© 1997 American Heart Association, Inc.
Articles |
Integrin Signaling Transduces Shear Stress–Dependent Vasodilation of Coronary Arterioles
the Department of Veterinary Biomedical Sciences (J.M.M.), University of Missouri, Columbia; the Department of Physiology (W.M.C.), Medical College of Wisconsin, Milwaukee; and the Microcirculation Research Institute (M.J.D.), Department of Medical Physiology, Texas A&M University Health Science Center, College Station.
Correspondence to Dr William M. Chilian, Department of Physiology, Medical College of Wisconsin, PO Box 26509, 8701 Watertown Plank Rd, Milwaukee, WI 53226-0509. E-mail chilian@post.its.mcw.edu
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Abstract |
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A direct relationship exists between shear stress and endothelium-dependent NO-mediated vasodilation of blood vessels. The transduction of shear stress to the biochemical signals resulting in the production of NO is, however, unknown. We tested the hypothesis that integrin binding to Arg-Gly-Asp (RGD) peptide sequences in extracellular matrix proteins is a critical step in initiation of the signaling sequence whereby shear stress activates endothelial tyrosine kinase(s) and induces vasodilation of isolated arterioles. Isolated coronary arterioles were exposed to increasing shear stress under control conditions and in the presence of a synthetic peptide, GRGDNP, to competitively inhibit integrin binding to extracellular matrix proteins containing RGD peptide sequences. Intraluminal GRGDNP (0.1, 0.5, and 1.0 mmol/L) inhibited shear stress–induced vasodilation in a concentration-dependent manner. Application of GRGDNP had no effect on endothelium-dependent relaxation to substance P (10-12 to 10-8 mol/L). An inactive structural analogue, GRGESP, did not alter shear stress–induced vasodilation. To further elucidate the integrin involved in shear stress–induced vasodilation, we administered a blocking antibody to the integrin ß3 chain (F11) and observed significant attenuation of the vasodilation. Shear stress was also associated with an increase in tyrosine kinase activity, as assessed by anti-phosphotyrosine binding. Application of GRGDNP significantly decreased anti-phosphotyrosine binding during shear stress, suggesting a link between tyrosine kinase activation and integrin signaling during this vasodilatory response. Taken together, these results indicate that integrin-matrix interactions, possibly at focal adhesions, are of cardinal importance in the signaling pathway of shear stress–induced vasodilation.
Key Words: tyrosine kinase • endothelium • GRGDNP • nitric oxide • ß3 integrin
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Introduction |
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Increases in intraluminal flow (shear stress) elicit robust vasodilation in coronary resistance vessels.1 This vasodilatory response is a seminal component of coronary vasomotor adjustments to many physiological stimuli, including reactive hyperemia2 and functional hyperemia.3 The dilation occurs through endothelial release of NO.4 However, the sensory transduction mechanism and the intracellular signaling pathway by which shear stress stimulates release of NO in endothelial cells remain to be defined.
Recent data indicate that tyrosine kinase activation is necessary for shear stress–induced vasodilation to occur in coronary arterioles.5 In mechanotransduction pathways of other cell types, tyrosine kinases are involved at sites of integrin ligation to the extracellular matrix, the focal adhesions.6 7 8 9 We reasoned that integrins may serve as mechanotransducers of shear forces into biochemical signals, because they are colocalized with cytoskeletal proteins in focal adhesions,10 11 providing a tensile link between the surface of the cell and integrin attachment to the matrix.12 13 Although shear stress alters topography and stiffness of the luminal surface of endothelial cells,10 it is important to note that focal adhesions on the abluminal surface increase their rates of association/dissociation with the extracellular matrix in response to luminal shear stress.14 Therefore, we hypothesized that shear stress induces vasodilation of isolated coronary arterioles through interaction between integrins and extracellular matrix proteins. Specifically, we tested the hypothesis that competitive inhibition of integrin binding to extracellular matrix proteins, which contain the RGD (Arg-Gly-Asp) sequence, blocks shear stress–induced vasodilation and activation of tyrosine kinase(s).
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Materials and Methods |
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Isolation and Cannulation of Coronary Arterioles
Pigs (n=45) of either sex weighing 7 to 18 kg were sedated with rompun (2.25 mg/kg IM) and ketamine (25 mg/kg IM), anesthetized with sodium pentobarbital (30 mg/kg IV), and administered heparin (1000 U/kg IV). Pigs were then intubated and ventilated with room air. A left thoracotomy was performed, and the heart was fibrillated, excised, and placed in cold saline (4°C).
Coronary arterioles were dissected and cannulated according to the method of Kuo et al.4 Physiological saline solution containing 3% gelatin and india ink was infused into the left anterior descending artery, the circumflex artery, and right coronary artery to visualize coronary arterioles. Arterioles (intraluminal diameter, 70 to 130 µm) were dissected free from the myocardium under a dissection microscope and then transferred to a Lucite chamber for cannulation with micropipettes (diameter, 50 to 60 µm) of matched tip resistance.
Evaluation of Shear Stress–Induced Vasodilatory Responses
The microvessel chamber was transferred to the stage of an inverted microscope (Zeiss Axiovert 100) equipped with a video camera and video caliper system for determination of intraluminal diameter. Arterioles were pressurized to 60 cm H2O with two independent hydrostatic pressure reservoirs. Leaks were detected by pressurizing arterioles, then closing the valves to the reservoirs, and verifying that intraluminal pressure remained constant. Arterioles that exhibited leaks were discarded. Arterioles were bathed in PSS containing (mmol/L) NaCl 145.0, KCl 4.7, CaCl2 2.0, MgSO4 1.17, NaH2PO4 1.2, glucose 5.0, pyruvate 2.0, EDTA 0.02, and MOPS buffer 3.0, along with 1 g/100 mL BSA. The PSS was adjusted to a pH of 7.4 and warmed to 37±1°C. During an initial equilibration period, the arterioles were allowed to develop spontaneous tone. Some vessels (n=5) were constricted with 40 mmol/L KCl to produce tone equivalent to that occurring spontaneously in order to demonstrate that shear stress–dependent responses occur regardless of the type of tone (spontaneous or pharmacological). Whether tone development occurred spontaneously or was induced pharmacologically, vasodilatory responses were determined only in vessels that constricted to 
75% of resting diameter.
Upon displaying a steady level of spontaneous tone, arterioles were exposed to graded increases in shear stress in the absence of changes in intraluminal pressure. This was accomplished by the method described previously in this laboratory.1 4 Alteration of the heights of two independent pressure reservoirs in equal and opposite directions generated pressure differences (
P=4, 10, 20, 40, or 60 cm H2O) between the cannulating pipettes without changing mean intraluminal pressure. Previous work performed in this laboratory demonstrated that intraluminal pressure was not altered by this method of generating intraluminal flow.4 Actual flow rates were computed from measurements of vessel radius and the velocity of red blood cells injected into the vessel lumen. The relationship between P and flow rate was calibrated for each pair of micropipettes used for a given size of vessel. Shear stress was calculated as follows: 
=4
Q/
r3, where is viscosity (0.8 cp), Q is volumetric flow rate, and r is the steady state vessel radius.15
After determination of the shear stress–diameter relationship, the vessel was allowed to equilibrate for 20 minutes in the absence of shear stress and to redevelop baseline tone. After this period, vasodilatory responses to increasing concentrations of substance P (1x10-12 to 1x10-8 mol/L) were determined at constant intraluminal pressure.
Responses to shear stress and substance P were then reassessed after 30 minutes of incubation with GRGDNP (0.1, 0.5, or 1.0 mmol/L, applied intraluminally), an antagonist of integrin binding to matrix proteins at the RGD site. GRGDNP is one of the more efficacious antagonists of this interaction and is resistant to the actions of carboxypeptidases.16 Similarly, responses to shear stress and substance P were determined in the absence and presence of a scrambled form of the peptide, GRGESP (0.5 mmol/L), as an inactive control. After completion of the entire protocol, a single concentration of sodium nitroprusside (100 µmol/L) was administered to determine maximal arteriolar diameter.
In a separate series of vessels, shear stress–induced vasodilation was evaluated before and after 30 minutes of intraluminal exposure to a soluble synthetic form of pronectin (pronectin F, 40 µmol/L), a monoclonal blocking antibody to the integrin ß3 subunit (F11, 25 µg/mL), or a nonspecific mouse myeloma (mouse myeloma IgG1, 25 µg/mL). In these protocols, shear stress–induced vasodilation was first determined, and then the vessel was allowed to redevelop tone at zero flow. Pronectin F, F11, or mouse myeloma IgG1 was then added intraluminally, and 30 minutes was allowed for reestablishment of vascular tone. Administration of pronectin F consistently reduced or eliminated spontaneous tone, making it necessary to induce tone with the thromboxane analogue U46619 (0.1 to 1.0 µmol/L). The level of induced tone matched the level of spontaneous tone present in the vessel at the beginning of the protocol (±5%), and we have previously shown that shear stress–induced dilation still occurs after pharmacological induction of tone.4 The responses to shear stress were then redetermined.
Distribution of GRGDNP
Arterioles were prepared, cannulated, and pressurized in the same manner as arterioles used to study vasodilatory responses to shear stress. Once the vessels had developed spontaneous tone, FITC-labeled GRGDNP (1.0 mmol/L, Phoenix Pharmaceuticals, Inc) was infused intraluminally. The vessel was incubated with the labeled peptide for 30 minutes. The vessel was then removed from the cannulating pipettes, cut open on its longitudinal axis, and placed on a coverslip with the endothelium exposed. After fixation with PBS containing (mmol/L) KCl 2.7, KH2PO4 1.5, NaCl 137.0, and Na2HPO4 8.0, along with 2% paraformaldehyde (2 g/100 mL), the vessel was washed three times with PBS and mounted on a microscope slide with DABCO mounting solution.
The endothelial layer was visualized using a laser confocal microscope (Noran Instruments), in conjunction with fluorescence techniques for FITC (excitation, 480 nm; band pass, 515 nm). After visualization of the endothelium, a series of Z-plane images were obtained (30 to 35 sections; thickness, 0.3 to 0.5 µm). Each Z-plane image represented the average of 256 frames. Using Metamorph (Universal Imaging Corp), the series was reconstructed into a single three-dimensional image and then rotated 90° to allow visualization of the luminal and abluminal sides of the endothelium. Fluorescence intensity was measured across the endothelium (from the lumen to the abluminal side in four preparations) for each reconstruction.
Immunocytochemical Detection of Tyrosine Phosphorylation
Coronary arterioles were isolated, cannulated, and allowed to develop spontaneous tone at a constant intraluminal pressure of 60 cm H2O. Arterioles were then exposed to flow in the absence (n=6) or the presence of 1.0 mmol/L GRGDNP (n=6). A pressure difference of 20 cm H2O was established across the arteriole for 10 minutes. For each experimental condition (flow alone or flow in the presence of GRGDNP), basal tyrosine phosphorylation was determined in control arterioles from the same heart, which were cannulated, pressurized, and allowed to develop spontaneous tone over the same time course but were not exposed to intraluminal flow. At the end of this protocol, 100 µmol/L sodium orthovanadate was added as a phosphatase inhibitor. Arterioles were quickly removed from the cannulating pipettes and opened longitudinally. The arterioles were pinned out and fixed with 2% paraformaldehyde (in PBS). Fixation was followed by two washes with PBS containing 0.1 mmol/L glycine and a rinse with PBS. Arterioles were then permeabilized with 0.1% Triton X-100 in PBS, rinsed three times with PBS, and incubated with FITC-conjugated monoclonal anti-phosphotyrosine (diluted 50-fold in PBS containing 0.9% sodium citrate, 2% goat serum, 1% albumin, 0.05% Triton X-100, and 0.025 NaN3) for 45 minutes. The antibody solution was removed by three washes with PBS containing 0.9% sodium citrate and 0.05% Triton X-100. The arterioles were then transferred to a coverslip and mounted on a microscope slide with DABCO mounting solution and viewed through an inverted microscope (Zeiss Axiovert 100).
A clear focus of the endothelial cell layer of each vessel was obtained under bright-field illumination. An intensified CCD camera (Hamamatsu) was then used to acquire four or five fluorescence images from each vessel using excitation and emission wavelengths of 485 and 535 nm, respectively. Background fluorescence was subtracted, and the resulting images were captured and stored on a computer (Metamorph). A random grid, generated by image-processing software (NIH Image, version 1.57), was superimposed over each image. To evaluate the occurrence of discretely labeled cells, three individuals who were blind to the experimental treatments were asked to count the number of points at which labeled cells intersected with the grid lines. The values obtained from the three observers were summed and averaged for each image.
Chemicals
GRGDNP and GRGESP were purchased from GIBCO-BRL. FITC-labeled GRGDNP [FITC-(CH2)6-GRGDNP] was purchased from Phoenix Pharmaceuticals, Inc. Soluble pronectin (pronectin F) was purchased from Protein Polymer Technologies, Inc. Albumin was purchased from Amersham Life Science. Monoclonal anti-CD61 (F11) was purchased from Pharmingen. Mouse myeloma IgG1 was purchased from Zymed Laboratories, Inc. Monoclonal anti-phosphotyrosine and all other chemicals were purchased from Sigma Chemical Co.
Statistical Analysis
Diameter changes were expressed as a percent of the maximal dilation measured in response to 100 µmol/L sodium nitroprusside. Differences within and between groups were determined using ANOVA for repeated measures with Fisher's least significant difference multiple-range tests when appropriate. The effect of flow in the absence and in the presence of GRGDNP on anti-phosphotyrosine labeling (as determined by the scoring procedure described above) was determined using the Wilcoxon paired-sample test. Significance was defined as P<.05.
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Results |
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Coronary arterioles were isolated from a total of 45 pigs. The average intraluminal diameter of arterioles in which vasodilatory responses were studied was 101±4 µm. Slightly larger vessels (average intraluminal diameter, 151±5 µm) were isolated for antibody staining to ensure application of the antibody to a large surface of endothelium that was undamaged by cutting the vessel along its longitudinal axis. These larger vessels also exhibited shear stress–induced vasodilation; diameters increased by
29% during exposure to shear stress.
Under control conditions, shear stress induced a vasodilation equal to 65±5% of that produced by 100 µmol/L sodium nitroprusside. A concentration of 0.1 mmol/L GRGDNP slightly attenuated shear stress–induced vasodilation, but the higher concentrations, 0.5 and 1.0 mmol/L (Fig 1
), significantly inhibited the response. At the highest shear stress, in the presence of 1.0 mmol/L GRGDNP, coronary arteriolar dilation was reduced to only 12±11% of maximal dilation. Although incubation with GRGDNP produced loss of tone in some vessels, the majority were unaffected. Of 24 vessels studied, four lost significant tone (>10%) and were not included in the analysis. Six of the remaining vessels showed slight dilation to the antagonist (5% to 10%), but the level of tone remained stable. These vessels were included in the final analyses.
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Shear stress–induced vasodilation was partially restored after removal of GRGDNP but was not as vigorous as that seen under control conditions. In five vessels, shear stress (1.2, 2.4, and 6.2 dyne/cm2) produced 46±7%, 68±9%, and 73±6% dilation, respectively. At the same levels of shear stress in the presence of the antagonist, dilation was only 4±3%, 8±7%, and 29±9%, respectively. A 30-minute period of washout partially restored shear stress–induced vasodilation to 6±3%, 39±7%, and 53±5%.
The scrambled inactive peptide GRGESP had no effect on the shear stress–induced response (Fig 2). This result suggests that the inhibitory effects of the receptor antagonist GRGDNP were due to specific interactions with integrins that recognize the RGD sequence.
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Application of GRGDNP did not depress endothelium-dependent dilation to substance P (Fig 3). Under control conditions, increasing concentrations of substance P increased diameter by 34±6%. Even at the highest concentration of GRGDNP (1.0 mmol/L), a similar increase in diameter (30±10%) was produced by substance P. These results confirm that application of the RGD-containing peptide did not cause nonspecific inhibition of all endothelium-dependent NO-mediated vasodilation.
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The ß3 integrin–blocking antibody, F11, significantly inhibited shear stress–induced vasodilation (Fig 4A). However, unlike treatment with the RGD-containing peptide, F11 did not alter basal tone. The antibody reduced the vasodilation to shear stress by 50%; at the highest shear stress (6.2±0.6 dyne/cm2), the dilation was reduced from 74±11% to 40±5%. Twenty minutes after removal of the antibody, a portion of the shear stress–induced response was restored. Because F11 is a mouse IgG1 (
isoform), we used a mouse myeloma antibody of the same isoform (mouse myeloma IgG1, isoform) as a negative control for nonspecific antibody effects. The mouse myeloma antibody did not alter shear stress–induced responses (Fig 4B).
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Intraluminal administration of pronectin F (apparent molecular mass, 110 kD) at a concentration of 40 µmol/L, which would contain approximately the same number of RGD binding sites as 0.5 mmol/L GRGDNP, caused the vessels to relax. During the 30-minute incubation period, the vessels dilated to 95% of maximal diameter, making it necessary to pharmacologically constrict the vessels with the thromboxane mimetic U46619. However, after preconstriction to 70% of resting diameter, the vessels still dilated in response to shear stress. Fig 5 illustrates that the vasodilatory responses to shear stress were similar in the absence and presence of soluble pronectin (P=.39).
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Confocal views of the luminal and abluminal sides of the endothelium labeled with FITC-GRGDNP are presented in Fig 6. Panels A and B represent 0.3-µm sections obtained from the abluminal and luminal sides, respectively. Note that the fluorescence intensity of the abluminal image is brighter than that of the luminal side. The average of several images from four arterioles revealed that the fluorescence intensities were 55% greater on the abluminal versus luminal side of the endothelium.
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Anti-phosphotyrosine labeling of endothelial cells was increased by 10 minutes of exposure to shear stress. This increase was blocked by treatment with GRGDNP. Fig 7 shows representative images from arterioles stained with anti-phosphotyrosine under baseline conditions (in the absence of shear stress) (panel A), after 10 minutes of exposure to shear stress (panel B), and after 10 minutes of exposure to shear stress in the presence of 1.0 mmol/L GRGDNP (panel C).
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Discussion |
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The important finding of the present study is that inhibition of integrin binding to extracellular matrix proteins that contain the RGD amino acid sequence blunts shear stress–induced vasodilation of coronary arterioles and blocks the increase in anti-phosphotyrosine labeling of endothelial cells in arterioles exposed to intraluminal flow. These results suggest that shear stress–induced vasodilation is mediated, at least in part, by integrin binding to extracellular matrix proteins containing this peptide sequence. A ß3 integrin antibody inhibited vasodilation to shear stress, suggesting a specific role for ß3 integrins in transducing the response to shear stress. Inhibition of the shear stress–induced increase in anti-phosphotyrosine labeling by treatment with GRGDNP also suggests that integrin binding is linked to tyrosine kinase activation, a signaling mechanism that we have previously shown to be involved in shear stress–induced vasodilation.5
Integrins provide a mechanical link between the intracellular environment and the extracellular matrix. Recent studies have investigated physiological roles for integrin binding and formation of focal adhesions in cultured endothelial cells.10 12 13 14 17 18 19 20 21 Data from those studies have demonstrated that focal adhesions and integrin interactions with extracellular matrix proteins are vital to cell adhesion14 and control of cell shape13 in endothelial cells grown in static culture. Davies et al14 have shown that exposure of endothelial cells to 10-dyne/cm2 unidirectional shear stress produces realignment of focal adhesions in the direction of flow. Ishida et al17 have recently reported that activation of ß1 integrins enhanced flow-induced increases in mitogen-activated protein (MAP) kinase activity in human umbilical vein endothelial cells. Our data indicate that integrin binding is involved in the signaling pathway by which shear stress causes vasodilation in coronary arterioles.
The synthetic peptide GRGDNP produced concentration-dependent inhibition of shear stress–induced vasodilation in isolated coronary arterioles. These results suggest that the RGD peptide interacts competitively with the RGD binding sites on integrins and that, similar to receptor-mediated responses, inhibition is related to the number of sites that are inactivated by antagonist binding. Critical to this conclusion are the results obtained using confocal microscopy, which show that the RGD antagonist has access to the abluminal side of the endothelium and thus can antagonize integrin-matrix interactions. It is also important to emphasize that shear stress–induced vasodilation was partially restored by removal of GRGDNP, indicating that the addition of GRGDNP did not irrevocably alter the shear stress–sensitive mechanisms of the arteriolar endothelium. The reversibility of this effect of the RGD peptide suggests that integrins play a dynamic role in mediating the response to shear stress.
The scrambled inactive form of the peptide, GRGESP, did not alter the flow-induced vasodilatory responses, suggesting that the inhibition produced by the GRGDNP was specifically due to its ability to compete with integrin binding to extracellular matrix proteins. The experiments using GRGESP followed the same time course as the experiments involving GRGDNP, indicating that the effects seen in the presence of GRGDNP were not due to repeated exposure to flow over an extended time period. This result is critical to our conclusions because it shows that the antagonism by GRGDNP was specific to shear stress–dependent vasodilation and not attributed to nonspecific depression of NO production by the endothelium or altered reactivity of vascular smooth muscle to NO. Thus, integrins may participate in mediating shear stress–induced vasodilation through a specific signaling pathway that is distinct from agonist-induced endothelium-dependent vasodilation.
Intraluminal administration of GRGDNP appeared to specifically target endothelium-dependent shear stress–induced vasodilation because agonist-induced nitroxidergic vasodilation was not affected by the antagonist. Mogford et al22 reported that extraluminal application of 10 µmol/L GRGDNP caused smooth muscle dilation in skeletal muscle arterioles. We do not believe that our results are inconsistent with this observation for the following reasons: In the present study, intraluminal application preferentially targets the endothelium because of the high intraluminal concentration of the drug. Because of the permeability barrier imposed by the endothelium, we suspect that only a fraction of the antagonist diffuses to the media. Even after the compound has diffused from the lumen, it is further diluted by the volume of the bath (in our experiments we have estimated this dilution to be 200-fold). Thus, we believe that the concentration of GRGDNP at smooth muscle cells is too low to be consistently vasoactive, and this is why we have found the effects of the peptide to be confined predominantly to the endothelium during intraluminal administration. Because the permeability barrier offered by the endothelial cell-cell junctions would serve to restrict diffusion, we do not know the exact concentration of GRGDNP at the abluminal side of the endothelial cells. Therefore, the effective dose of the antagonist at the integrin-matrix bond cannot be determined, and a direct comparison between the concentrations used in the present study and those used by Mogford et al22 cannot be made. We can state with conviction that luminal doses of antagonist specifically blocked shear stress–induced vasodilation.
Integrins may regulate cell function through distinct effects of receptor aggregation, receptor occupancy, or the combination of aggregation and receptor occupancy.18 23 24 It is possible that the inhibitory effects of the RGD peptide are specifically due to its ability to block receptor occupancy of a specific integrin ligand involved in the shear stress signaling pathway. Schwartz and Denninghoff18 have demonstrated that an endothelial cell integrin that mediates a rise in intracellular calcium made little contribution to adhesion to fibronectin. Miyamoto et al23 have shown that there are distinct effects upon integrin cytoskeletal function that are mediated by ligand occupancy, by integrin aggregation, or by a combination of occupancy and aggregation. It is possible that an integrin located luminally in endothelial cells acts as a shear stress sensor without being involved in abluminal attachment to the extracellular matrix. To demonstrate that the actions of the GRGDNP are attributed to actions on the abluminal surface of endothelial cells rather than to the binding of integrins on the luminal surface, we evaluated shear stress–dependent vasodilation under control conditions and after intraluminal administration of pronectin F. This large synthetic molecule, which incorporates 13 copies of RGD cell attachment sequences from fibronectin between structural peptide segments (apparent molecular mass, 110 kD),25 would be largely confined to the luminal space because of the permeability barrier imposed by the arteriolar endothelium.26 In cell culture, this polymer promotes rapid cell spreading (within 20 minutes) and attachment of a variety of cell types, including endothelial cells.27 28 29 Our finding that shear stress–induced vasodilation was not altered by pronectin F suggests that the inhibitory effects of the RGD peptide are due to actions at the abluminal surface of the endothelial cells and that shear stress–induced signaling is mediated through integrins located abluminally. Pronectin F treatment inhibited spontaneous tone, suggesting that some endothelial element (presumably an integrin receptor) present on the luminal surface was activated by this compound. It is possible that pronectin F stimulated release of a vasodilatory factor upon activation of an abluminal surface receptor. However, the response to shear stress was not altered, suggesting that the abluminal actions of pronectin F were not sufficient to inhibit shear stress–induced dilation in a manner similar to that seen with the RGD peptide.
Additionally, the present data suggest a specific role for ß3 integrins in mediating shear stress–induced vasodilation. The 
vß3 integrin is present in endothelial cells19 30 and participates in binding to vitronectin, fibronectin, and fibrinogen.18 20 Spreading of endothelial cells plated in wells coated with LM609, an antibody to the vß3 complex, is associated with a rise in intracellular calcium.31 However, the role of other integrins in mediating this response cannot be ruled out, because the inhibition of the shear stress–induced vasodilation was not complete. Ishida et al17 have shown that both flow and ß1 integrin activation by the antibody 8A2 produced tyrosine phosphorylation of focal adhesion kinase (FAK) and stimulated MAP kinase in human umbilical vein endothelial cells. Simultaneous exposure of human umbilical vein endothelial cells to flow and 8A2 produced additive increases in MAP kinase. However, the MAP kinase activation elicited by 8A2 alone was not as great as the activation that occurred in response to flow. These investigators proposed that the cellular responses that resulted from stimulation with 8A2 represented only a portion of the cellular events stimulated by flow and that other signaling mechanisms may have occurred simultaneously during flow activation of endothelial cells. The present data suggest that other integrins, such as the ß3 integrins, are involved in the transduction of shear stress. A combination of signaling mechanisms (both integrin-mediated and non–integrin-mediated) may be necessary to elicit release of NO in response to shear stress.
In a previous study, we showed that the tyrosine kinase inhibitors genistein and piceatannol produced significant inhibition of shear stress–induced vasodilation in coronary arterioles.5 Additionally, exposure to shear stress increased anti-phosphotyrosine labeling of arteriolar endothelial cells. This effect was blocked by genistein. In human umbilical vein endothelial cells, exposure to flow for several minutes produced a time-dependent increase in the tyrosine phosphorylation of a number of substrates, including MAP kinase and FAK.17 21 In the present study, shear stress also increased anti-phosphotyrosine labeling of the endothelium, and this effect was absent in vessels exposed to shear stress in the presence of the RGD-containing peptide, GRGDNP. We did not study the time course of the increase in tyrosine phosphorylation; however, maximal shear stress–induced vasodilatory responses in these arterioles generally occurred within 30 seconds and were sustained during continuous exposure to flow over a period of 10 to 15 minutes. Because of our previous results showing that inhibition of tyrosine kinase blocks shear stress–dependent dilation, we believe that the activation of this signaling enzyme occurs concomitantly with initiation of shear and is causally related to the subsequent dilation. Taken together, our previous and present results support our contention that integrin signaling, mediated via tyrosine kinase activation, is a critical link in the mechanotransduction of shear stress–induced vasodilation.
The present data indicate that integrin binding is involved in the signaling pathway by which shear stress causes vasodilation in coronary arterioles. Our findings tie together several observations in the literature. Hecker et al32 observed that the glycocalyx is involved in sensing flow (shear stress), because its removal by neuraminidase blocked flow-dependent vasodilation. Wang et al12 reported that ß1 integrins can act as cell surface mechanosensors that are linked to actin stress fibers in the cytoskeleton. Furthermore, Ishida et al17 recently reported that activation of ß1 integrins enhanced flow-induced increases in MAP kinase activity in human umbilical vein endothelial cells. Taken together, these results can be synthesized into a scheme in which shear forces are transmitted from the glycocalyx, through the cytoskeleton, to the focal adhesions of endothelial cells. According to this scheme, transmission of shear forces to the abluminal side of endothelial cells should commence integrin-mediated shear stress–induced vasodilation. Our results further suggest that integrin binding could be linked to tyrosine kinase activation and initiation of an intracellular signaling cascade leading to endothelium-dependent release of NO and subsequent vasodilation.
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Acknowledgments |
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This study was supported by National Heart, Lung, and Blood Institute awards HL-32788 and HL-51748 to Dr Chilian, HL-46502 to Dr Davis, and F32 HL-08975-02 to Dr Muller and an American Heart Association Established Investigator Award to Dr Davis. The authors gratefully acknowledge the technical contributions made by Judy A. Davidson to the work described in this manuscript.
Received November 8, 1996; accepted December 5, 1996.
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