Name: induction of adhesion-dependent signals using low-intensity ultrasound
Uploaded: 2012-05-08T13:00:00
Duration: 8 min 51 s
Description: Watch this Scientific Journal Video about Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound at JoVE.com

This work was supported by Wellcome Trust grant 088419 to MDB and sponsorship by Smith &amp; Nephew UK Ltd.

1. Coating Surfaces with Matrix Ligand
<ol>
	<li>Ultrasound will be applied to cells in 3.5-cm wells, either as individual dishes or as a 6-well plate. For biochemical assays, coat ligand directly onto the plastic. For immunofluorescence, boil glass coverslips three times in MilliQ water and place 4 coverslips into the bottom of each well.</li>
	<li>Glass surfaces must be derivatized to ensure efficient ligand coating. Dissolve sulpho-m-maleimidobenzoyl-N-hydrosuccinimide ester in PBS at 1 mM. Add 2 ml to each well and incubate for 30 minutes at room temperature.</li>
	<li>Wash the derivatized surfaces 3 times with PBS.</li>
	<li>Coat either derivatized glass or untreated plastic with integrin ligand. Apply 2 ml of 10 ug/ml integrin ligand9, dissolved in PBS containing Ca2+ and Mg2+ to each well and incubate overnight at 4 &deg;C.</li>
	<li>Prepare monoparticulate BSA by heating 1% BSA, dissolved in PBS, to 85 &deg;C for 13 minutes. Allow to cool and then pass through a 0.45-&mu;m filter.</li>
	<li>Wash ligand-coated surfaces 3 times with PBS and block with the monoparticulate BSA for 30 minutes.</li>
</ol>
2. Preparation of Cells
<ol>
	<li>To control the matrix environment throughout the experiment, protein synthesis should be blocked by addition of 25 &mu;g/ml cycloheximide to 80% confluent fibroblasts for 2 hours.</li>
	<li>Rinse cycloheximide-treated cells with PBS and detach from the culture flask using 0.5 mg/ml trypsin/EDTA.</li>
	<li>Harvest detached cells by centrifugation at 500x g for 5 minutes.</li>
	<li>Resuspend cells in DMEM/25 mM HEPES, 25 &mu;g/ml cycloheximide.</li>
	<li>Count cell density of the suspension using a haemocytometer and dilute to 105 ml-1.</li>
	<li>Incubate cells at 37 &deg;C for 20 minutes to recover surface integrin.</li>
	<li>Rinse the ligand-coated, BSA-blocked wells 3 times with PBS.</li>
	<li>Seed 2 ml of the cell suspension into each well.</li>
	<li>Allow cells to spread for 2 hours at 37 &deg;C, 5% CO2.</li>
</ol>
3. Ultrasound Stimulation
<ol>
	<li>Warm the ultrasound emitter array and coupling gel to 37 &deg;C.</li>
	<li>Apply a pea-sized blob of coupling gel to each emitter.</li>
	<li>Test that each emitter is functional using a ultrasound indicator diode detector.</li>
	<li>Place the wells on the emitter array. Return array to 37 &deg;C, 5% CO2 and allow temperature to equilibrate for 10 minutes.</li>
	<li>Switch on the ultrasound signal.</li>
	<li>The ultrasound signal will deactivate after 20 minutes, mimicking the therapeutic regime. If shorter stimulation times are required, plates should be removed from the array at the appropriate time-points. In all cases use unstimulated plates as the negative control.</li>
</ol>
4. Focal Adhesion Analysis by Immunofluorescence
<ol>
	<li>For analysis of focal adhesion formation, apply the 20-minute ultrasound signal and then allow structures to develop for a further 40 minutes by maintaining cells at 37 &deg;C, 5% CO2.</li>
	<li>Aspirate media and fix cells by applying 2 ml 4% paraformaldehyde in PBS. Incubate for 14 minutes at room temperature.</li>
	<li>Aspirate paraformaldehyde and rinse three times with PBS, applying the PBS gently down the edge of the well to avoid generation of shear forces.</li>
	<li>Transfer each coverslip from the 3.5 cm well to a 24-well plate for subsequent immunofluorescent staining.</li>
	<li>Quench residual paraformaldehyde by applying 0.5 ml 0.1 M glycine in PBS to each of the 24 wells. Incubate for 20 minutes at room temperature.</li>
	<li>Aspirate glycine and rinse three times with PBS.</li>
	<li>Permeabilise cells by applying 0.5 ml 0.5% Triton X-100 (w/v) in PBS. Incubate for 4 minutes at room temperature.</li>
	<li>Aspirate Triton X-100 and rinse three times with PBS.</li>
	<li>Block by applying 0.5 ml 3% (w/v) BSA in PBS. Incubate overnight at 4 &deg;C.</li>
	<li>Stain vinculin-containing focal adhesion with hVIN-1 antibody diluted 1:400 in BSA block. Incubate for one hour at room temperature.</li>
	<li>Rinse three times with PBS.</li>
	<li>Apply fluorophore (DyLight 488)-conjugated secondary antibody (diluted 1:200) and stain actin with TRITC-conjugated phalloidin (diluted 1:200) in BSA block. Incubate for 30 minutes at room temperature.</li>
	<li>Rinse three times with PBS, and once with water.</li>
	<li>Mount on a glass slide in an inverted position using Prolong Gold mountant.</li>
</ol>
5. Quantification of Rac1 Activation by Pull-down Assay
<ol>
	<li>For analysis of Rac1 activation, apply the 20-minute ultrasound signal to cells at 37 &deg;C, 5% CO2. When assessing response times longer than 20 minutes, apply the ultrasound signal for 20 minutes and maintain cells at 37 &deg;C, 5% CO2 for remaining time to allow the response to develop.</li>
	<li>Aspirate media and place wells on ice. Rinse wells with 2 ml cold PBS and aspirate. Ensure any residual PBS is removed by leaving wells tilted for 1 minute followed by thorough aspiration.</li>
	<li>Lyse cells on ice with 35 &mu;l lysis buffer (10% glycerol, 20 mM Hepes pH 7.4, 140 mM NaCl, 1% NP40, 0.5% Sodium Deoxycholate, 4 mM EGTA, 4 mM EDTA, 1 &times; cOmplete protease inhibitor) per well. In this example, 6 wells are used per time point to generate 210 &mu;l of cell lysate. More or fewer wells can be used but total lysate volume should total ~200 &mu;l.</li>
	<li>Scrape cells thoroughly with cell scraper to ensure complete cell lysis and leave wells tilted for 1 minute to allow lysate to pool.</li>
	<li>Transfer lysate to a ice-cold 1.5-ml microfuge tube (pooling lysates from same treatment regime) and spin at 21000 &times; g for 2 minutes at 4 &deg;C to pellet cellular debris.</li>
	<li>Transfer 180 &mu;l of lysate to ice-cold 1.5-ml microfuge tubes containing 130 &mu;l of lysis buffer and 25 &mu;l PAK-glutathione agarose beads10. Keep remaining crude lysate at -20 &deg;C for subsequent gel separation.</li>
	<li>Capture active Rac1 on the PAK-glutathione agarose beads by rotating tubes for 40 minutes at 4 &deg;C.</li>
	<li>Harvest agarose bead conjugates by centrifugation at 2000 &times; g for 1 minute at 4 &deg;C.</li>
	<li>Wash bead conjugates three times with 500 &mu;l lysis buffer, harvesting beads by centrifugation at 2000x g and discarding supernatant after each wash. Carefully remove any remaining lysis wash buffer with a 200 &mu;l pipette.</li>
	<li>Elute each sample by adding 35 &mu;l SDS-PAGE loading buffer and incubate at 85 &deg;C on a shaking heat block for 5 minutes. Remove beads by centrifugation at 21000 &times; g for 1 minute.</li>
	<li>Resolve bead eluate by SDS-PAGE, transfer to nitrocellulose and probe for Rac1.</li>
</ol>
6. Representative Results
In this protocol we describe the induction of vinculin-stained focal adhesions and Rac1 activity by ultrasound. For the focal adhesion experiment, the baseline position is that fibroblasts spread on a ligand of &alpha;5&beta;1-integrin do not form vinculin-containing adhesions (Fig. 3A) unless a second fibronectin receptor, syndecan-4, is engaged by addition of soluble ligand (Fig. 3B) 3,4. However, stimulation with ultrasound induces focal adhesion formation to the same extent as engagement of syndecan-4 (Fig. 3C), indicating that ultrasound stimulation can substitute for certain components of the fibronectin-dependent signaling pathway 5. The key hurdle with this protocol is eliminating focal adhesion formation in the absence of stimulant. Incomplete inhibition of protein synthesis by cycloheximide will allow matrix deposition that is sufficient for the cells to auto-stimulate. Overcrowding of cells can also lead to low-level focal adhesion formation and a poor response to ultrasound as crosstalk between cell-cell and cell-matrix contacts will complicate an experiment that is designed to isolate a single stimulus. As a development of the basic assay we show the same experiment, repeated with fibroblasts lacking syndecan-4 (Fig. 4). These fibroblasts still respond to ultrasound (Fig. 4C), but fail to respond to syndecan-4 ligand (Fig. 4B). The experiment using Sdc4 -/- fibroblasts demonstrates that ultrasound can indeed bypass the need for certain fibronectin receptors, and illustrates how the simple protocol can be developed to gain extra information about the signaling pathway.
For the biochemical experiment we demonstrate that ultrasound can induce activation of Rac1, using a pull-down assay that is an adaptation of the method described by Del Pozo et al. 10. Precipitation of active Rac1 0, 10, 30 and 60 minutes after initiation of ultrasound stimulation reveals that Rac1 activity peaks at 10-30 minutes, before returning to the baseline (Fig. 5). Blotting crude lysates for vinculin ensures equivalent loading between time points. The result resembles activation of Rac1 by engagement of syndecan-43, but is slightly more protracted than activation by fibronectin. Therefore 8-10 repeats are typically required to achieve significant data. Activation of Rac1 is responsible for commitment of cells to focal adhesion formation, and nascent focal adhesions start to form at 10-30 minutes, coinciding with Rac1 activation. Once initiated, those adhesions will continue to mature into the large adhesion plaques shown in Figures 3 and 4.
<img alt="Figure 1" src="/files/ftp_upload/4024/4024fig1.jpg" /> 
Figure 1. The ultrasound wave form. Ultrasound comprises an intermittent 1.5 MHz signal that due to low amplitude (30 mW/cm2, spatial average, temporal average) has no heating effect.
<img alt="Figure 2" src="/files/ftp_upload/4024/4024fig2.jpg" /> 
Figure 2. Schematic representation of the workflow for preparation and stimulation of cells with ultrasound.
<img alt="Figure 3" src="/files/ftp_upload/4024/4024fig3.jpg" /> 
Figure 3. Ultrasound induction of focal adhesion formation in fibroblasts. Fibroblasts were spread on the integrin-binding fragment of fibronectin (A) before stimulation with the syndecan-4-binding fragment of fibronectin (B) or ultrasound (C). Following 60-minute stimulation, cells were fixed, stained for vinculin and actin, and imaged by epifluorescence. Bar = 10 &mu;m.
<img alt="Figure 4" src="/files/ftp_upload/4024/4024fig4.jpg" /> 
Figure 4. Ultrasound induction of focal adhesion formation in fibroblasts lacking syndecan-4. Sdc4 -/- fibroblasts were spread on the integrin-binding fragment of fibronectin (A) before stimulation with the syndecan-4-binding fragment of fibronectin (B) or ultrasound (C). Following 60-minute stimulation, cells were fixed, stained for vinculin and actin, and imaged by epifluorescence. Although Sdc4 -/- fibroblasts were fibronectin-insensitive, ultrasound still induced focal adhesion formation, indicating that ultrasound bypasses matrix receptor engagement. Bar = 10 &mu;m.
<img alt="Figure 5" src="/files/ftp_upload/4024/4024fig5.jpg" /> 
Figure 5. Activation of Rac1 by ultrasound. Cells were stimulated for 0, 10, 30 and 60 minutes with 6 wells used per time point. The amount of GTP-Rac1 present in samples from a Rac1 pull-down assay time course was visualized by incubation with anti-Rac1 antibody on a western blot (top image). Quantification of the band intensity (average of 8-10 replicates) reveals increased Rac1 activity resulting from ultrasound signal at 10 and 30 minutes, before returning to baseline levels at 60 minutes (graph). Error bars represent s.e.m.

<ol>
	<li>Bass, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+syndecan-4+hair+trigger+initiates+wound+healing+through+caveolin-+and+RhoG-regulated+integrin+endocytosis.">A syndecan-4 hair trigger initiates wound healing through caveolin- and RhoG-regulated integrin endocytosis.</a> Dev. Cell. 21, 681-693 (2011).</li><li>Echtermeyer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+wound+repair+and+impaired+angiogenesis+in+mice+lacking+syndecan-4.">Delayed wound repair and impaired angiogenesis in mice lacking syndecan-4.</a> J. Clin. Invest. 107, R9-R14 (2001).</li><li>Bass, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syndecan-4-dependent+Rac1+regulation+determines+directional+migration+in+response+to+the+extracellular+matrix.">Syndecan-4-dependent Rac1 regulation determines directional migration in response to the extracellular matrix.</a> J. Cell Biol. 177, 527-538 (2007).</li><li>Woods, A., Couchman, J. R., Johansson, S., Hook, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+and+cytoskeletal+organisation+of+fibroblasts+in+response+to+fibronectin+fragments.">Adhesion and cytoskeletal organisation of fibroblasts in response to fibronectin fragments.</a> Embo. J. 5, 665-670 (1986).</li><li>Mahoney, C. M., Morgan, M. R., Harrison, A., Humphries, M. J., Bass, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+ultrasound+bypasses+canonical+syndecan-4+signaling+to+activate+rac1.">Therapeutic ultrasound bypasses canonical syndecan-4 signaling to activate rac1.</a> J. Biol. Chem. 284, 8898-8909 (2009).</li><li>Claes, L., Willie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enhancement+of+bone+regeneration+by+ultrasound.">The enhancement of bone regeneration by ultrasound.</a> Prog. Biophys. Mol. Biol. 93, 384-398 (2007).</li><li>Heckman, J. D., Ryaby, J. P., McCabe, J., Frey, J. J., Kilcoyne, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acceleration+of+tibial+fracture-healing+by+non-invasive,+low-intensity+pulsed+ultrasound.">Acceleration of tibial fracture-healing by non-invasive, low-intensity pulsed ultrasound.</a> J. Bone Joint Surg. Am. 76, 26-34 (1994).</li><li>Kristiansen, T. K., Ryaby, J. P., McCabe, J., Frey, J. J., Roe, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+healing+of+distal+radial+fractures+with+the+use+of+specific,+low-intensity+ultrasound.+A+multicenter,+prospective,+randomized,+double-blind,+placebo-controlled+study.">Accelerated healing of distal radial fractures with the use of specific, low-intensity ultrasound. A multicenter, prospective, randomized, double-blind, placebo-controlled study.</a> J. Bone Joint Surg. Am. 79, 961-973 (1997).</li><li>Danen, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+the+synergy+site+for+cell+adhesion+to+fibronectin+depends+on+the+activation+state+of+integrin+alpha+5+beta+1.">Requirement for the synergy site for cell adhesion to fibronectin depends on the activation state of integrin alpha 5 beta 1.</a> J. Biol. Chem. 270, 21612-21618 (1995).</li><li>Del Pozo, M. A., Price, L. S., Alderson, N. B., Ren, X. D., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+to+the+extracellular+matrix+regulates+the+coupling+of+the+small+GTPase+Rac+to+its+effector+PAK.">Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK.</a> Embo. J. 19, 2008-2014 (2000).</li><li>Makarem, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competitive+binding+of+vascular+cell+adhesion+molecule-1+and+the+HepII/IIICS+domain+of+fibronectin+to+the+integrin+alpha+4+beta+1.">Competitive binding of vascular cell adhesion molecule-1 and the HepII/IIICS domain of fibronectin to the integrin alpha 4 beta 1.</a> J. Biol. Chem. 269, 4005-4011 (1994).</li></ol>

Andrew Harrison is an employee of Smith &amp; Nephew UK Limited.

In this protocol we describe the method by which a treatment that is normally applied to human patients can be used in cell-based experiments. The ultimate goal is to understand the molecular mechanism of ultrasound action so that the therapy can be refined. In this protocol, we use mouse embryonic fibroblasts (MEFs) as a model cell system, but ultrasound has been also been found to be effective in primary human foreskin fibroblasts 5, mesenchymal stem cells, osteoblasts and chondrocytes 6. We use Rac1 activation as an example biochemical assay, but the method could be used equally to test regulation of protein phosphorylation or formation of protein complexes by immunoprecipitation. For the immunofluorescent example we demonstrate an effect ultrasound on focal adhesion formation, but redistribution of any molecule could be examined. For example, one could test recruitment of cytosolic factors to the plasma membrane, colocalisation of proteins in trafficking vesicles or examine the effect of ultrasound on mitotic spindle organization. So far we have confined ourselves to testing fibronectin-dependent pathways, by the effect of ultrasound on cells on collagen, or in the presence of growth factors could be tested to build up a picture of how ultrasound affects cell behavior in a complex environment that more closely resembles an in vivo situation. A greater challenge will be to test the effect of ultrasound using time-lapse imaging, where the presence of the emitter blocking the light path and vibrations from the ultrasound present additional technical obstacles. However, the fact that therapeutic application requires only 20 minutes of stimulation per day suggests that analysis of cell behavior after a burst of stimulation may well be productive.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>sulpho-m-maleimidobenzoyl-N-hydrosuccinimide 			ester</td>
 <td>FisherPerbio</td>
 <td>PN22312</td>
 <td>25 mM stocks dissolved in water can 			be stored at -20&deg;C</td>
 </tr>
 <tr>
 <td>6-well tissue culture plate</td>
 <td>Corning</td>
 <td>3516</td>
 <td>Plastic from other companies can coat 			poorly</td>
 </tr>
 <tr>
 <td>13-mm glass coverslip</td>
 <td>Scientific Laboratory Supplies 			Ltd.SLS</td>
 <td>MIC3336</td>
 <td>Glass from other companies can coat 			poorly</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Sigma</td>
 <td>D8537</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PBS, Ca2+ 			Mg2+</td>
 <td>Sigma</td>
 <td>D8662</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50K integrin ligand (fibronectin 			fragment)</td>
 <td>Construct description and preparation 			in 9, 11</td>
 <td>&nbsp;</td>
 <td>Alternative matrix ligands could be 			used to interrogate other pathways</td>
 </tr>
 <tr>
 <td>BSA</td>
 <td>Sigma</td>
 <td>A3059</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cycloheximide</td>
 <td>Sigma</td>
 <td>C7698</td>
 <td>Can be stored as 10mg/ml stock in 			water</td>
 </tr>
 <tr>
 <td>DMEM/25 mM HEPES</td>
 <td>Sigma</td>
 <td>D6171</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Exogen 4000+</td>
 <td>Smith &amp; Nephew Inc</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ultrasound coupling 			gel</td>
 <td>Smith &amp; Nephew Inc</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>SAFHS indicator</td>
 <td>Smith &amp; Nephew Inc</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>hVIN-1</td>
 <td>Sigma</td>
 <td>V9264</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DyLight 488-conjugated anti-mouse</td>
 <td>Stratech Scientific</td>
 <td>715-485-150</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TRITC-labelled phalloidin</td>
 <td>Sigma</td>
 <td>P1951</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Prolong Gold antifade reagent</td>
 <td>Invitrogen</td>
 <td>P36930</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>cOmplete protease inhibitor (EDTA 			free)</td>
 <td>Roche</td>
 <td>056 489 001</td>
 <td>100&times; stock made up from tablet stored at -20&deg;C</td>
 </tr>
 <tr>
 <td>PAK-glutathione agarose beads</td>
 <td>Construct description and preparation 			in 10</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glycerol</td>
 <td>Fisher</td>
 <td>G/0650/17</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hepes</td>
 <td>Apollo Scientific</td>
 <td>BI8181</td>
 <td>Make 1M stock and pH to 7.4</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Fisher</td>
 <td>S/0160/65</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NP40</td>
 <td>Sigma</td>
 <td>I3021</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium Deoxycholate</td>
 <td>Sigma</td>
 <td>D6750</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>EGTA</td>
 <td>Sigma</td>
 <td>E4378</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Sigma</td>
 <td>E5134</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

induction of adhesion-dependent signals using low-intensity ultrasound

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from regulation of cell migration and proliferation, to secretion and even differentiation. The signals underlying each of these complex processes arise from the molecular interactions of extracellular matrix receptors on the surface of the cell. Integrins are the prototypic receptors and provide a mechanical link between extracellular matrix and the cytoskeleton, as well as initiating some of the adhesion-dependent signaling cascades. However, it is becoming increasingly apparent that additional transmembrane receptors function alongside the integrins to regulate both the integrin itself and signals downstream. The most elegant of these examples is the transmembrane proteoglycan, syndecan-4, which cooperates with &alpha;5&beta;1-integrin during adhesion to fibronectin. In vivo models demonstrate the importance of syndecan-4 signaling, as syndecan-4-knockout mice exhibit healing retardation due to inefficient fibroblast migration1,2. In wild-type animals, migration of fibroblasts toward a wound is triggered by the appearance of fibronectin that leaks from damaged capillaries and is deposited by macrophages in injured tissue. Therefore there is great interest in discovering strategies that enhance fibronectin-dependent signaling and could accelerate repair processes.
The integrin-mediated and syndecan-4-mediated components of fibronectin-dependent signaling can be separated by stimulating cells with recombinant fibronectin fragments. Although integrin engagement is essential for cell adhesion, certain fibronectin-dependent signals are regulated by syndecan-4. Syndecan-4 activates the Rac1 protrusive signal3, causes integrin redistribution1, triggers recruitment of cytoskeletal molecules, such as vinculin, to focal adhesions4, and thereby induces directional migration3. We have looked for alternative strategies for activating such signals and found that low-intensity pulsed ultrasound (LIPUS) can mimic the effects of syndecan-4 engagement5. In this protocol we describe the method by which 30 mW/cm2, 1.5 MHz ultrasound, pulsed at 1 kHz (Fig. 1) can be applied to fibroblasts in culture (Fig. 2) to induce Rac1 activation and focal adhesion formation. Ultrasound stimulation is applied for a maximum of 20 minutes, as this combination of parameters has been found to be most efficacious for acceleration of clinical fracture repair6. The method uses recombinant fibronectin fragments to engage &alpha;5&beta;1-integrin, without engagement of syndecan-4, and requires inhibition of protein synthesis by cycloheximide to block deposition of additional matrix by the fibroblasts., The positive effect of ultrasound on repair mechanisms is well documented7,8, and by understanding the molecular effect of ultrasound in culture we should be able to refine the therapeutic technique to improve clinical outcomes.

Watch this Scientific Journal Video about Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound at JoVE.com

Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound

This protocol describes the stimulation of cultured fibroblasts with low-intensity pulsed ultrasound, which drives focal adhesion formation and Rac1 activation by mimicking engagement of the transmembrane matrix receptor, syndecan-4. This approach allows investigation of a successful clinical technique at the cellular level, thereby providing opportunities for refinement of the therapy.

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from ...

induction-of-adhesion-dependent-signals-using-low-intensity-ultrasound

Research

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Bioengineering

Contrast Ultrasound Targeted Treatment of Gliomas in Mice via Drug-Bearing Nanoparticle Delivery and Microvascular Ablation

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the microvasculature are controlled by varying ultrasound pulsing parameters.  Specifically, we are testing whether such approaches may be used to treat malignant brain tumors through drug delivery and microvascular ablation.  Preliminary studies have been performed to determine whether targeted drug-bearing nanoparticle delivery can be facilitated by the ultrasound mediated destruction of "composite" delivery agents comprised of 100nm poly(lactide-co-glycolide) (PLAGA) nanoparticles that are adhered to albumin shelled microbubbles. We denote these agents as microbubble-nanoparticle composite agents (MNCAs). When targeted to subcutaneous C6 gliomas with ultrasound, we observed an immediate 4.6-fold increase in nanoparticle delivery in MNCA treated tumors over tumors treated with microbubbles co-administered with nanoparticles and a 8.5 fold increase over non-treated tumors. Furthermore, in many cancer applications, we believe it may be desirable to perform targeted drug delivery in conjunction with ablation of the tumor microcirculation, which will lead to tumor hypoxia and apoptosis. To this end, we have tested the efficacy of non-theramal cavitation-induced microvascular ablation, showing that this approach elicits tumor perfusion reduction, apoptosis, significant growth inhibition, and necrosis. Taken together, these results indicate that our ultrasound-targeted approach has the potential to increase therapeutic efficiency by creating tumor necrosis through microvascular ablation and/or simultaneously enhancing the drug payload in gliomas.

Insonation of microbubbles is a promising strategy for tumor ablation at reduced time-averaged acoustic powers, as well as for the targeted delivery of therapeutics. The purpose of the present study is to develop low duty cycle ultrasound pulsing strategies and nanocarriers to maximize non-thermal microvascular ablation and payload delivery to subcutaneous C6 gliomas.

We are developing minimally-invasive contrast agent microbubble based therapeutic approaches in which the permeabilization and/or ablation of the ...

Introduction to the Ultrasound Targeted Microbubble Destruction Technique

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid microbubble contrast agents7-9. In mice these vector-carrying microbubbles can be administered intravenously or directly to the left ventricle of the heart. In larger animals they can also be infused through an intracoronary catheter. The subsequent delivery from the circulation to a target organ occurs by acoustic cavitation at a resonant frequency of the microbubbles. It seems likely that the mechanical energy generated by the microbubble destruction results in transient pore formation in or between the endothelial cells of the microvasculature of the targeted region10. As a result of this sonoporation effect, the transfection efficiency into and across the endothelial cells is enhanced, and transgene-encoding vectors are deposited into the surrounding tissue. Plasmid DNA remaining in the circulation is rapidly degraded by nucleases in the blood, which further reduces the likelihood of delivery to non-sonicated tissues and leads to highly specific target-organ transfection.

Ultrasound Targeted Microbubble Destruction (UTMD) can be used to direct site-specific delivery of bioactive molecules, including therapeutic genes, to target organs accessible to ultrasound, such as the heart and liver1-6.

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid ...

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 &#181;m. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic ...

Real-time Monitoring of High Intensity Focused Ultrasound (HIFU) Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An oscillatory motion is generated at the focus of a 93-element and 4.5 MHz center frequency HIFU transducer by applying a 25 Hz amplitude-modulated signal using a function generator. A 64-element and 2.5 MHz imaging transducer with 68kPa peak pressure is confocally placed at the center of the HIFU transducer to acquire the radio-frequency (RF) channel data. In this protocol, real-time monitoring of thermal ablation using HIFU with an acoustic power of 7 W on canine livers in vitro is described. HIFU treatment is applied on the tissue during 2 min and the ablated region is imaged in real-time using diverging or plane wave imaging up to 1,000 frames/second. The matrix of RF channel data is multiplied by a sparse matrix for image reconstruction. The reconstructed field of view is of 90&#176; for diverging wave and 20 mm for plane wave imaging and the data are sampled at 80 MHz. The reconstruction is performed on a Graphical Processing Unit (GPU) in order to image in real-time at a 4.5 display frame rate. 1-D normalized cross-correlation of the reconstructed RF data is used to estimate axial displacements in the focal region. The magnitude of the peak-to-peak displacement at the focal depth decreases during the thermal ablation which denotes stiffening of the tissue due to the formation of a lesion. The displacement signal-to-noise ratio (SNRd) at the focal area for plane wave was 1.4 times higher than for diverging wave showing that plane wave imaging appears to produce better displacement maps quality for HMIFU than diverging wave imaging.

Real-time Monitoring of High Intensity Focused Ultrasound HIFU Ablation of In Vitro Canine Livers Using Harmonic Motion Imaging for Focused Ultrasound HMIFU

This article describes real-time monitoring of HIFU ablation in canine liver with high frame rate ultrasound imaging using diverging and plane wave imaging. Harmonic Motion Imaging for Focused Ultrasound is used to image the decrease of acoustic radiation force induced displacement in the ablated region.

Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a technique that can perform and monitor high-intensity focused ultrasound (HIFU) ablation. An ...

Transmission of Multiple Signals through an Optical Fiber Using Wavefront Shaping

The transmission of multiple independent optical signals through a multimode fiber is accomplished using wavefront shaping in order to compensate for the light distortion during the propagation within the fiber. Our methodology is based on digital optical phase conjugation employing only a single spatial light modulator, where the optical wavefront is individually modulated at different regions of the modulator, one region per light signal. Digital optical phase conjugation approaches are considered to be faster than other wavefront shaping approaches, where (for example) a complete determination of the wave propagation behavior of the fiber is performed. In contrast, the presented approach is time-efficient since it only requires one calibration per light signal. The proposed method is potentially appropriate for spatial division multiplexing in communications engineering. Further application fields are endoscopic light delivery in biophotonics, especially in optogenetics, where single cells in biological tissue have to be selectively illuminated with high spatial and temporal resolution.

We demonstrate the transmission of multiple independent signals through a multimode fiber using wavefront shaping employing a single spatial light modulator. By modulating the wavefront for each signal individually, spatially separated foci are transmitted. Potential applications are multiplexed data transfer in communications engineering and endoscopic light delivery in biophotonics.

The transmission of multiple independent optical signals through a multimode fiber is accomplished using wavefront shaping in order to compensate for the ...

Manufacturing Abdominal Aorta Hydrogel Tissue-Mimicking Phantoms for Ultrasound Elastography Validation

Ultrasound (US) elastography, or elasticity imaging, is an adjunct imaging technique that utilizes sequential US images of soft tissues to measure the tissue motion and infer or quantify the underlying biomechanical characteristics. For abdominal aortic aneurysms (AAA), biomechanical properties such as changes in the tissue's elastic modulus and estimates of the tissue stress may be essential for assessing the need for the surgical intervention. Abdominal aortic aneurysms US elastography could be a useful tool to monitor AAA progression and identify changes in biomechanical properties characteristic of high-risk patients.
A preliminary goal in the development of an AAA US elastography technique is the validation of the method using a physically relevant model with known material properties. Here we present a process for the production of AAA tissue-mimicking phantoms with physically relevant geometries and spatially modulated material properties. These tissue phantoms aim to mimic the US properties, material modulus, and geometry of the abdominal aortic aneurysms. Tissue phantoms are made using a polyvinyl alcohol cryogel (PVA-c) and molded using 3D printed parts created using computer aided design (CAD) software. The modulus of the phantoms is controlled by altering the concentration of PVA-c and by changing the number of freeze-thaw cycles used to polymerize the cryogel. The AAA phantoms are connected to a hemodynamic pump, designed to deform the phantoms with the physiologic cyclic pressure and flows. Ultra sound image sequences of the deforming phantoms allowed for the spatial calculation of the pressure normalized strain and the identification of mechanical properties of the vessel wall. Representative results of the pressure normalized strain are presented.

Here we describe a method to manufacture aneurysmal, aortic tissue-mimicking phantoms for the use in testing ultrasound elastography. The combined use of computer-aided design (CAD) and 3-dimensional (3D) printing techniques produce aortic phantoms with predictable, complex geometries to validate the elastographic imaging algorithms with controlled experiments.

Ultrasound (US) elastography, or elasticity imaging, is an adjunct imaging technique that utilizes sequential US images of soft tissues to measure the ...

Functional Transcranial Doppler Ultrasound for Monitoring Cerebral Blood Flow

Functional transcranial Doppler ultrasound (fTCD) is the use of transcranial Doppler ultrasound (TCD) to study neural activation occurring during stimuli such as physical movement, activation of tactile sensors in the skin, and viewing images. Neural activation is inferred from an increase in the cerebral blood flow velocity (CBFV) supplying the region of the brain involved in processing sensory input. For example, viewing bright light causes increased neural activity in the occipital lobe of the cerebral cortex, leading to increased blood flow in the posterior cerebral artery, which supplies the occipital lobe. In fTCD, changes in CBFV are used to estimate changes in cerebral blood flow (CBF).
With its high temporal resolution measurement of blood flow velocities in the major cerebral arteries, fTCD complements other established functional imaging techniques. The goal of this Methods paper is to give step-by-step instructions for using fTCD to perform a functional imaging experiment. First, the basic steps for identifying the middle cerebral artery (MCA) and optimizing the signal will be described. Next, placement of a fixation device for holding the TCD probe in place during the experiment will be described. Finally, the breath-holding experiment, which is a specific example of a functional imaging experiment using fTCD, will be demonstrated.

Functional transcranial Doppler ultrasound complements other functional imaging modalities, with its high temporal resolution measurement of stimulus-induced changes in cerebral blood flow within the basal cerebral arteries. This Methods paper gives step-by-step instructions for using functional transcranial Doppler ultrasound to perform a functional imaging experiment.&#160;

Functional transcranial Doppler ultrasound (fTCD) is the use of transcranial Doppler ultrasound (TCD) to study neural activation occurring during stimuli ...

Contrast-Enhanced Subharmonic Aided Pressure Estimation (SHAPE) Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound imaging are gas-filled, encapsulated microbubbles (diameter &#60; 10 &#956;m) that traverse the entire vasculature and enhance signals by up to 30 dB. These microbubbles also produce nonlinear oscillations at frequencies ranging from the subharmonic (half of the transmit frequency) to higher harmonics. The subharmonic amplitude has an inverse linear relationship with the ambient hydrostatic pressure. Here an ultrasound system capable of performing real-time, subharmonic aided pressure estimation (SHAPE) is presented. During ultrasound contrast agent infusion, an algorithm for optimizing acoustic outputs is activated. Following this calibration, subharmonic microbubble signals (i.e., SHAPE) have the highest sensitivity to pressure changes and can be used to noninvasively quantify pressure. The utility of the SHAPE procedure for identifying portal hypertension in the liver is the emphasis here, but the technique has applicability across many clinical scenarios.

Contrast-Enhanced Subharmonic Aided Pressure Estimation SHAPE Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

A protocol for noninvasively estimating ambient pressures utilizing subharmonic ultrasound imaging of infused contrast microbubbles (following appropriate calibration) is described with examples from human patients with chronic liver disease.

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound ...

Multi-timescale Microscopy Methods for the Characterization of Fluorescently-labeled Microbubbles for Ultrasound-Triggered Drug Release

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic toxicity and increases circulation time of the drugs. In a novel approach to microbubble-assisted drug delivery, nanoparticles are incorporated in or on microbubble shells, enabling local and triggered release of the nanoparticle payload with ultrasound. A thorough understanding of the release mechanisms within the vast ultrasound parameter space is crucial for efficient and controlled release. This set of presented protocols is applicable to microbubbles with a shell containing a fluorescent label. Here, the focus is on microbubbles loaded with poly(2-ethyl-butyl cyanoacrylate) polymeric nanoparticles, doped with a modified Nile Red dye. The particles are fixed within a denatured casein shell. The microbubbles are produced by vigorous stirring, forming a dispersion of perfluoropropane gas in the liquid phase containing casein and nanoparticles, after which the microbubble shell self-assembles. A variety of microscopy techniques are needed to characterize the nanoparticle-stabilized microbubbles at all relevant timescales of the nanoparticle release process. Fluorescence of the nanoparticles enables confocal imaging of single microbubbles, revealing the particle distribution within the shell. In vitro ultra-high-speed imaging using bright-field microscopy at 10 million frames per second provides insight into the bubble dynamics in response to ultrasound insonation. Finally, nanoparticle release from the bubble shell is best visualized by means of fluorescence microscopy, performed at 500,000 frames per second. To characterize drug delivery in vivo, the triggered release of nanoparticles within the vasculature and their extravasation beyond the endothelial layer is studied using intravital microscopy in tumors implanted in dorsal skinfold window chambers, over a timescale of several minutes. The combination of these complementary characterization techniques provides unique insight into the behavior of microbubbles and their payload release at a range of time and length scales, both in vitro and in vivo.

The presented protocols can be used to characterize the response of fluorescently-labeled microbubbles designed for ultrasound-triggered drug delivery applications, including their activation mechanisms as well as their bioeffects. This paper covers a range of in vitro and in vivo microscopy techniques performed to capture the relevant length and timescales.

Microbubble contrast agents hold great promise for drug delivery applications with ultrasound. Encapsulating drugs in nanoparticles reduces systemic ...

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. These include the limited number of cadaveric pancreas donors and the long-term use of immunosuppressants. Mesenchymal stem cells (MSCs) have been considered to be a potential candidate as an alternative source of islet-like cell generation. Our previous reports have successfully illustrated the establishment of induction protocols for differentiating human dental pulp stem cells (hDPSCs) to insulin-producing cells (IPCs). However, the induction efficiency varied greatly. In this paper, we demonstrate the comparison of hDPSCs pancreatic induction efficiency via integrative (microenvironmental and genetic manipulation) and non-integrative (microenvironmental manipulation) induction protocols for delivering hDPSC-derived IPCs (hDPSC-IPCs). The results suggest distinct induction efficiency for both the induction approaches in terms of 3-dimensional colony structure, yield, pancreatic mRNA markers, and functional property upon multi-dosage glucose challenge. These findings will support the future establishment of a clinically applicable IPCs and pancreatic lineage production platform.

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

This protocol presents a comparison between two different induction protocols for differentiating human dental pulp stem cells (hDPSCs) toward pancreatic lineages in vitro: the integrative protocol and the non-integrative protocol. The integrative protocol generates more insulin producing cells (IPCs).

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...