through a5b1 integrin. Using this approach, it was further suggested that the strength of binding between single fibronectin–integrin interactions was 39 8 pN. Using an AFM probe with an attached microsphere coated with fibronectin, withdrawal of the probe can be used to pull on the membrane and underlying cytoskeleton. In experiments such as these, the magnitude of the applied force is related to the distance of retraction and bending (spring constant dependent) of the AFM cantilever (Box 2). Biofunctionalizing the probe with selected ECM proteins that interact differentially with members of the integrin family enables the stretch stimulus (force step) to be applied through various putative mechanosensory pathways (Box 1). Wider use of AFM and combining it with other imaging and cellular approaches The potential use of AFM in the study of vascular cells extends beyond interactions between ECM proteins and cell surface integrins. Appropriately functionalized probes have the capability to enable the study of many cell surface molecules of proposed importance to vascular cell function and mechanotransduction, including ion channels [52] and connexins [53,54]. At a more macroscopic level, the interaction of white cells with vascular endothelial cells has been studied by functionalizing AFM probes with leukocytes [48]. A powerful future direction for imaging-based studies of mechanotransduction will be the evolution of combining nanoscale-force application systems (including AFM, laser tweezers and magnetized microbeads) with other imaging approaches: for example, wide-field fluorescence, confocal and multiphoton microscopy, and FRET and TIRF microscopy. Crucial to this is that combining AFM with optical approaches improves the use of AFM without compromising www.sciencedirect.com
Vol.28 No.7
313
Figure 2. Combined imaging modes for simultaneously measuring changes in cytosolic Ca2+ concentration and cell stiffness. Studies were performed on cultured cremaster muscle arteriolar smooth muscle cells. Changes in intracellular Ca2+ concentration were estimated using fluorescence ratio imaging with cells loaded with the Ca2+-sensitive indicator fura 2. Changes in cell stiffness were followed using AFM by taking repetitive force measurements at a single site on the cell surface. An increase in [Ca2+]i, as indicated by the 340:380-nm excitation fluorescence ratio, was followed by an increase in stiffness consistent with the occurrence of contraction. Changes in cell height (not shown) measured by AFM also followed the changes in [Ca2+]i (Z.S. and G.A.M., unpublished).
its features such as its nanoscale resolution. Combined systems enable, for example, the measurement of changes in [Ca2+]i, which can be correlated with indices of contraction as determined by AFM (Figure 2). Furthermore, combined imaging modes promise the study of focal adhesions and cytoskeletal proteins, including during the application of nanoscale or picoscale forces. Importantly, such responses can be followed over time so that dynamic studies of mechanotransduction can be performed. Similarly, although AFM can be used to apply controlled forces to the upper surface of the cell, TIRF microscopy can be employed simultaneously to examine adhesion sites on the basal surface of the cell. Because it is not practical to provide an exhaustive review of the literature, several examples of AFM combined with optical-imaging approaches are provided in Table 1. The studies listed in Table 1 are limited to those involving the Table 1. Examples of AFM combined with optical-imaging approaches Combination of imaging approaches AFM and confocal microscopy
AFM and dual-photon microscopy AFM and TIRF AFM and FRET AFM and fluorescent Ca2+ imaging a
Cell system studied
Refs
Osteoblasts a Bacterial membranes Jurkhat T lymphoma Tumor mast cells Chloroplasts Fibroblasts Titin molecules a VSM cells a Titin molecules a Skeletal myoblasts a Osteoblasts a
[58] [59]
Studies specifically relevant to mechanotransduction.
[60] [61] [62] [62] [63] [64] [63] [65] [58]
314
Opinion
TRENDS in Pharmacological Sciences
simultaneous use of multiple imaging modes rather than correlative studies in which various imaging approaches were performed in parallel experiments. Concluding remarks The use of nanomechanical approaches such as AFM and optical-imaging techniques has the potential to make significant advances in the understanding of vascular cell mechanotransduction. In particular, such techniques show promise for isolating specific cellular mechanosensors and delineating the signaling pathways that are subsequently activated. Data from such techniques will provide for new avenues of research that can be applied to more-intact preparations, including isolated arterioles and vascular networks. Knowledge of the behavior of single cells in response to controlled nanoscale or picoscale forces might then provide the basis for understanding how these preparations respond to complex mechanical stimuli such as those provided by an alteration in intraluminal pressure or shear stress. Ultimately, it will be important to put the pieces together to understand the physiological role of the myogenic response within a complex vascular network. References 1 Davies, P.F. (1995) Flow-mediated endothelial mechanotransduction. Physiol. Rev. 75, 519–560 2 McCue, S. et al. (2004) Shear-induced reorganization of endothelial cell cytoskeleton and adhesion complexes. Trends Cardio. Med. 14, 143– 151 3 Li, Y.S. et al. (2005) Molecular basis of the effects of shear stress on vascular endothelial cells. J. Biomech. 38, 1949–1971 4 Davies, P.F. et al. (2005) Shear stress biology of the endothelium. Ann. Biomed. Eng. 33, 1714–1718 5 Bayliss, W.M. (1902) On the local reactions of the arterial wall to changes of internal pressure. J. Physiol. 28, 220–231 6 Johnson, P.C. (1980) The myogenic response. In Handbook of Physiology, the Cardiovascular System, Vascular Smooth Muscle, pp. 409–442, American Physiological Society 7 Davis, M.J. and Hill, M.A. (1999) Signaling mechanisms underlying the vascular myogenic response. Physiol. Rev. 79, 387–423 8 Schubert, R. and Mulvany, M.J. (1999) The myogenic response: established facts and attractive hypotheses. Clin. Sci. (Lond.) 96, 313–326 9 Zou, H. et al. (1995) Role of myosin phosphorylation and [Ca2+]i in myogenic reactivity and arteriolar tone. Am. J. Physiol. 269, H1590– H1596 10 Knot, H.J. and Nelson, M.T. (1998) Regulation of arterial diameter and wall [Ca2+] in cerebral arteries of rat by membrane potential and intravascular pressure. J. Physiol. 508, 199–209 11 Gokina, N.I. and Osol, G. (2002) Actin cytoskeletal modulation of pressure-induced depolarization and Ca2+ influx in cerebral arteries. Am. J. Physiol. 282, H1410–H1420 12 Lagaud, G. et al. (2002) Pressure-dependent myogenic constriction of cerebral arteries occurs independently of voltage-dependent activation. Am. J. Physiol. 283, H2187–H2195 13 Schubert, R. et al. (2002) Rho kinase inhibition partly weakens myogenic reactivity in rat small arteries by changing calcium sensitivity. Am. J. Physiol. 283, H2288–H2295 14 Flavahan, N.A. et al. (2005) Imaging remodeling of the actin cytoskeleton in vascular smooth muscle cells after mechanosensitive arteriolar constriction. Am. J. Physiol. 288, H660–H669 15 Keller, M. et al. (2006) Sphingosine kinase functionally links elevated transmural pressure and increased reactive oxygen species formation in resistance arteries. FASEB J. 20, 702–704 16 Vogel, V. and Sheetz, M. (2006) Local force and geometry sensing regulate cell functions. Nature Rev. 7, 265–275 www.sciencedirect.com
Vol.28 No.7
17 Alenghat, F.J. and Ingber, D.E. (2002) Mechanotransduction: all signals point to cytoskeleton, matrix, and integrins. Sci. STKE 2002, PE6 18 Wang, Y. et al. (2005) Visualizing the mechanical activation of Src. Nature 434, 1040–1045 19 Kotlikoff, M.I. (2007) Genetically encoded Ca2+ indicators: using genetics and molecular design to understand complex physiology. J. Physiol. 578, 55–67 20 Welsh, D.G. et al. (2002) Transient receptor potential channels regulate myogenic tone of resistance arteries. Circ. Res. 90, 248– 250 21 Slish, D.F. et al. (2002) Diacylglycerol and protein kinase C activate cation channels involved in myogenic tone. Am. J. Physiol. 283, H2196– H2201 22 Earley, S. et al. (2004) Critical role for transient receptor potential channel TRPM4 in myogenic constriction of cerebral arteries. Circ. Res. 95, 922–929 23 Bolz, S.S. et al. (2003) Sphingosine kinase modulates microvascular tone and myogenic responses through activation of RhoA/Rho kinase. Circulation 108, 342–347 24 Martinez-Lemus, L.A. et al. (2003) Integrins as unique receptors for vascular control. J. Vasc. Res. 40, 211–233 25 Mogford, J.E. et al. (1996) Vascular smooth muscle avb3 integrin mediates arteriolar vasodilation in response to RGD peptides. Circ. Res. 79, 821–826 26 D’Angelo, G. et al. (1997) Integrin-mediated reduction in vascular smooth muscle [Ca2+]i induced by RGD-containing peptide. Am. J. Physiol 272, H2065–H2070 27 Wu, X. et al. (1998) Modulation of calcium current in arteriolar smooth muscle by avb3 and a5b1 integrin ligands. J. Cell Biol. 143, 241–252 28 Wu, X. et al. (2001) Regulation of the L-type calcium channel by a5b1 integrin requires signaling between focal adhesion proteins. J. Biol. Chem. 276, 30285–30292 29 Gui, P. et al. (2006) Integrin receptor activation triggers converging regulation of Cav1.2 calcium channels by c-Src and protein kinase A pathways. J. Biol. Chem. 281, 14015–14025 30 Yip, K.P. and Marsh, D.J. (1997) An Arg-Gly-Asp peptide stimulates constriction in rat afferent arteriole. Am. J. Physiol. 273, F768–F776 31 Hein, T.W. et al. (2001) Integrin-binding peptides containing RGD produce coronary arteriolar dilation via cyclooxygenase activation. Am. J. Physiol. 281, H2378–H2384 32 Wang, N. et al. (1993) Mechanotransduction across the cell surface and through the cytoskeleton. Science 260, 1124–1127 33 Guilford, W.H. et al. (1995) Locomotive forces produced by single leukocytes in vivo and in vitro. Am. J. Physiol. 268, C1308–C1312 34 Pommerenke, H. et al. (1996) Stimulation of integrin receptors using a magnetic drag force device induces an intracellular free calcium response. Eur. J. Cell Biol. 70, 157–164 35 Schmidt, C.E. et al. (1993) Integrin–cytoskeletal interactions in migrating fibroblasts are dynamic, asymmetric, and regulated. J. Cell Biol. 123, 977–991 36 Sheetz, M.P. and Dai, J. (1996) Modulation of membrane dynamics and cell motility by membrane tension. Trends Cell Biol. 6, 85–89 37 Giannone, G. et al. (2003) Talin1 is critical for force-dependent reinforcement of initial integrin–cytoskeleton bonds but not tyrosine kinase activation. J. Cell Biol. 163, 409–419 38 Davis, M.J. et al. (1992) Stretch-activated single-channel and whole cell currents in vascular smooth muscle cells. Am. J. Physiol. 262, C1083– C1088 39 Wu, X. and Davis, M.J. (2001) Characterization of stretch-activated cation current in coronary smooth muscle cells. Am. J. Physiol. 280, H1751–H1761 40 Langton, P.D. (1993) Calcium channel currents recorded from isolated myocytes of rat basilar artery are stretch sensitive. J. Physiol. 471, 1–11 41 Welsh, D.G. et al. (2000) Swelling-activated cation channels mediate depolarization of rat cerebrovascular smooth muscle by hyposmolarity and intravascular pressure. J. Physiol. 527, 139–148 42 Radmacher, M. (1997) Measuring the elastic properties of biological samples with the AFM. IEEE Eng. Med. Biol. Mag. 16, 47–57 43 Hassan, A.E. et al. (1998) Relative microelastic mapping of living cells by atomic force microscopy. Biophys. J. 74, 1564–1578
Opinion
TRENDS in Pharmacological Sciences
44 Sun, Z. et al. (2004) Vascular smooth muscle cells (VSMC) mechanically respond to application of force to the fibronectin–a5b1 integrin bond. FASEB J. 18, A665 45 Lehenkari, P.P. and Horton, M.A. (1999) Single integrin molecule adhesion forces in intact cells measured by atomic force microscopy. Biochem. Biophys. Res. Comm. 259, 645–650 46 Sun, Z. et al. (2005) Mechanical properties of the interaction between fibronectin and a5b1-integrin on vascular smooth muscle cells studied using atomic force microscopy. Am. J. Physiol. 289, H2526– H2535 47 Moy, V.T. et al. (1994) Intermolecular forces and energies between ligands and receptors. Science 266, 257–259 48 Zhang, X. et al. (2004) Atomic force microscopy measurement of leukocyte–endothelial interaction. Am. J. Physiol. 286, H359– H367 49 Hill, M.A. et al. (2000) Transient increases in diameter and [Ca2+]i are not obligatory for myogenic constriction. Am. J. Physiol. 278, H345– H352 50 Noria, S. et al. (2004) Assembly and reorientation of stress fibers drives morphological changes to endothelial cells exposed to shear stress. Am. J. Pathol. 164, 1211–1223 51 McCue, S. et al. (2006) Shear stress regulates forward and reverse planar cell polarity of vascular endothelium in vivo and in vitro. Circ. Res. 98, 939–946 52 Schar-Zammaretti, P. et al. (2002) Potassium-selective atomic force microscopy on ion-releasing substrates and living cells. Anal. Chem. 74, 4269–4274 53 Lal, R. and Lin, H. (2001) Imaging molecular structure and physiological function of gap junctions and hemijunctions by multimodal atomic force microscopy. Microsc. Res. Tech. 52, 273–288 54 Liu, F. et al. (2006) Nanomechanics of hemichannel conformations: connexin flexibility underlying channel opening and closing. J. Biol. Chem. 281, 23207–23217
Vol.28 No.7
55 Kotecha, N. and Hill, M.A. (2005) Myogenic contraction in rat skeletal muscle arterioles: smooth muscle membrane potential and Ca2+ signaling. Am. J. Physiol. 289, H1326–H1334 56 Zou, H. et al. (2000) Temporal aspects of Ca2+ and myosin phosphorylation during myogenic and norepinephrine-induced arteriolar constriction. J. Vasc. Res. 37, 556–567 57 Jaggar, J.H. et al. (1998) Ca2+ channels, ryanodine receptors and Ca2+-activated K+ channels: a functional unit for regulating arterial tone. Acta. Physiol. Scand. 164, 577–587 58 Charras, G.T. and Horton, M.A. (2002) Single cell mechanotransduction and its modulation analyzed by atomic force microscope indentation. Biophys. J. 82, 2970–2981 59 Kassies, R. et al. (2005) Combined AFM and confocal fluorescence microscope for applications in bio-nanotechnology. J. Microsc. 217, 109–116 60 Lulevich, V. et al. (2006) Cell mechanics using atomic force microscopybased single-cell compression. Langmuir 22, 8151–8155 61 Frankel, D.J. et al. (2006) Revealing the topography of cellular membrane domains by combined atomic force microscopy/ fluorescence imaging. Biophys. J. 90, 2404–2413 62 Gradinaru, C.C. et al. (2004) Simultaneous atomic-force and two-photon fluorescence imaging of biological specimens in vivo. Ultramicroscopy 99, 235–245 63 Kellermayer, M.S. and Grama, L. (2002) Stretching and visualizing titin molecules: combining structure, dynamics and mechanics. J. Muscle Res. Cell Motil. 23, 499–511 64 Trache, A. and Meininger, G.A. (2005) Atomic force–multi-optical imaging integrated microscope for monitoring molecular dynamics in live cells. J. Biomed. Opt. 10, 064023 65 Formigli, L. et al. (2005) Sphingosine 1-phosphate induces cytoskeletal reorganization in C2C12 myoblasts: physiological relevance for stress fibres in the modulation of ion current through stretch-activated channels. J. Cell Sci. 118, 1161–1171
Free journals for developing countries The WHO and six medical journal publishers have launched the Health InterNetwork Access to Research Initiative, which enables nearly 70 of the world’s poorest countries to gain free access to biomedical literature through the internet. The science publishers, Blackwell, Elsevier, Harcourt Worldwide STM group, Wolters Kluwer International Health and Science, Springer-Verlag and John Wiley, were approached by the WHO and the British Medical Journal in 2001. Initially, more than 1500 journals were made available for free or at significantly reduced prices to universities, medical schools, and research and public institutions in developing countries. In 2002, 22 additional publishers joined, and more than 2000 journals are now available. Currently more than 70 publishers are participating in the program. Gro Harlem Brundtland, the former director-general of the WHO, said that this initiative was ‘‘perhaps the biggest step ever taken towards reducing the health information gap between rich and poor countries’’.
For more information, visit www.who.int/hinari www.sciencedirect.com
315
TRENDS in Pharmacological Sciences
Vol.28 No.7
New technologies for dissecting the arteriolar myogenic response Michael A. Hill, Zhe Sun, Luis Martinez-Lemus and Gerald A. Meininger Dalton Cardiovascular Research Center and Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, MO 65211, USA
The arteriolar myogenic response is crucial for the setting of vascular resistance and for providing a level of tone upon which vasodilators can act. Despite its physiological importance, questions remain regarding the underlying signaling mechanisms of the arteriolar myogenic response. Does an increase in pressure within an arteriole exert its effects via the extracellular matrix, an action on cell membranes and/or deformation of cytoskeletal structures? Recent advances in methodology, particularly involving sophisticated imaging approaches, are enabling the study of forces at single-cell and even subcellular levels. Atomic force microscopy (AFM) not only enables detection of cell morphology and stiffness but also allows discrete forces to be applied to single smooth muscle cells and subsequent responses to be observed. Importantly, the repertoire of approaches involving AFM can be expanded by using it in combination with other imaging approaches – including fluorescence imaging for cellular signals such as Ca2+, and total internal reflectance fluorescence, fluorescence resonance energy transfer and confocal microscopy for probing cellular contact function. Combinations of these advanced imaging and nanomechanical approaches will be instructive to studies of intact vessels and the circulatory system in general.
events has not been attained. With regard to arteriolar constriction resulting from an increase in intraluminal pressure (the ‘myogenic response’), the initial description of this vascular response is generally credited to Bayliss in 1902 [5]. Bayliss observed a link between reduced intravascular pressure and increased regional blood flow, which he attributed to the reduced distending pressure acting on the blood vessel wall. Since then, advances in understanding have come mainly from studies of whole animals, exteriorized organs, microvascular preparations and isolated arterioles (for review, see Refs [6–8]). These approaches have provided convincing evidence that pressure-induced vasoconstriction contributes to the setting of peripheral vascular resistance, the autoregulation of blood flow and the regulation of capillary pressure. Mechanistically and conceptually, in its simplest form, increased smooth muscle cell membrane tension (resulting from an increase in intraluminal pressure) is thought to lead to membrane depolarization, voltage-gated Ca2+ entry, activation of actomyosin interaction and contraction [7–10] (see Figure Ia in Box 1). Recently, the contributions of Ca2+ sensitization of the contractile proteins and cytoskeletal rearrangement have been proposed as additional Glossary
Introduction Vascular cells are continually exposed to mechanical forces, as typified by the transmural forces exerted by blood pressure and the shear stresses generated by the flow of blood (Figure 1). Importantly, cells of the vascular wall (both smooth muscle and endothelial) are able to respond to these stimuli by acutely activating signaling pathways to modulate vessel diameter while, in the longer term, altering the structure of the vessel wall, in part by modulation of biosynthetic pathways. Through the ability of these mechanically initiated signaling pathways, both short- and long-term alterations of vasomotor tone, local control of blood flow and vascular resistance can be affected. Although this article focuses predominately on pressure–stretch activation of vascular smooth muscle, the reader is referred to recent reviews of shear-stress-induced activation of endothelial cells [1–4]. Despite the importance of mechanically activated mechanisms of control of the microcirculation and vessel wall, a complete knowledge of the underlying cellular Corresponding author: Hill, M.A. ([email protected]). Available online 18 June 2007. www.sciencedirect.com
Atomic force microscopy (AFM): method enabling the application and measurement of forces in the pN–nN range by precisely controlling the deformation of a thin cantilever. The cantilever carries a tip that is either sharp or a surface such as a bead, and can be functionalized with biological molecules that act as binding partners for the cell or surface being studied. Fluorescence (Forster) resonance energy transfer (FRET): method using the non-radiative transfer of energy from a donor to an acceptor molecule. Energy transfer relies heavily on the distance separating the donor and acceptor molecules. Interference reflection microscopy (IRM): an optical technique for detecting the topography of the side of a cell in contact with a planar substrate. IRM uses the zero-order reflection interference pattern of the cell basal plane to measure the separation (<100 nm) and areas of contact (focal adhesions or close contacts) between the cell and a glass substrate. Laser tweezer: method that enables the application and measurement of forces in the pN range by manipulating a bead using laser beams (optical trap). Mechanical activity: force generated through (smooth muscle) cell contractility or relaxation. Paramagnetic bead: method enabling the application of force, through torque, on a pN–nN scale. Torque is provided by a magnetic field or magnetic needle. RGD peptides: synthetic amino acid sequences containing arginine-glycineaspartate, which are analogs of sequences found in ECM proteins. Binding of the RGD sequence to integrins initiates signaling cascades. Stiffness: Young’s modulus, a measure of the elasticity of a cell in response to the loading of a compression force. Total internal reflectance fluorescence (TIRF): a microscopy technique that excites the cell-labeling fluorophores with an evanescent wave (near-field standing wave exhibiting exponential decay), enabling quantitative measurement of fluorescent labeling within 200-nm distance from a glass substrate.
0165-6147/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2007.05.006
Opinion
TRENDS in Pharmacological Sciences
Vol.28 No.7
309
Figure 1. Mechanical forces and the vascular wall. The arteriolar wall is continually exposed to transmural and shearing forces as a result of the closed nature of the circulatory system. Complexity of the vessel wall occurs as a result of junctional contacts, both between vascular cells and with the ECM proteins. Gap junctional communication enables the vessel wall to act as a functional syncytium, facilitating the transfer of signals along – and perhaps through – the cells of the vessel wall. Contacts between cells and ECM proteins provide a mechanism for ‘sensing’ deformation caused by mechanical forces and, perhaps, initiating signal transduction mechanisms. Abbreviation: MEGJ, myoendothelial gap junction. Modified, with permission, from Ref. [24].
mechanisms that can modulate this pressure-dependent response [11–15]. Despite these advances in understanding, however, the exact molecular mechanisms through which a change in intraluminal pressure is sensed have remained elusive. In particular, the presence of a pressuresensitive mechanosensor, or mechanically activated receptor, is yet to be conclusively demonstrated. It is our opinion, however, that developments in novel and sophisticated imaging techniques [e.g. atomic force microscopy (AFM) (see Glossary), fluorescence resonance energy transfer
(FRET), total internal reflectance fluorescence (TIRF) microscopy, interference reflection microscopy (IRM) and multi-photon microscopy] – together with nanoscale- and picoscale-force measuring devices such as AFM, laser tweezers and paramagnetic microspheres, and approaches from disciplines such as nanomedicine and proteomics – are providing new tools for achieving this goal. These developments in imaging and nanoscale-force measurement are enabling, in particular, the study of candidate mechanosensors, including the axis formed by
Box 1. Signaling events underlying the arteriolar myogenic response Increased intraluminal pressure within an arteriole is thought to be detected by either cell stretch or an increase in wall tension [6,7]. This is conveyed through a direct effect on mechanically sensitive protein elements such as ion channels or the membrane lipid bilayer itself, or through interactions between extracellular elements (e.g. ECM proteins) and cell surface ‘receptors’ such as integrins. Activation of these pathways then leads to the opening of non-selective cation channels, membrane depolarization and the opening of voltage-gated Ca2+ entry [10,55]. Increased [Ca2+]i results in Ca2+/calmodulindependent activation of myosin light-chain kinase, phosphorylation of the 20-kDa myosin regulatory light chains and contractile interaction of actin and myosin [9,56] (Figure Ia). In addition to these events, it is evident that the mechanical stimulus drives other signaling Ca2+dependent and -independent events, including Ca2+ release, kinase activation and, perhaps, phosphatase inhibition. These pathways might contribute to modulation of the contractile response through alterations in Ca2+ sensitivity, Ca2+ handling, activation of ion channels such as BKCa and cytoskeletal rearrangements [11–14,57]. www.sciencedirect.com
Although Figure Ia presents these events in a linear fashion, culminating in contraction, it is equally – if not more – likely that the mechanical stimulation that occurs during an increase in intravascular pressure results in parallel activation of multiple, perhaps interacting, pathways (Figure Ib). Such a situation would be expected if mechanical deformation of the membrane, as would occur during cell stretch, simultaneously applies force to varying mechanosensors or proteins whose activity is modulated by nanoscale forces. Interaction between such pathways would invariably occur on multiple levels. For example, myogenic contraction might decrease tension and the resultant driving force for Ca2+ entry, leading to a biphasic global Ca2+ signal; decreased tension might also relieve a stimulus for adaptive remodeling and structural modification of the vessel wall; Ca2+ entry, in addition to contraction, might activate opposing hyperpolarizing events, as proposed for the BKCa channel. Importantly, the separation of these events requires the ability to apply nanoscale forces precisely to the membrane, perhaps through specific ligand–receptor interactions.
310
Opinion
TRENDS in Pharmacological Sciences
Vol.28 No.7
Figure I. Signaling events underlying the arteriolar myogenic response. Mechanosensors can include different ECM-protein–membrane interactions, mechanosensitive ion channels, mechanosensitive proteins and enzymes, and mechanically activated cytoskeletal rearrangements (denoted by * in the figure).
www.sciencedirect.com
Opinion
TRENDS in Pharmacological Sciences
extracellular matrix (ECM), cell membrane integrins and the cytoskeleton [16,17]. Importantly, this will enable our thinking about mechanotransduction to be further expanded from a purely linear set of events (see Figure Ia in Box 1) to consider more fully that physical forces can activate more than one pathway (see Figure Ib in Box 1). In this regard, it is clear from basic studies of mechanotransduction that cells of the vascular wall interact in a complex manner with their surrounding environment. Thus, it is conceivable that signals generated in response to physical forces within the vessel wall can be conducted in parallel through multiple cellular elements undergoing strain or deformation. Conceptually, these affected elements could include specific ECM proteins (e.g. fibronectin, collagen and laminin), cell surface adhesion receptor links, mechanosensitive ion channels and enzymes, and perturbation of membrane lipid or cytoskeletal components. Further benefits of some of these techniques include the ability to follow dynamic processes in (near) real-time and to consider important parameters such as the directionality of a given stimulus and cell polarity. Importantly, many of these processes cannot currently be studied using classical in vivo microcirculatory and isolated vessel techniques. The use of these imaging techniques is also enhanced by the ability to manipulate cells and vascular preparations genetically to enable the expression of fluorescently labeled reporter molecules or mutated proteins or to alter the expression of given signaling molecules. In this regard, many studies have shown successful transfection of cultured vascular cells and even intact vascular preparations. This has enabled the introduction of reporter molecules for fluorescence imaging, fluorescence-based measurements of ions such as Ca2+ and FRET partners for dynamically following candidate signaling molecules in mechanotransduction [18,19]. Transfection in intact arterioles has been used for regulating the activity of kinases, including Rho and sphingosine kinase, and the introduction of oligonucleotides and small interfering (si)RNA has been used to decrease the expression of candidate signaling molecules (e.g. for TrpC6 and TrpM4) [20–23]. Collectively, these imaging and molecular approaches provide investigators with novel platforms from which to complement pharmacological studies and, ultimately, to improve the understanding of cellular events associated with vascular cell mechanotransduction. Interactions between the ECM and vascular smooth muscle cells Recent attention has been given to the hypothesis that mechanical stimuli exert their effects on vascular cells via the interaction of ECM proteins with cell surface ‘receptors’ such as integrins [17,24]. Integrins represent a heterogeneous family of receptors that exist as heterodimers formed from the 14 a- and seven b-subunits expressed in vascular smooth muscle [24]. It has been shown that synthetic RGD peptides and anti-integrin function-blocking antibodies can inhibit myogenic vascular tone and pressure-induced myogenic signaling [24]. Specifically, in arterioles taken from skeletal muscle, synthetic RGD peptides lower intracellular Ca2+ concentrations ([Ca2+]i) in vascular smooth muscle, leading to vasodilatation via a mechanism www.sciencedirect.com
Vol.28 No.7
311
involving the avb3 integrin, whereas application of a non-vasodilatory concentration of RGD or anti-b3 antibody to the same arteriolar preparation inhibits acute pressureinduced vasoconstriction [24–26]. Such effects of integrinmediated signaling might result from interactions with smooth muscle cell ion channels that regulate vasomotor activity [27–29]. Complexity, however, exists because heterogeneity in integrin-mediated responses occurs between tissues (e.g. skeletal muscle compared with coronary and renal arterioles, and conduit compared with microvessels [24,30,31]). Additionally, the mechanisms by which binding to integrins elicits a particular vascular response can depend on cell-specific integrin signaling (e.g. specifically affecting either smooth muscle or endothelial cells) and which of the various integrin dimers are involved. Clearly, these wholevessel and pharmacological approaches, although providing data to support the hypothesis that an ECM–integrin axis is involved in myogenic signaling, do not enable the specificity of this interaction to be studied at the molecular level. This, therefore, currently limits the ability to understand precisely events such as integrin-mediated mechanotransduction in the arteriolar myogenic response. More than one way to apply a force: twisting, pulling and swelling Several nanoscale approaches have proved valuable in studying the effects of mechanical forces on cells, including those of the vasculature: for example, via the movement of cell-attached magnetic beads [32–34] and with laser tweezers [35–37]. In the former, movement and, hence, stretch can be applied by a magnetic field creating torque on matrixprotein-coated beads, or a directional stimulus can be applied using a magnetic needle. In the latter, proteincoated beads bound to cell surface receptors are trapped in an energy well formed by an infrared laser beam. Displacement of the laser beam can then be used to apply a controlled force to the cell. Stretching of single vascular smooth muscle cells has also been accomplished using modified patch pipettes [38,39]. In electrophysiological studies using this approach, cells were anchored at one site and the lateral movement of a second pipette was used to apply a stretch (100–130% of resting-cell length) stimulus. A third patch pipette was then used to record ion channel events using the whole-cell-recording mode. In these studies, membrane stretch was shown to activate non-selective cation currents, with subsequent opening of voltage-gated Ca2+ channels and large-conductance K+ channels. The effect of membrane stretch has also been studied under conditions of hypo-osmotic cell swelling [40,41], again linking mechanical stimulation to the opening of ion channels. These methods are typically used to study whole-cell stretch and do not currently enable the contribution of single molecules (e.g. integrin–ECM interactions) to be directly studied, although pharmacological approaches can be used (e.g. the addition of synthetic RGD peptides and function-blocking antibodies). Probing interactions between the ECM and vascular smooth muscle cells: AFM The advent of techniques such as biological AFM seems to provide a method for approaching the problem with
312
Opinion
TRENDS in Pharmacological Sciences
the required level of specificity: that is, where singlematrix-molecule–integrin interactions can be discerned. AFM is proving to be a flexible technique that, although first used for topographical studies, can provide information on the mechanical properties of cells, including stiffness, mechanical activity and ligand–receptor interactions, and can be used to measure and apply discrete nanoscale and picoscale forces [42–44]. The ability to measure and apply force, combined with the ability to ‘biofunctionalize’ the AFM tip with protein molecules [45–48], enables the study of mechanical activation via the ECM-protein–integrin axis (Box 2). The ability of AFM to apply forces on a nanoscale also lends itself to examining whether smooth muscle
Vol.28 No.7
mechanotransduction is equally effective when stimuli are applied to different regions of the cell membrane, raising issues such as whether the cell experiences stretch equally as well along its long and short axes. For smooth muscle cells resident in the arteriolar wall, an increase in intraluminal pressure passively stretches cells along their long axis because of their largely circumferential arrangement. However, in certain situations (e.g. during traumatic injury), vessels might be exposed to considerable longitudinal stretch that applies a mechanical force across the short axes of the smooth muscle cells. Interestingly, arterioles subjected to controlled in vitro stretch along these two directions show markedly different [Ca2+]i responses [49].
Box 2. Modes of AFM: the ability to apply controlled nanoscale and picoscale forces AFM (Figure I) provides a flexible approach for the topographical mapping of the cell surface, the measurement of forces associated with binding events, the estimation of biophysical properties of the cytoskeleton – including elasticity – and the application of mechanical stimuli [26,42–44]. These studies can be performed on intact cells, without the need for fixation, and in physiological solutions. AFM is
accomplished through the interaction of a fine tip attached to a cantilever of known spring constant that, for topographical imaging, can be raster-scanned across the surface of a given specimen (hence, AFM is a mode of scanning probe microscopy). Measured spring deflection–indentation at each point enables the reconstruction of images or force maps via digital techniques [42].
Figure I. Example modes of AFM operation, from simple scanning to situations in which the tip or probe is biofunctionalized to increase its specificity of interaction with the cell surface. (a)(i) A native, or non-functionalized, AFM probe enables the collection of detailed topographic information from live cells. (ii) Detail of the underlying cytoskeleton. (iii) A calculated topography map showing the relative height of the cell. Such images are usually achieved in the contact or tapping mode operation of AFM, where the probe is controlled to scan across the cell surface with a preset contact force (ii). (b)(i) AFM probes functionalized with ligand molecules to enable the detection of individual ligand–receptor interactions on the surface of a cell. In force mode operation, the probe is first controlled to contact the cell surface to enable the formation of ligand–receptor bonds. (ii) The probe is then lifted from the cell surface to break the ligand–receptor bonds physically. (iii) The data obtained can be used to resolve the characteristic binding force of a specific ligand–receptor interaction. Thus, for fibronectin–integrin interactions, we have calculated a binding force of 39 pN. (c)(i) Tips on AFM probes can be replaced by microspheres, which have a much larger surface area and can be coated with a variety of ligand molecules. (ii) Bringing the fibronectin-coated microspheres into contact with the vascular smooth muscle cell (VSMC) surface induces the formation of focal-adhesion-like structures, including the formation of integrin clusters and cytoskeletal connections. The AFM probe can then be controlled to apply either pulling or pushing forces to the VSMC through this molecular structure. The cellular response to the forces can be measured by using the AFM to monitor the up-and-down movement of the microsphere. (iii) As contact time continues, an increasing force is required to detach FN-coated microspheres from the VSMC surface, indicating that the molecular structures formed around the microspheres strengthen with time. Panels (a)(iii), (b)(iii) and (c)(iii) reproduced, with permission, from Ref. [46]. www.sciencedirect.com
Opinion
TRENDS in Pharmacological Sciences
As a further example of a relationship between cellular polarity and mechanical signals, endothelial cells align both their long axis and their cytoskeletal apparatus with the direction of flow-induced shear stress [50,51], whereas vascular smooth muscle cells organize themselves perpendicular to mechanical stress fields (Figure 1). It is not surprising that regions of the cell membrane might show differing responses to mechanical stimuli given the relatively recent appreciation of specialized signaling domains and complexes such as cholesterol-rich structures (e.g. caveolae and rafts), focal adhesions, adherens junctions and connexins. Thus, vascular cells might show a degree of polarity with respect to how they respond to mechanical forces: an issue that could be investigated further using nanomechanical approaches. Sun et al. [46] used AFM probes that were biofunctionalized with fibronectin to examine the characteristics of the binding of this ECM protein with cell surface integrins. In these studies, the fibronectin-coated probe was lowered onto cultured arteriolar smooth muscle cells and allowed to adhere for varying lengths of time, after which the probe was retracted from the cell surface and the rupture force was recorded (Box 2). The specificity of binding was shown by the use of anti-integrin antibodies and RGD peptides, with the authors concluding that the fibronectin binding was mediated largely through a5b1 integrin. Using this approach, it was further suggested that the strength of binding between single fibronectin–integrin interactions was 39 8 pN. Using an AFM probe with an attached microsphere coated with fibronectin, withdrawal of the probe can be used to pull on the membrane and underlying cytoskeleton. In experiments such as these, the magnitude of the applied force is related to the distance of retraction and bending (spring constant dependent) of the AFM cantilever (Box 2). Biofunctionalizing the probe with selected ECM proteins that interact differentially with members of the integrin family enables the stretch stimulus (force step) to be applied through various putative mechanosensory pathways (Box 1). Wider use of AFM and combining it with other imaging and cellular approaches The potential use of AFM in the study of vascular cells extends beyond interactions between ECM proteins and cell surface integrins. Appropriately functionalized probes have the capability to enable the study of many cell surface molecules of proposed importance to vascular cell function and mechanotransduction, including ion channels [52] and connexins [53,54]. At a more macroscopic level, the interaction of white cells with vascular endothelial cells has been studied by functionalizing AFM probes with leukocytes [48]. A powerful future direction for imaging-based studies of mechanotransduction will be the evolution of combining nanoscale-force application systems (including AFM, laser tweezers and magnetized microbeads) with other imaging approaches: for example, wide-field fluorescence, confocal and multiphoton microscopy, and FRET and TIRF microscopy. Crucial to this is that combining AFM with optical approaches improves the use of AFM without compromising www.sciencedirect.com
Vol.28 No.7
313
Figure 2. Combined imaging modes for simultaneously measuring changes in cytosolic Ca2+ concentration and cell stiffness. Studies were performed on cultured cremaster muscle arteriolar smooth muscle cells. Changes in intracellular Ca2+ concentration were estimated using fluorescence ratio imaging with cells loaded with the Ca2+-sensitive indicator fura 2. Changes in cell stiffness were followed using AFM by taking repetitive force measurements at a single site on the cell surface. An increase in [Ca2+]i, as indicated by the 340:380-nm excitation fluorescence ratio, was followed by an increase in stiffness consistent with the occurrence of contraction. Changes in cell height (not shown) measured by AFM also followed the changes in [Ca2+]i (Z.S. and G.A.M., unpublished).
its features such as its nanoscale resolution. Combined systems enable, for example, the measurement of changes in [Ca2+]i, which can be correlated with indices of contraction as determined by AFM (Figure 2). Furthermore, combined imaging modes promise the study of focal adhesions and cytoskeletal proteins, including during the application of nanoscale or picoscale forces. Importantly, such responses can be followed over time so that dynamic studies of mechanotransduction can be performed. Similarly, although AFM can be used to apply controlled forces to the upper surface of the cell, TIRF microscopy can be employed simultaneously to examine adhesion sites on the basal surface of the cell. Because it is not practical to provide an exhaustive review of the literature, several examples of AFM combined with optical-imaging approaches are provided in Table 1. The studies listed in Table 1 are limited to those involving the Table 1. Examples of AFM combined with optical-imaging approaches Combination of imaging approaches AFM and confocal microscopy
AFM and dual-photon microscopy AFM and TIRF AFM and FRET AFM and fluorescent Ca2+ imaging a
Cell system studied
Refs
Osteoblasts a Bacterial membranes Jurkhat T lymphoma Tumor mast cells Chloroplasts Fibroblasts Titin molecules a VSM cells a Titin molecules a Skeletal myoblasts a Osteoblasts a
[58] [59]
Studies specifically relevant to mechanotransduction.
[60] [61] [62] [62] [63] [64] [63] [65] [58]
314
Opinion
TRENDS in Pharmacological Sciences
simultaneous use of multiple imaging modes rather than correlative studies in which various imaging approaches were performed in parallel experiments. Concluding remarks The use of nanomechanical approaches such as AFM and optical-imaging techniques has the potential to make significant advances in the understanding of vascular cell mechanotransduction. In particular, such techniques show promise for isolating specific cellular mechanosensors and delineating the signaling pathways that are subsequently activated. Data from such techniques will provide for new avenues of research that can be applied to more-intact preparations, including isolated arterioles and vascular networks. Knowledge of the behavior of single cells in response to controlled nanoscale or picoscale forces might then provide the basis for understanding how these preparations respond to complex mechanical stimuli such as those provided by an alteration in intraluminal pressure or shear stress. Ultimately, it will be important to put the pieces together to understand the physiological role of the myogenic response within a complex vascular network. References 1 Davies, P.F. (1995) Flow-mediated endothelial mechanotransduction. Physiol. Rev. 75, 519–560 2 McCue, S. et al. (2004) Shear-induced reorganization of endothelial cell cytoskeleton and adhesion complexes. Trends Cardio. Med. 14, 143– 151 3 Li, Y.S. et al. (2005) Molecular basis of the effects of shear stress on vascular endothelial cells. J. Biomech. 38, 1949–1971 4 Davies, P.F. et al. (2005) Shear stress biology of the endothelium. Ann. Biomed. Eng. 33, 1714–1718 5 Bayliss, W.M. (1902) On the local reactions of the arterial wall to changes of internal pressure. J. Physiol. 28, 220–231 6 Johnson, P.C. (1980) The myogenic response. In Handbook of Physiology, the Cardiovascular System, Vascular Smooth Muscle, pp. 409–442, American Physiological Society 7 Davis, M.J. and Hill, M.A. (1999) Signaling mechanisms underlying the vascular myogenic response. Physiol. Rev. 79, 387–423 8 Schubert, R. and Mulvany, M.J. (1999) The myogenic response: established facts and attractive hypotheses. Clin. Sci. (Lond.) 96, 313–326 9 Zou, H. et al. (1995) Role of myosin phosphorylation and [Ca2+]i in myogenic reactivity and arteriolar tone. Am. J. Physiol. 269, H1590– H1596 10 Knot, H.J. and Nelson, M.T. (1998) Regulation of arterial diameter and wall [Ca2+] in cerebral arteries of rat by membrane potential and intravascular pressure. J. Physiol. 508, 199–209 11 Gokina, N.I. and Osol, G. (2002) Actin cytoskeletal modulation of pressure-induced depolarization and Ca2+ influx in cerebral arteries. Am. J. Physiol. 282, H1410–H1420 12 Lagaud, G. et al. (2002) Pressure-dependent myogenic constriction of cerebral arteries occurs independently of voltage-dependent activation. Am. J. Physiol. 283, H2187–H2195 13 Schubert, R. et al. (2002) Rho kinase inhibition partly weakens myogenic reactivity in rat small arteries by changing calcium sensitivity. Am. J. Physiol. 283, H2288–H2295 14 Flavahan, N.A. et al. (2005) Imaging remodeling of the actin cytoskeleton in vascular smooth muscle cells after mechanosensitive arteriolar constriction. Am. J. Physiol. 288, H660–H669 15 Keller, M. et al. (2006) Sphingosine kinase functionally links elevated transmural pressure and increased reactive oxygen species formation in resistance arteries. FASEB J. 20, 702–704 16 Vogel, V. and Sheetz, M. (2006) Local force and geometry sensing regulate cell functions. Nature Rev. 7, 265–275 www.sciencedirect.com
Vol.28 No.7
17 Alenghat, F.J. and Ingber, D.E. (2002) Mechanotransduction: all signals point to cytoskeleton, matrix, and integrins. Sci. STKE 2002, PE6 18 Wang, Y. et al. (2005) Visualizing the mechanical activation of Src. Nature 434, 1040–1045 19 Kotlikoff, M.I. (2007) Genetically encoded Ca2+ indicators: using genetics and molecular design to understand complex physiology. J. Physiol. 578, 55–67 20 Welsh, D.G. et al. (2002) Transient receptor potential channels regulate myogenic tone of resistance arteries. Circ. Res. 90, 248– 250 21 Slish, D.F. et al. (2002) Diacylglycerol and protein kinase C activate cation channels involved in myogenic tone. Am. J. Physiol. 283, H2196– H2201 22 Earley, S. et al. (2004) Critical role for transient receptor potential channel TRPM4 in myogenic constriction of cerebral arteries. Circ. Res. 95, 922–929 23 Bolz, S.S. et al. (2003) Sphingosine kinase modulates microvascular tone and myogenic responses through activation of RhoA/Rho kinase. Circulation 108, 342–347 24 Martinez-Lemus, L.A. et al. (2003) Integrins as unique receptors for vascular control. J. Vasc. Res. 40, 211–233 25 Mogford, J.E. et al. (1996) Vascular smooth muscle avb3 integrin mediates arteriolar vasodilation in response to RGD peptides. Circ. Res. 79, 821–826 26 D’Angelo, G. et al. (1997) Integrin-mediated reduction in vascular smooth muscle [Ca2+]i induced by RGD-containing peptide. Am. J. Physiol 272, H2065–H2070 27 Wu, X. et al. (1998) Modulation of calcium current in arteriolar smooth muscle by avb3 and a5b1 integrin ligands. J. Cell Biol. 143, 241–252 28 Wu, X. et al. (2001) Regulation of the L-type calcium channel by a5b1 integrin requires signaling between focal adhesion proteins. J. Biol. Chem. 276, 30285–30292 29 Gui, P. et al. (2006) Integrin receptor activation triggers converging regulation of Cav1.2 calcium channels by c-Src and protein kinase A pathways. J. Biol. Chem. 281, 14015–14025 30 Yip, K.P. and Marsh, D.J. (1997) An Arg-Gly-Asp peptide stimulates constriction in rat afferent arteriole. Am. J. Physiol. 273, F768–F776 31 Hein, T.W. et al. (2001) Integrin-binding peptides containing RGD produce coronary arteriolar dilation via cyclooxygenase activation. Am. J. Physiol. 281, H2378–H2384 32 Wang, N. et al. (1993) Mechanotransduction across the cell surface and through the cytoskeleton. Science 260, 1124–1127 33 Guilford, W.H. et al. (1995) Locomotive forces produced by single leukocytes in vivo and in vitro. Am. J. Physiol. 268, C1308–C1312 34 Pommerenke, H. et al. (1996) Stimulation of integrin receptors using a magnetic drag force device induces an intracellular free calcium response. Eur. J. Cell Biol. 70, 157–164 35 Schmidt, C.E. et al. (1993) Integrin–cytoskeletal interactions in migrating fibroblasts are dynamic, asymmetric, and regulated. J. Cell Biol. 123, 977–991 36 Sheetz, M.P. and Dai, J. (1996) Modulation of membrane dynamics and cell motility by membrane tension. Trends Cell Biol. 6, 85–89 37 Giannone, G. et al. (2003) Talin1 is critical for force-dependent reinforcement of initial integrin–cytoskeleton bonds but not tyrosine kinase activation. J. Cell Biol. 163, 409–419 38 Davis, M.J. et al. (1992) Stretch-activated single-channel and whole cell currents in vascular smooth muscle cells. Am. J. Physiol. 262, C1083– C1088 39 Wu, X. and Davis, M.J. (2001) Characterization of stretch-activated cation current in coronary smooth muscle cells. Am. J. Physiol. 280, H1751–H1761 40 Langton, P.D. (1993) Calcium channel currents recorded from isolated myocytes of rat basilar artery are stretch sensitive. J. Physiol. 471, 1–11 41 Welsh, D.G. et al. (2000) Swelling-activated cation channels mediate depolarization of rat cerebrovascular smooth muscle by hyposmolarity and intravascular pressure. J. Physiol. 527, 139–148 42 Radmacher, M. (1997) Measuring the elastic properties of biological samples with the AFM. IEEE Eng. Med. Biol. Mag. 16, 47–57 43 Hassan, A.E. et al. (1998) Relative microelastic mapping of living cells by atomic force microscopy. Biophys. J. 74, 1564–1578
Opinion
TRENDS in Pharmacological Sciences
44 Sun, Z. et al. (2004) Vascular smooth muscle cells (VSMC) mechanically respond to application of force to the fibronectin–a5b1 integrin bond. FASEB J. 18, A665 45 Lehenkari, P.P. and Horton, M.A. (1999) Single integrin molecule adhesion forces in intact cells measured by atomic force microscopy. Biochem. Biophys. Res. Comm. 259, 645–650 46 Sun, Z. et al. (2005) Mechanical properties of the interaction between fibronectin and a5b1-integrin on vascular smooth muscle cells studied using atomic force microscopy. Am. J. Physiol. 289, H2526– H2535 47 Moy, V.T. et al. (1994) Intermolecular forces and energies between ligands and receptors. Science 266, 257–259 48 Zhang, X. et al. (2004) Atomic force microscopy measurement of leukocyte–endothelial interaction. Am. J. Physiol. 286, H359– H367 49 Hill, M.A. et al. (2000) Transient increases in diameter and [Ca2+]i are not obligatory for myogenic constriction. Am. J. Physiol. 278, H345– H352 50 Noria, S. et al. (2004) Assembly and reorientation of stress fibers drives morphological changes to endothelial cells exposed to shear stress. Am. J. Pathol. 164, 1211–1223 51 McCue, S. et al. (2006) Shear stress regulates forward and reverse planar cell polarity of vascular endothelium in vivo and in vitro. Circ. Res. 98, 939–946 52 Schar-Zammaretti, P. et al. (2002) Potassium-selective atomic force microscopy on ion-releasing substrates and living cells. Anal. Chem. 74, 4269–4274 53 Lal, R. and Lin, H. (2001) Imaging molecular structure and physiological function of gap junctions and hemijunctions by multimodal atomic force microscopy. Microsc. Res. Tech. 52, 273–288 54 Liu, F. et al. (2006) Nanomechanics of hemichannel conformations: connexin flexibility underlying channel opening and closing. J. Biol. Chem. 281, 23207–23217
Vol.28 No.7
55 Kotecha, N. and Hill, M.A. (2005) Myogenic contraction in rat skeletal muscle arterioles: smooth muscle membrane potential and Ca2+ signaling. Am. J. Physiol. 289, H1326–H1334 56 Zou, H. et al. (2000) Temporal aspects of Ca2+ and myosin phosphorylation during myogenic and norepinephrine-induced arteriolar constriction. J. Vasc. Res. 37, 556–567 57 Jaggar, J.H. et al. (1998) Ca2+ channels, ryanodine receptors and Ca2+-activated K+ channels: a functional unit for regulating arterial tone. Acta. Physiol. Scand. 164, 577–587 58 Charras, G.T. and Horton, M.A. (2002) Single cell mechanotransduction and its modulation analyzed by atomic force microscope indentation. Biophys. J. 82, 2970–2981 59 Kassies, R. et al. (2005) Combined AFM and confocal fluorescence microscope for applications in bio-nanotechnology. J. Microsc. 217, 109–116 60 Lulevich, V. et al. (2006) Cell mechanics using atomic force microscopybased single-cell compression. Langmuir 22, 8151–8155 61 Frankel, D.J. et al. (2006) Revealing the topography of cellular membrane domains by combined atomic force microscopy/ fluorescence imaging. Biophys. J. 90, 2404–2413 62 Gradinaru, C.C. et al. (2004) Simultaneous atomic-force and two-photon fluorescence imaging of biological specimens in vivo. Ultramicroscopy 99, 235–245 63 Kellermayer, M.S. and Grama, L. (2002) Stretching and visualizing titin molecules: combining structure, dynamics and mechanics. J. Muscle Res. Cell Motil. 23, 499–511 64 Trache, A. and Meininger, G.A. (2005) Atomic force–multi-optical imaging integrated microscope for monitoring molecular dynamics in live cells. J. Biomed. Opt. 10, 064023 65 Formigli, L. et al. (2005) Sphingosine 1-phosphate induces cytoskeletal reorganization in C2C12 myoblasts: physiological relevance for stress fibres in the modulation of ion current through stretch-activated channels. J. Cell Sci. 118, 1161–1171
Free journals for developing countries The WHO and six medical journal publishers have launched the Health InterNetwork Access to Research Initiative, which enables nearly 70 of the world’s poorest countries to gain free access to biomedical literature through the internet. The science publishers, Blackwell, Elsevier, Harcourt Worldwide STM group, Wolters Kluwer International Health and Science, Springer-Verlag and John Wiley, were approached by the WHO and the British Medical Journal in 2001. Initially, more than 1500 journals were made available for free or at significantly reduced prices to universities, medical schools, and research and public institutions in developing countries. In 2002, 22 additional publishers joined, and more than 2000 journals are now available. Currently more than 70 publishers are participating in the program. Gro Harlem Brundtland, the former director-general of the WHO, said that this initiative was ‘‘perhaps the biggest step ever taken towards reducing the health information gap between rich and poor countries’’.
For more information, visit www.who.int/hinari www.sciencedirect.com
315