Published online before print April 9, 2009, doi: 10.1161/CIRCRESAHA.108.192930
© 2009 American Heart Association, Inc.
Cellular Biology |
TRPV4 Channels Mediate Cyclic Strain–Induced Endothelial Cell Reorientation Through Integrin-to-Integrin Signaling
From the Vascular Biology Program (C.K.T., B.M., A.R., A.M., K.G., A.L.B., D.E.I.) and Departments of Pathology (D.E.I.), Surgery (C.K.T., B.M., A.M., K.G., D.E.I.), and Medicine (B.M.), Harvard Medical School and Children’s Hospital, Boston; and Wyss Institute for Biologically Inspired Engineering and Harvard School of Engineering and Applied Sciences (D.E.I.), Cambridge, Mass.
Correspondence to Donald E. Ingber, MD, PhD, Vascular Biology Program, KFRL 11.127,300 Longwood Ave, Children’s Hospital/Harvard Medical School, Boston, MA 02115. E-mail donald.ingber{at}childrens.harvard.edu
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Abstract |
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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 (transient receptor potential vanilloid 4) ion channels that, in turn, stimulate phosphatidylinositol 3-kinase–dependent activation and binding of additional β1 integrin receptors, which promotes cytoskeletal remodeling and cell reorientation. Inhibition of integrin activation using blocking antibodies and knock down of TRPV4 channels using specific small interfering RNA 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.
Key Words: mechanical strain • integrin • TRPV4 • endothelial cell • reorientation • cytoskeleton
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Introduction |
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Mechanical forces regulate vascular growth and development by influencing endothelial cell growth, survival, differentiation and migration.1,2 Local mechanical cues conveyed by extracellular matrix (ECM) attributable to cyclic deformation of blood vessels, hemodynamic forces, or cell-generated traction forces are also potent inducers of directional capillary blood vessel growth and vascular remodeling in vitro and in vivo.3–10 For example, the initial step in neovascularization involves reorientation of a subset of capillary endothelial (CE) cells that spread and migrate perpendicular to the main axis of the preexisting vessel toward the angiogenic stimulus11; however, the molecular mechanism responsible for this CE cell reorientation response is unknown. Many cell types, including large vessel endothelial cells, realign perpendicular to the direction of the applied force when they experience cyclic stretching (mechanical strain).12–15 In the case of macrovascular endothelium, this reorientation response can be prevented by treatment with chemical inhibitors of stretch-activated (SA) ion channels.15 But neither the identity of these channels nor the mechanism by which they elicit cell reorientation is known.
Endothelial cells express most members of the transient receptor potential (TRP) family of ion channels16–18 and TRP vanilloid (TRPV)4 has been reported to mediate flow-induced vasodilation in large vessel endothelium.19–22 Here, we show that calcium influx through TRPV4 channels stimulated by mechanically stretching CE cells through their integrin–extracellular matrix (ECM) adhesions promotes cell reorientation by activating phosphatidylinositol 3-kinase (PI3K), thereby stimulating activation of additional β1 integrin receptors. This mechanism is distinct from that used by macrovascular endothelium to sense fluid shear stresses, which is mediated by a mechanosensory complex containing platelet endothelial cell adhesion molecule 1, vascular endothelial growth factor receptor, and VE-cadherin.23
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Materials and Methods |
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Cell Culture
CE cells were isolated from bovine adrenal cortex, cloned, and passaged as described previously.24 Frozen aliquots of these cells (passage, <15), which we have confirmed retain their functionality and differentiation potential,3 were maintained at 37°C in 10% CO2 on gelatin-coated tissue culture dishes in low glucose DMEM (Invitrogen) supplemented with 10% FCS (Hyclone), 10 mmol/L HEPES (JRH-Biosciences), and L-glutamine (292 µg/mL), penicillin (100 U/mL), streptomycin (100 µg/mL) (GPS), as described.25 Human microvascular endothelial cells from dermis (Cambrex, Walkersville, Md) were cultured in EBM-2 (Cambrex), supplemented with 5% FBS, and growth factors (basic fibroblast growth factor, insulin-like growth factor, vascular endothelial growth factor) according to the instructions of the manufacturer.
Mechanical Strain Application
CE cells cultured on fibronectin-coated 6-well Uniflex (Flex Cell International) plates for 24 hours were subjected to uniaxial cyclic stretch (10% elongation; 1 Hz frequency) for 1 to 2 hours using a Flexercell Tension Plus System (Flex Cell International).26 In some experiments, CE cells were plated on fibronectin-coated 6-well Bioflex (Flex Cell International) for 1 hour and subjected to static stretch (15% elongation) for 1 to 15 minutes. Control cells were maintained under identical conditions in the absence of strain application.
Measurement of Cell Orientation
To measure the orientation of cells in cyclic strain experiments, fluorescent images of cells were traced to measure angle with the direction of cyclic strain using ImageJ software (NIH) and reported as percentage cells aligned at 90±30°. For each condition, 5 to 6 fields were evaluated, with approximately 15 to 30 cells per field. Statistical differences between experimental groups were determined using the Student’s t test. All data were obtained from at least 3 separate experiments and are expressed as means±SEM.
Small Interfering RNA Knock Down of TRPV Channels
Smart pool small interfering (si)RNAs (10 nmol/L) of TRPV2, TRPV4 (both from Dharmacon), TRP channel (TRPC)1 (Ambion), or control (Qiagen) siRNAs was transfected into CE cells using siLentFect reagent (Bio-Rad) as described.27 Three days later, cells were used for calcium imaging or reorientation experiments. The knock down of TRPV channel expression was assessed using RT-PCR with species-specific primers and Western blotting.
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Results |
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Capillary Cell Reorientation Induced by Cyclic Strain
Directional CE cell motility and angiogenesis have been shown to be stimulated by mechanical strain (distortion) in ECM gels and living tissues.7–10 To begin to analyze the molecular mechanism by which mechanical strain influences CE cell orientation, we cultured bovine CE cells on flexible fibronectin-coated substrates and subjected them to 10% uniaxial cyclic strain (1 Hz) using a FlexerCell Tension Plus system. Fluorescence microscopic analysis of cells labeled with Alexa 488–phalloidin combined with computerized morphometry revealed that stress fibers thickened in these cells, and most (
80%) realigned perpendicular to the main axis of the applied strain within 2 hours after force application (Figure 1A and 1B). Stress fiber realignment was accompanied by redistribution and reorientation of focal adhesions containing vinculin (Figure 1C), focal adhesion kinase, and talin (not shown), which appeared in close association with the ends of newly aligned stress fibers (Figure 1C).
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Strain-Induced Capillary Cell Reorientation Requires β1 Integrin Activation
The effects of fluid shear on large vessel endothelium28 and mechanical strain on fibroblasts29 are mediated by stress-dependent activation of integrin receptors within minutes after force application. When CE cells cultured on flexible fibronectin-coated substrates were exposed to static stretch (15% elongation), β1 integrin activation increased within 1 minute after force application, as indicated by increased phosphorylation of the T788/789 site of the β1 integrin cytoplasmic tail in Western blots (Figure 2A), which has been shown to correlate with integrin activation.30–32 Immunofluorescence staining using 12G10 antibodies that only recognize the activated conformation of β1 integrins33,34 also showed increased clustering of activated β1 integrins within large streak-like focal adhesions at the cell periphery within 15 minutes after static strain application (Figure 2B). The ability of the 12G10 antibody to detect activated β1 integrins in our CE cells was confirmed using flow cytometry, which demonstrated a significantly increased 12G10 signal after globally activating integrins by treatment with manganese (Figure I in the online data supplement, available at http://circres.ahajournals.org). Static stretch-induced activation of integrin signaling was confirmed independently by demonstrating increased phosphorylation of mitogen-activated protein kinase (extracellular signal-regulated kinase [ERK]1/2) (Figure 2C) and focal adhesion kinase (Online Figure II) within 5 to 15 minutes after exposure to mechanical strain. Application of uniaxial cyclic strain (10%; 1 Hz) also induced β1 integrin activation within minutes, as measured by enhanced binding to the fibronectin fragment glutathione S-transferase (GST)-FNIII8–11 (Figure 2D and Online Figure III, B) and to the 12G10 antibody, which only ligate the activated form of the β1 integrin receptor (Figure 2E),35 as well as by increased T788/789 phosphorylation of β1 integrin (Online Figure III, C). Cyclic strain also increased β1 integrin activation in human CE cells, as measured by enhanced binding of GST-FNIII8–11 (Online Figure III, B), and, thus, this appears to be a generalized response in CE cells.
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To explore whether this mechanical strain–induced wave of β1 integrin activation is required for CE cell reorientation, cells were preincubated with function-blocking anti–β1 integrin (P5D2) antibody for 30 minutes, and then the cells were subjected to uniaxial cyclic strain (10%) for 2 hours. Treatment with this inhibitory antibody, but not isotype-matched control IgG, inhibited strain-induced cell realignment by almost 70% (P<0.001) (Figure 2F), and it prevented reorientation of stress fibers and focal adhesions (Online Figure IV). Before stretching, we did not find any changes in cell morphology or actin staining in antibody-treated cells, confirming that binding of these antibodies did not affect existing adhesions. These results indicate that application of mechanical strain to CE cells through existing integrins that are bound to substrate-immobilized ECM molecules (and hence activated) induces focal adhesion remodeling, stress fiber realignment, and cell reorientation through a mechanism that requires activation of additional β1 integrin receptors.
PI3K Is Upstream of β1 Integrin Activation in This Mechanical Signaling Cascade
PI3K has been implicated in the activation of β3 integrins by fluid shear stress in large vessel endothelium23; however, it also can act downstream of integrin activation.36 To explore whether PI3K is involved in early mechanical signaling in microvascular endothelium, CE cells were transfected with green fluorescent protein (GFP) fused to an AKT-PH domain that translocates to the plasma membrane when it binds to the PI3K product, phosphatidylinositol 3-phosphate.37 Bright linear GFP-AKT-PH staining was detected at the peripheral membrane within 1 minute after application of static stretch (15%), whereas it remained diffusely distributed throughout the cytoplasm in control (unstrained) CE cells (Figure 3A). Quantification of GFP-AKT-PH translocation by 2 independent parameters (fraction of GFP-AKT-PH in total perimeter of the membrane or GFP-fluorescence intensity ratio between membrane and cytosol) revealed a significant increase in response to mechanical strain that was inhibited by treatment with the PI3K inhibitor LY294002 (Figure 3B). Static stretch also activated PI3K, as determined by enhanced phosphorylation of its downstream target AKT at Ser473 within minutes after force application, as detected in Western blots (Figure 3C). Moreover, stretch-induced translocation of GFP-AKT-PH to the membrane and AKT phosphorylation were both abolished by inhibiting PI3K with LY294002 (Figure 3A and 3D). LY294002 treatment also prevented β1 integrin activation (Figure 3D and 3E) and suppressed focal adhesion kinase activation (Online Figure II). Thus, force application through ECM–integrin adhesions activates additional cell surface β1 integrin receptors by stimulating PI3K.
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Strain-Induced Cell Reorientation Is Mediated by Stress-Activated Ion Channels
SA ion channels have been implicated in force-dependent alignment of large vessel endothelial cells.15 Direct force application to cell surface β1 integrins using magnetic tweezers also results in rapid (within 2 to 5 seconds) calcium influx in our bovine CE cells, and this response can be blocked using the general SA channel inhibitor, gadolinium chloride.25,38 To confirm that mechanical strain activates SA channels in these CE cells, cells adherent to flexible ECM substrates were loaded with the calcium reporter dye Fluo-4, subjected to static stretch (15% elongation) and calcium influx was measured using microfluorimetry.25 Stretching CE cells for as little as 3 seconds induced rapid calcium influx, and this response could be almost completely abolished by treatment with gadolinium chloride (Online Figure III, A). Pretreatment of bovine and human CE cells for 30 minutes with gadolinium chloride also significantly inhibited β1 integrin activation in response to static stretch, as measured by decreased binding of GST-FNIII8–11 and reduced β1 integrin phosphorylation (Online Figure III, B and C). In addition, blocking SA channels with gadolinium chloride inhibited PI3K activity, as measured by membrane translocation of GFP-AKT-PH (Online Figure III, D). Finally, the cell and cytoskeletal reorientation normally induced by cyclic strain were greatly suppressed in the presence of this SA ion channel blocker (Online Figure III, E). Thus, mechanical stretch-dependent activation of mechanosensitive calcium channels appears to be required for activation of both PI3K and β1 integrins, as well as subsequent cytoskeletal reorientation in CE cells.
TRPV4 Channels Mediate Strain-Induced Capillary Cell Reorientation
We then set out to identify the specific type of mechanosensitive ion channel that mediates the effects of mechanical strain on CE cell orientation. TRPV4 is an interesting potential candidate because it mediates cell sensitivity to osmotic stresses39 and shear stress–induced vasodilation.19To determine whether TRPV4 is the candidate mechanosensitive channel, first we measured its expression in CE cells. Western blot analysis showed a strong band around 85 kDa (and a fainter band at 100 kDa) in both bovine and human CE cells (Figure 4A). RT-PCR analysis also confirmed the presence of TRPV4 mRNA in both bovine and human CE cells (Figure 4B and Online Figure V). We then found that a specific activator of TRPV4 channels, 4-
-phorbol-12,13-didecanoate (4--PDD),40 induced a robust calcium signal in bovine and human CE cells, thus suggesting that both cell types express functional TRPV4 channels (Online Figure VI). Next, we measured TRPV4 channel activation directly by whole-cell clamp using bovine CE cells transiently transfected with TRPV4-EGFP that gave robust TRPV4 currents in response to 4--PDD, and we found that substitution of N-methyl-D-glucamine for cations in the bathing solution, inhibited activation of inward, but not outward, currents by 4--PDD in these cells (Online Figure VII). We used this approach because TRPV4-like currents in primary endothelial cells are small, transient, and difficult to characterize, as previously described22 and as we observed as well. Thus, taken together, these findings strongly suggest that CE cells express functional TRPV4 channels, although at a low level.
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To confirm that calcium influx through TRPV4 channels mediates the effects of cyclic strain on CE cell orientation, we knocked down the expression of TRPV4 in bovine and human CE cells using specific siRNA; sham siRNA and siRNA directed against the closely related channel TRPV2 were used as controls. Sequence analysis of smart pool siRNAs confirmed that both siRNA sequences exhibit 80% to 100% homology with bovine and human TRPV4. RT-PCR analysis revealed that TRPV2 and TRPV4 mRNA levels were knocked down by 70% and 90% in bovine and human CE cells, respectively, using this approach, whereas use of a sham control siRNA had no effect (Figure 4B and Online Figure V). We found that TRPV4 protein expression was also knocked down by 60% and 80% in bovine and human CE cells, respectively (Figure 4C and 4D and Online Figure V).
Importantly, microfluorimetric analysis revealed that application of static stretch (15%) for 4 seconds induced a large wave of calcium influx in bovine CE cells transfected with control siRNAs, whereas this response was significantly inhibited (P<0.02) in cells treated with TRPV4 siRNA (Figure 4E and 4F). In contrast, use of siRNA directed against the closely related SA channel TRPV2 had no effect (Figure 4E and 4F). siRNA knock down of TRPV4 also inhibited cyclic strain–induced activation of β1 integrins, AKT, and ERK1/2, further confirming that TRPV4 activation is upstream of integrin activation (Figure 5). Pretreatment of CE cells with the general TRPV inhibitor ruthenium red41 or with TRPV4 siRNA also significantly suppressed calcium signaling and cell reorientation induced by application of cyclic strain in CE cells, whereas addition of siRNA against 2 different related SA channels, TRPV2 or TRPC1 (Online Figure V), was ineffective (Figure 6A through 6D). This inhibition was specific for reorientation as transfection of cells with TRPV4 siRNA did not alter the number of viable adherent CE cells when they were cultured on standard tissue culture substrates (Online Figure VIII). Moreover, we found that application of similar cyclic strain, in the presence or absence of ruthenium red, did not effect CE cell proliferation or apoptosis, as measured by Ki 67 staining and poly(ADP-ribosyl) polymerase cleavage (Online Figure IX). Taken together, these results indicate that TRPV4 channels are mechanosensitive calcium channels in CE cells that are activated by mechanical strain applied through the integrin-mediated cell–ECM adhesions and that calcium influx through these channels is required for downstream signaling events that drive the cell and cytoskeletal reorientation response triggered by cell stretching.
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) and presence (
) of the TRPV inhibitor ruthenium red (RR) (*P<0.02). C, Phase-contrast photomicrographs of CE cells showing the effects of cyclic strain on cell reorientation in the absence and presence of ruthenium red. Arrow indicates the direction of applied strain. Note that ruthenium red inhibits cyclic strain–induced cell reorientation. Scale bar=50 µm. D, Percentage of cells oriented 90±30° degrees (aligned) relative to the direction of applied strain in control (white bars) and strain-exposed (black bars) human CE cells treated with the indicated siRNA. Note that TRPV4 siRNA–treated cells failed to reorient fully compared to TRPV2- or TRPC1-treated cells (*P<0.0025). |
Discussion |
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In this study, we showed that application of mechanical strain to bound integrins on the CE cell surface stimulates calcium influx through mechanosensitive TRPV4 ion channels, which activates additional β1 integrins and subsequent downstream cytoskeletal reorientation responses. Although cyclic strain induces reorientation of large vessel endothelial cells and this process has been shown to be mediated by activation of SA channels,15 the present study is the first to analyze this process in microvascular CE cells and to determine the specific molecular identity of these channels. Our work shows the TRPV4 is at least one of the SA channels that is required for activation of β1 integrins and subsequent reorientation of CE cells in response to mechanical strain.
Cell stretching and strain application to integrins have both been implicated as critical regulators of endothelial cell proliferation, migration, and angiogenesis in the past,3,5,6,9,42–44 but how these mechanical signals control vascular development is not known. The present findings provide direct evidence to show that mechanical strain activates β1 integrins in bovine and human CE cells and that this is required for downstream cell and cytoskeletal remodeling events that mediate cell reorientation critical for directional cell motility. Given that we exposed cells to both static and cyclic stretch and similar results were obtained using multiple different assays and probes to assess β1 integrin activation, we believe that these findings unequivocally confirm that mechanical strain activates β1 integrins in CE cells.
The most important finding of this study is the identification of TRPV4 as the SA channel responsible for β1 integrin activation in response to mechanical strain application to microvascular cells. We make this conclusion based on the following observations: (1) bovine and human CE cells functionally express TRPV4 channels that are activated by the selective TRPV activator 4-PDD; (2) the TRPV4 blocker ruthenium red inhibits calcium influx and cell reorientation in response to mechanical strain; and (3) siRNA knock down of TRPV4, but not TRPV2 or TRPC1, inhibits strain-induced calcium influx and capillary cell reorientation. Among all known TRP channels, only TRPV4 has been reported to be mechanosensitive in that it transduces osmotic signals39 and plays a role in shear stress–induced vasodilation.19,20 TRPV4 is also important for the mechanical behavior of Caenorhabditis elegans,45 and mice lacking TRPV4 are insensitive to normal levels of noxious mechanical stimuli.46 Here, we show that activation of TRPV4 by mechanical distortion of cell–ECM adhesions plays a critical role in control of downstream signaling pathways that mediate cell reorientation and vascular development in response to mechanical strain of integrin-mediated cell–ECM adhesions.
Although TRPV2 and TRPC1 channels were shown to mediate stretch-induced calcium signaling when overexpressed in CHO cells and oocytes,47,48 we found that knocking down of either TRPV2 and TRPC1 expression using siRNAs did not influence calcium influx or cytoskeletal reorientation in response to mechanical strain, suggesting that these candidate SA channels do not appear to contribute to SA calcium entry in CE cells.49 TRPV4 channel activation by mechanical strain could be mediated through its interaction with integrins. Other types of TRP channels, such as polycystins and ENaC channels, coimmunoprecipitate with β1 integrins,50,51 and TRPV4 has been found to coimmunoprecipitate with 2 integrins,52 suggesting that it resides in a common mechanosignaling complex with these ECM receptors.
Regardless of the precise mechanism by which TRPV4 channels sense changes in the forces that are balanced across integrins, our findings show that strain-induced calcium influx through these channels activates PI3K.53 PI3K, in turn, activates additional integrins and related downstream signaling molecules that result in activation of Rho and its target ROCK (Rho-associated kinase),13,26 which promote focal adhesion and stress fiber remodeling. The fact that this structural remodeling occurs in a highly oriented manner that is perpendicular to the applied tension field in nonconfluent cells, provides additional evidence to suggest that these events occur locally at the cell surface–ECM interface, where forces are exerted, rather than homogenously throughout the cytoplasm or within lateral membrane junctional complexes that form between cells in a confluent endothelial cell monolayer, as is required for shear stress sensation.23
These findings are important because CE cell reorientation plays a crucial role in the directional migration and oriented sprouting that drive angiogenesis. Ion signaling through SA channels has been previously shown to be important for both cell migration44 and reorientation15 in response to stress. However, these SA channels were never identified, and the importance of this type of mechanotransduction response for angiogenesis has not been explored previously. The possibility that TRP channels might be involved in vascular development has been raised in the past54,55; however, there has been no evidence to suggest that they play a direct role in endothelial cell reorientation. Importantly, our data show that TRPV4 channels are the SA channels that mediate CE cell responses to mechanical forces and cell–ECM interactions, which are critical for control of cell migration and tube formation during capillary development. Mechanically gated TRPV4 channels therefore appear to mediate a novel stretch-sensitive "integrin-to-integrin" mechanical signaling that is required for CE cell reorientation during angiogenesis, and, thus, these channels may represent new targets for future therapeutic intervention in angiogenesis-dependent diseases, such as cancer, arthritis, and macular degeneration.
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Acknowledgments |
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We thank Scott Ramsey and David Clapham (Children’s Hospital, Boston) for performing patch-clamp experiments and their helpful comments in preparing the manuscript; Richard Clark and X.-D. Ren (Stony Brook University, New York) for providing the GST-FN III8–11 domain; and Dr Martin Schwartz (University of Virginia, Charlottesville) for the GFP-AKT-PH construct.
Sources of Funding
This work was supported by NIH grants CA55833 and CA45548 and American Heart Association Grant 0635095N.
Disclosures
None.
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Footnotes |
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Original received December 19, 2008; revision received March 6, 2009; accepted March 30, 2009.
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