Role of the endothelial caveolae microdomain in shear stress–mediated coronary vasorelaxation

In this study, we determined the role of caveolae and the ionic mechanisms that mediate shear stress–mediated vasodilation (SSD). We found that both TRPV4 and SK channels are targeted to caveolae in freshly isolated bovine coronary endothelial cells (BCECs) and that TRPV4 and KCa2.3 (SK3) channels are co-immunoprecipitated by anti-caveolin-1 antibodies. Acute exposure of BCECs seeded in a capillary tube to 10 dynes/cm2 of shear stress (SS) resulted in activation of TRPV4 and SK currents. However, after incubation with HC067047 (TRPV4 inhibitor), SK currents could no longer be activated by SS, suggesting SK channel activation by SS was mediated through TRPV4. SK currents in BCECs were also activated by isoproterenol or by GSK1016790A (TRPV4 activator). In addition, preincubation of isolated coronary arterioles with apamin (SK inhibitor) resulted in a significant diminution of SSD whereas preincubation with HC067047 produced vasoconstriction by SS. Exposure of BCECs to SS (15 dynes/cm2 16 h) enhanced the production of nitric oxide and prostacyclin (PGI2) and facilitated the translocation of TRPV4 to the caveolae. Inhibition of TRPV4 abolished the SS-mediated intracellular Ca2+ ([Ca2+]i) increase in BCECs. These results indicate a dynamic interaction in the vascular endothelium among caveolae TRPV4 and SK3 channels. This caveolae–TRPV4–SK3 channel complex underlies the molecular and ionic mechanisms that modulate SSD in the coronary circulation.

In this study, we determined the role of caveolae and the ionic mechanisms that mediate shear stress-mediated vasodilation (SSD). We found that both TRPV4 and SK channels are targeted to caveolae in freshly isolated bovine coronary endothelial cells (BCECs) and that TRPV4 and KCa2.3 (SK3) channels are co-immunoprecipitated by anti-caveolin-1 antibodies. Acute exposure of BCECs seeded in a capillary tube to 10 dynes/ cm 2 of shear stress (SS) resulted in activation of TRPV4 and SK currents. However, after incubation with HC067047 (TRPV4 inhibitor), SK currents could no longer be activated by SS, suggesting SK channel activation by SS was mediated through TRPV4. SK currents in BCECs were also activated by isoproterenol or by GSK1016790A (TRPV4 activator). In addition, preincubation of isolated coronary arterioles with apamin (SK inhibitor) resulted in a significant diminution of SSD whereas preincubation with HC067047 produced vasoconstriction by SS. Exposure of BCECs to SS (15 dynes/cm 2 16 h) enhanced the production of nitric oxide and prostacyclin (PGI 2 ) and facilitated the translocation of TRPV4 to the caveolae. Inhibition of TRPV4 abolished the SS-mediated intracellular Ca 2؉ ([Ca 2؉ ] i ) increase in BCECs. These results indicate a dynamic interaction in the vascular endothelium among caveolae TRPV4 and SK3 channels. This caveolae-TRPV4 -SK3 channel complex underlies the molecular and ionic mechanisms that modulate SSD in the coronary circulation.
Endothelial cells (ECs) 2 help maintain vascular homeostasis by sensing and integrating hemodynamic and neurohumoral stimuli and by generating and releasing vasoactive molecules to modulate vascular function (1,2). The unique location of vascular ECs exposes them to three types of mechanical forces: hydrostatic pressure, wall tension, and shear stress (SS). Of these, SS appears to be the most important because it regulates cellular function and gene expression and is a determinant of blood vessel formation, maintenance, and remodeling (3). SS is a potent endothelium-dependent physiological vasodilatory signal that regulates most tissue beds and involves major endothelium-dependent vasodilators, including nitric oxide (NO) (3), prostacyclin (PGI 2 ) (4), and endothelium-derived hyperpolarizing factors (EDHFs) (5). We have found that in mouse coronary arteries, shear stress-mediated vasodilation (SSD) involves all three major vasodilator pathways (6). The effects of SS on endothelial function are complex, involving many potential sensors and effectors (3,7,8). Previous studies have implicated caveolae as mechanical sensors that play a critical role in the EC response to SS (9,10). Recently, we have reported that SSD of mouse coronary arteries is endothelium-and caveolaedependent and is defective in caveolin-1 null (Cav-1 Ϫ/Ϫ ) mice (6). However, the molecular ionic mechanisms through which caveolae mediate SSD have not been directly delineated. The transient receptor potential (TRP) channels are particularly appealing as SS sensors. These channels are Ca 2ϩ -permeable cationic channels with polymodal activation properties and are well-adapted to function in cellular sensing as well as modulation of K ϩ channels and vasoreactivity (11,12). Among the TRP channels, TRPV4, TRPC4, and TRPC6 are implicated in the vasodilatory response to SS (13)(14)(15)(16). Compelling evidence exists that TRPV4 appears to be mechanistically important in endothelial mechanosensing of SS (14,15,(17)(18)(19)(20). Opening of TRPV4 leads to an increase in [Ca 2ϩ ] i, , an important signaling molecule that activates a wide range of Ca 2ϩ -dependent pathways, including nitric oxide synthase (eNOS), phospholipase A 2 , prostacyclin synthase, and calcium-activated potassium (K Ca ) channels. Among the K Ca channels, the endothelial intermediate conductance IK (KCa3.1) and small conductance calcium-activated potassium channels (SK) have been shown to mediate the effects of EDHF (21,22). Recently, SK and IK have been found to be involved in agonist-mediated vasodilation in mouse mesenteric arteries through TRPV4 activation (23). Whether these ion channels are also involved in SSD in coronary arteries and how TRPV4 is coupled to the K Ca channels is unclear.
A major impediment to progress in our understanding of the regulation of ion channel function in vascular endothelial cells is the change of phenotype of these cells under culture conditions. We have overcome this barrier by studying freshly isolated bovine coronary endothelial cells. We have also designed novel approaches to examine the regulation of endothelial ion channel by SS (see below). In this study, we found that TRPV4 and SK3 (KCa2.3) channels are both targeted to the caveolae microdomain and that SS facilitates the translocation of TRPV4 channels into caveolae, promoting a more robust interaction with K Ca channels. In addition, we were able to directly measure ionic currents in freshly isolated single ECs from coronary arteries in response to SS, demonstrating the regulation of TRPV4 and SK3 function by SS. These novel findings provide mechanistic insights into the molecular and ionic signaling mediating SSD in the coronary vasculature and have significant scientific and clinical implications.

Characterization of freshly isolated bovine coronary endothelial cell (BCEC) preparations
BCECs were identified by the presence of specific cell surface antigens and by cellular uptake of acetylated LDL (24). Freshly isolated BCECs were incubated with acetylated LDL conjugated with Alexa Fluor 488 (15 g/ml) (Thermo Fisher Scientific) in DMEM with 10% FBS at 37°C for 4 h. Cells were washed and fixed. Internalized acetylated LDL was detected by fluorescence microscopy. Freshly isolated BCEC preparations showed uptake of acetylated LDL in Ͼ90% of total cells. In contrast, vascular smooth muscle cells and HEK293 cells did not take up acetylated LDL under similar conditions (Fig. 1A). Further evaluation to confirm the purity of the BCEC preparations was performed by incubating the cells with FITC-conjugated bovine-specific monoclonal anti-CD31 antibodies (1:10 clone CO.3E1D4, Thermo Fisher Scientific) or with control nonimmune mouse IgG conjugated with FITC. Cells were then washed and suspended in 200 l PBS and analyzed by a FAC-SCalibur flow cytometer (BD Biosciences). Greater than 90% of the cells were positively labeled (Fig. 1B), indicating the freshly isolated cell preparations of bovine coronary arteries were predominantly BCECs.

Co-localization of SK and TRP channels in the caveolae of nonpassaged BCECs
We examined the subcellular distribution of TRPC4, TRPV4, IK, and SK channels in BCECs by sucrose gradient fractionation. Fig. 2A shows immunoblots of the 12 fractions from the gradient (1 being the lightest and 12 the heaviest). SK1 (KCa2.1), SK3, and TRPV4 proteins were detected in the lowbuoyant density, caveolae-rich membrane fractions, whereas SK2 (KCa2.2), IK, and TRPC4 were found only in the highdensity non-lipid raft membrane fractions (numbers 9 to 12). Co-immunoprecipitation (Co-IP) experiments of the lowbuoyant density fraction 5 showed that both SK3 and TRPV4 were precipitated by anti-Cav-1 antibody, but not by nonimmune IgG. In comparison, SK1 was absent in the anti-Cav-1 immunoprecipitates, suggesting that it was located in noncaveolar lipid rafts (Fig. 2B). Furthermore, reverse Co-IP using anti-TRPV4 and anti-SK3 antibodies showed the presence of Cav-1 in the immunoprecipitates (Fig. 2C). These results indicate that both SK3 and TRPV4 were co-localized in the caveola membrane microdomain, which may facilitate their interaction in BCECs because of the spatial proximity with each other.
The in situ proximity ligation assay (PLA) is a powerful technology that can be used to quantify and demonstrate the co-localization of proteins of interest to specific subcellular locations for inference of potential protein-protein interactions. We performed PLA in freshly isolated BCECs to confirm the co-localization of SK3 and TRPV4 in the caveolae of BCECs. As shown in Fig. 2D, the presence of fluorescence signals generated from labeled complementary oligonucleotide probes after a 100-min amplification reaction at 37°C indicates the close proximity of Cav-1 with TRPV4 and SK3, confirming our Co-IP results.

Functional characterization of K Ca channels in freshly isolated BCECs
We examined the function of K Ca channels in freshly isolated BCECs using isoproterenol (ISO), a known potent activator of K Ca channels (25)(26)(27). Fig. 3 shows the raw tracings of total whole-cell currents and the current-voltage (I-V) relationships recorded in the following sequence of conditions: baseline, 1 M ISO, followed by ISO with BK channel blocker (100 nM IBTX, a specific BK channel inhibitor), ISO with SK channel blocker (200 nM apamin (APA)), and then ISO with SK channel blocker plus IK channel blocker (200 nM Tram34). Under baseline conditions, the total whole-cell currents in freshly isolated BCECs were small (13.2 Ϯ 2.8 pA/pF at 150 mV, n ϭ 4). Application of ISO robustly increased current density by 5-fold, to 64.4 Ϯ 16.3 pA/pF at ϩ150 mV (n ϭ 4, p Ͻ 0.05 versus baseline). The ISO-induced currents were not inhibited by IBTX (71.58 Ϯ 14.07 pA/pF at ϩ150 mV, n ϭ 4, p ϭ not significant (N.S.) versus ISO), but were markedly inhibited by APA (23.3 Ϯ 6.7 pA/pF at ϩ150 mV, n ϭ 4, p Ͻ 0.05 versus ISO). Addition of Tram34 to APA did not produce any further current suppression (25.7 Ϯ 4.2 pA/pF at ϩ150 mV, n ϭ 4, p ϭ N.S. versus APA). In addition, activation of total currents by ISO was not inhibited by Tram34 alone, but by Tram34 plus APA (Fig. 4). Hence, our results suggest that SK channels are the major K Ca channels in freshly isolated BCECs and the ISO effect on increasing the K ϩ current is mediated through SK channel activation.

Activation of SK channels by acute exposure to SS
We further determined the effect of SS on SK channel activation. Nonpassaged BCECs were seeded in the distal end of a glass capillary tube, and the proximal end of the pipette was connected to a precision peristaltic pump to generate shear stress of 10 dynes/cm 2 (Fig. 5A). After a 2-h incubation for BCEC attachment to the capillary tube, total whole-cell currents were continuously recorded in perforated-patch configuration elicited with a voltage-ramp protocol of 100-ms duration from Ϫ100 mV to ϩ100 mV at 10-s intervals. Fig. 5B shows typical tracings of total currents at baseline, with 10 dynes/cm 2 of SS, and SS in the presence of 200 nM APA. Exposure to SS increased the outward currents at 100 mV (42.0 Ϯ 11.2 pA/pF versus 24.0 Ϯ 3.7 pA/pF at baseline, p Ͻ 0.05, n ϭ 4) but not the inward currents at Ϫ100 mV (7.6 Ϯ 1.3 pA/pF versus Ϫ4.0 Ϯ 1.7 pA/pF at baseline, p ϭ N.S., n ϭ 4). Application of 200 nM APA significantly blocked the effects of SS on outward current activation. The I-V curves of APA-sensitive currents at baseline and after exposure to SS are shown in Fig. 5C. These results show that SK currents were significantly activated by SS, and are a major mechanosensitive response element in the vascular endothelium.

Roles of TRPV4 in SK channel activation and SS-induced coronary vasodilation
Because TRPV4 is a major caveola-targeted ion channel in the BCECs, we studied the role of TRPV4 on SK channel regulation in response to SS. Fig. 6 represents the raw tracings of whole-cell currents elicited by voltage activation in freshly isolated BCECs before and after application of 150 nM GSK1016790A (GSK, a TRPV4 activator) and 200 nM APA. The currents were augmented 3.5-fold by GSK, from 28.2 Ϯ 10.6 pA/pF (n ϭ 3) at baseline to 98.1 Ϯ 13.4 pA/pF (n ϭ 3, p Ͻ 0.05 versus baseline), and most of the activated currents were suppressed by APA to 42.8 Ϯ 12.2 pA/pF (n ϭ 3, p Ͻ 0.05 versus GSK). These results suggest that activation of TRPV4 channels results in an increase in SK channel activity.
In conjunction with pharmacological intervention, SS at 10 dynes/cm 2 produced a robust enhancement in total currents as shown by continuous recordings in BCECs using a voltageramp protocol. The SS effects were significantly blocked by the TRPV4 inhibitor HC067047, as shown in Fig. 7A. Moreover, HC067047 had no effect on the endothelial tetraethylammoniumsensitive (10 mM) currents, but abolished the activation of APAsensitive currents by SS (10 dynes/cm 2 ) in freshly isolated BCECs (Fig. 7B). Taken together, these results indicate that augmentation of whole-cell currents by SS is mediated through SK channels and this is regulated by TRPV4 channel activity in the cell preparations examined.
The physiological roles of SK and TRPV4 channels in SSD were examined in isolated mouse coronary arteries. At baseline, SS was a potent stimulus for dilation in freshly isolated mouse coronary arteries producing 43.8 Ϯ 4.9% and 89.2 Ϯ 1.1% of maximal vasodilation by 10 and 25 dynes/cm 2 , respectively.

Figure 2. Subcellular distribution of SK, IK, and TRP channels in freshly isolated nonpassaged BCECs is shown.
A, the subcellular distribution of SK1, SK2, SK3, IK, TRPC4, and TRPV4 in freshly isolated BCECs was measured by density gradient fractionation, with fraction 1 being the lightest and fraction 12 the heaviest. Western blots against anti-SK1, anti-SK2, anti-SK3, anti-IK, anti-TRPV4, anti-TRPC4, and anti-Cav-1 antibodies show that SK1, SK3, and TRPV4 are detected in the low-buoyant density, caveolae-rich fractions, whereas SK2, IK, and TRPC4 are mainly found in the high-density non-lipid raft fractions and absent in the caveolae-rich fractions. B, immunoprecipitates of anti-Cav-1 antibody after incubation with the low-buoyant density fraction 5 were blotted against anti-SK1, anti-SK3, anti-IK, and anti-TRPV4 antibodies. Pulldowns using nonimmune IgG served as controls. C, immunoprecipitates of anti-TRPV4 or anti-SK3 antibodies from the low-buoyant density fraction 5 were blotted against anti-Cav-1 antibodies. Pulldown using nonimmune IgG served as controls. D, BCEC in situ proximity ligation assay (PLA). The fluorescent signal generated by labeled complementary oligonucleotide probes after a 100-min amplification reaction at 37°C in BCECs treated with mouse anti-Cav-1 and rabbit anti-SK3 or rabbit anti-TRPV4 antibodies. The nuclei were counterstained with DAPI, and the PLA signals were visualized at 40ϫ magnification under a Zeiss 510 Meta Confocal Laser Scanning Microscope equipped with DAPI/Texas Red filters and analyzed using Zeiss 510 software.

Activation of endothelial ion channels by shear stress
Preincubation with 200 nM APA significantly attenuated the SS effects, producing only 7.7 Ϯ 1.5% and 36.1 Ϯ 7.0% (n ϭ 5) vasodilation by 10 and 25 dynes/cm 2 of SS, respectively (n ϭ 4, p Ͻ 0.05 for both versus baseline), suggesting that SK currents play an important role in mediating SSD. Furthermore, after preincubation with 150 nM HC067047, SS at 10 dynes/cm 2 was abolished (8.1 Ϯ 5.7%, n ϭ 5, p Ͻ 0.05 versus baseline), whereas vasoconstriction was observed at 25 dynes/cm 2 (Ϫ10.7 Ϯ 12.0%, n ϭ 5, p Ͻ 0.05 versus baseline), suggesting that normal TRPV4 activity is critical in the regulation of SSD (Fig. 7C). Hence, these results show that the modulation of TRPV4 channels by SS, which in turn activates SK chan-nels, represents the major ionic mechanism underlying SSD in coronary arteries.

Translocation of TRPV4 channels to caveolae induced by SS in BCECs
To further delineate the molecular mechanisms through which modulation of endothelial ion channels results in SS-mediated coronary vasodilation, we examined the effects of SS on SK and TRPV4 subcellular distribution in BCEC using a coneplate viscometer (28). Sucrose gradient fraction measurements in BCECs showed that under baseline conditions, only 13.0% of total cellular TRPV4 channels were present in the low-buoyant Representative whole-cell currents were recorded from freshly isolated BCECs at baseline and after application of ISO, followed by IBTX (a specific BK channel inhibitor), APA (a specific SK channel inhibitor), and Tram34 (a specific IK channel inhibitor). K ϩ currents were small at baseline but were robustly activated by ISO. The ISO-induced whole-cell currents were not inhibited by IBTX, but were inhibited by APA, with no further suppression by Tram34 in the presence of APA. Group results with statistical analysis are illustrated in the I-V curves. †, p Ͻ 0.05 ISO versus baseline; *, p Ͻ 0.05 APA versus ISO; n ϭ 4, and error bars represent S.E.

Activation of endothelial ion channels by shear stress
density lipid raft fractions (Fig. 8, A and B). However, after an overnight exposure to SS at 15 dynes/cm 2 , 40.9% of the TRPV4 channels were found in the low-buoyant density fractions, a 3.2-fold increase (Fig. 8, A and B). Such change in TRPV4 subcellular distribution is not because of channel membrane trafficking from intracellular sites because the cell surface expression of TRPV4 proteins was unaltered by SS (Fig. 8C). In contrast, SS had no effect on the subcellular distribution of SK channels (data not shown). These results suggest that there is significant translocation of endothelial TRPV4 channels from the non-lipid raft plasma membranes to caveolae in response to SS and this phenomenon is ion channelspecific. Such findings also indicate that the regulation of TRPV4 activity and its downstream targets by SS is dynamic and complex.

SS increased intracellular Ca 2؉ as well as NO and PGI 2 generation in BCECs
It is well-known that NO and PGI 2 generation in the endothelium is Ca 2ϩ -dependent (29,30), contributing to SS-mediated coronary vasodilation in mice (6). We found that intracellular Ca 2ϩ in BCECs increased more than 5-fold on acute exposure to SS (11 dynes/cm 2 ) and the [Ca 2ϩ ] i changes were abolished by preincubation with 150 nM HC067047 (Fig. 9A). In addition, the production of vasodilators by BCECs was significantly enhanced by overnight SS at 15 dynes/cm 2 , with a 2-fold

Activation of endothelial ion channels by shear stress
increase in NOx and a 3.6-fold increase in 6-keto PGF1␣ (Fig. 9, B and C). These results indicate that SS is a potent stimulus for [Ca 2ϩ ] i increase with enhanced production of vasodilators by ECs.

Discussion
We have developed innovative techniques for this study and have made several important observations. First, SSD in coronary arteries is dependent on TRPV4 and SK channels. Second, acute SS is a potent activator of both TRPV4 and SK channels. Third, activation of SK channels by SS is a consequence of TRPV4 activation. Fourth, SS-mediated increase in [Ca 2ϩ ] i in endothelial cells is dependent on TRPV4. Fifth, both TRPV4 and SK3 channels are targeted to the caveola membrane microdomain. Sixth, SS induces translocation of TRPV4 into caveolae. These findings provide new insights into the molecular and ionic mechanisms in coronary endothelium that mediate SSD.
Research on endothelial ion channel regulation has been hindered by the change in EC phenotype under culture conditions. However, we have developed innovative approaches that overcome this obstacle. We have successfully obtained freshly isolated ECs from bovine coronary arteries for studying the effects of SS on endothelial ion channel regulation. These cells allow us to examine the acute effects of SS on endothelial ion channel and [Ca 2ϩ ] i regulation. We were able to study the direct effects of acute SS on TRPV4 and SK currents in freshly isolated BCECs using patch clamp techniques. These experiments were technically challenging but were feasible with high-fidelity recordings that provide novel mechanistic insights supported by direct evidence. We were able to isolate an adequate number of cells from each preparation to allow quantitative biochemical studies on prolonged SS-induced changes in vasodilator production and ion channel expression. The effects of acute SS on [Ca 2ϩ ] i regulation was determined using a custom-made chamber that allowed us to measure fluorescence signals in fura-2loaded freshly isolated BCECs and the effects of acute SS on [Ca 2ϩ ] i with and without pharmacological intervention. Our findings showed that the SS-induced Ca 2ϩ increase in BCECs is TRPV4 dependent, indicating it is the major Ca 2ϩ influx pathway in response to SS. The innovative approaches we employed are instrumental for providing unequivocal observations on SSmediated endothelial ion channel and vascular regulation.
We have recently reported that SSD is endothelium-and caveolae-dependent; absence of caveolae results in impaired SSD (6). The present study is an extension of these findings. We found that both TRPV4 and SK channels are co-localized in the caveola membrane microdomain in BCECs. Exposure to physiological levels of SS results in translocation of TRPV4 channels into the caveolae of BCECs, and such recruitment of TRPV4 channels in the caveolae is not because of the membrane trafficking of TRPV4 from intracellular sites (Fig. 8C). This phenomenon is ion channel-specific, as the subcellular distribution of SK is not affected by SS. Enhanced caveolae targeting of TRPV4 suggests that there is a greater SS-induced increase in [Ca 2ϩ ] i , as TRPV4 is the major Ca 2ϩ influx pathway in response to SS (Fig. 9A).

Figure 5. Activation of SK channels by acute exposure to shear stress (SS) in freshly isolated nonpassaged BCECs is shown.
A, illustration showing the patch clamp recording system for studying the acute effects of SS on ionic currents in freshly isolated BCECs. The distal end of a glass capillary tube was seeded with BCECs; the proximal end was connected to a precision peristaltic pump to generate 10 dynes/cm 2 of SS. B, representative whole-cell currents were recorded from BCECs in perforated-patch configuration using a voltage-ramp protocol of 100-ms duration from Ϫ100 mV to ϩ100 mV at 10-s intervals. Currents were continuously recorded at baseline, on exposure to 10 dynes/cm 2 SS, and with APA (200 nM) during SS. SS enhanced the total currents, which were markedly inhibited by 200 nM APA. C, the I-V curves of apamin-sensitive currents in BCECs at baseline and after activation by SS. *, p Ͻ 0.05 versus baseline; n ϭ 4, and error bars represent S.E.

Figure 6. Activation of TRPV4 channels increased SK currents in BCECs.
Whole-cell currents were elicited from freshly isolated BCECs at baseline and after exposure to GSK1016790A (a specific TRPV4 channel activator) and APA. The currents were markedly augmented by GSK, and most of the GSK-activated currents were sensitive to APA. Group results at all given testing potentials are shown in the I-V curves; n ϭ 3; *, p Ͻ 0.05 versus baseline, and error bars represent S.E.

Activation of endothelial ion channels by shear stress
The increase in endothelial [Ca 2ϩ ] i is critical in facilitating vasodilation through modulation of multiple signaling pathways (31). The activity of eNOS is tightly regulated by [Ca 2ϩ ] i and SS is the most potent activator of eNOS (32). Activation of phospholipase A 2 is also Ca 2ϩ -dependent, and it releases a major source of arachidonic acid-derived vasodilators (33). We have confirmed that the production of NO and PGI 2 in freshly isolated BCECs is significantly enhanced by SS (Fig. 9, B and C). Both eNOS and PGI 2 synthase (PGIS) are targeted to the caveolae of endothelial cells and their activities are profoundly regulated by SS. SS enhances eNOS activity and NO production by inducing eNOS phosphorylation that promotes eNOS subcellular translocation out of caveolae, releasing it from Cav-1 inhibition (34,35). Augmented PGIS activity by SS was associated with increased mRNA expression of PGIS and cyclooxygenase (COX) in vascular endothelial cells (36). Enhanced PGIS and COX activities, coupled with the Ca 2ϩ -de-pendent release of arachidonic acid through phospholipase A 2 (37), constitutes a major mechanism through which SS facilitates production of endothelium-derived relaxation factor (EDRF) and EDHF. Most importantly, the Ca 2ϩ influx mediated by TRPV4 is critical in SS-mediated activation of endothelial SK channels. Caveolae co-localization facilitates the interaction between TRPV4 and SK through spatial proximity. The fact that TRPV4 is recruited to caveolae in response to SS further enriches this interaction and guarantees that SK is more fully activated to perform its endothelium-derived hyperpolarization function. This important finding also explains the impaired SSD with loss of caveolae integrity.
Both TRPV4 and SK channels have been implicated as important ionic mechanisms in mediating SSD. Endothelial SK and IK channels are considered key candidates of EDHF in many vascular beds (38,39). In freshly isolated BCECs, we found that SK channels are targeted to caveolae, whereas IK channels are not (Fig. 2). We also found that SK3, but not SK1 or SK2, is present in caveolae (Fig. 2). These results are similar to those observed from human microvascular ECs (40). Moreover, activation of SK channels by [Ca 2ϩ ] i provides a negative feedback mechanism in regulating endothelial cell membrane potentials by producing endothelial membrane hyperpolarization, which in turn hyperpolarizes the adjacent smooth muscle cells through myoendothelial gap junctions, leading to vasorelaxation. We found that the activation of SK channels by SS is TRPV4-dependent (Fig. 7). TRPV4 appears to be mechanistically important in endothelial mechanosensing of SS (14, 15, Figure 7. Roles of TRPV4 in SS-mediated SK channel activation and coronary vasodilation are shown. A, raw tracings of whole-cell K ϩ currents elicited by a voltage-ramp protocol from BCECs seeded in the capillary tube before and after exposure to 10 dynes/cm 2 SS and HC067047 (a specific TRPV4 inhibitor). B, the I-V curves show that HC067047 has no direct effects on the TEA-sensitive K ϩ currents in BCECs, but eliminates the APA-sensitive currents activated by 10 dynes/cm 2 SS. C, dilation of isolated mouse coronary arteries was significantly increased by physiological SS (10 to 25 dynes/cm 2 ). After preincubation with APA, SS-induced coronary vasodilation was significantly attenuated. After preincubation with HC0607047, SS failed to produce vasodilation; instead, vasoconstriction ensued. *, p Ͻ 0.05 versus controls, and error bars represent S.E.  [17][18][19][20]. In this study, we showed that TRPV4 plays a central role in SS-mediated [Ca 2ϩ ] i increase in BCEC (Fig. 9), in the activation of SK channels by SS (Fig. 7, A and B), and in SSD (Fig. 7C). Our results are consistent with previous reports on the role of endothelial TRPV4 channels in vasodilation in the resistance vessels of the mesenteric circulation (23).

Activation of endothelial ion channels by shear stress
In summary, we used a combination of innovative approaches to examine the molecular and ionic mechanisms that mediate SSD. Using freshly isolated BCECs, we were able to demonstrate the physical and ionic coupling between TRPV4 and SK3 in caveolae. SS-induced recruitment of TRPV4 channels to caveolae is an exciting finding that further supports the caveolae as an important rendezvous point for the assembly of critical elements of SSD, including eNOS, PGIS, TRPV4, and SK3. In addition, TRPV4 represents the major SS-induced Ca 2ϩ influx pathway in BCEC. It facilitates the production of endothelium-derived relaxation factors such as NO and PGI 2 , as well as endothelium-derived hyperpolarization, through activation of SK channels.

Bovine coronary endothelial cell isolation
Fresh bovine hearts were obtained from a local slaughterhouse and immediately placed on ice-cold PBS. Two to three coronary arteries were dissected from the heart, washed three times with ice-cold PBS. The last wash was with PBS supplemented with penicillin (100 IU/ml) and streptomycin (100 g/ml). The lumens of the coronary arteries were washed once with 0.05% trypsin-EDTA (Thermo Fisher Scientific) and filled with this enzyme solution for 15 min at 37°C in a cell incubator. Endothelial cells were recovered by flushing the vessel lumen with 20 ml of DMEM supplemented with 10% FBS, penicillin (100 IU/ml), and streptomycin (100 g/ml). The BCEC-containing medium was centrifuged for 5 min at 1200 rpm (200 ϫ g). The cells were resuspended in EBM-2 Endothelial Basal Medium with growth factors and 10% FBS as described in the EGM-2 BulletKit (Lonza, Walkersville, MD). All protocols were approved by the Institutional Animal Care and Use Committee and the Institutional Biosafety Committee at Mayo Clinic, Rochester, MN.

Whole-cell patch clamp recordings
Freshly isolated BCECs were seeded in the distal end of a glass capillary pipette (1 mm ϫ 1 mm size), the roof (ϳ1 mm) of which was removed at the distal end. BCEC attachment was allowed with a 1-to 2-h incubation at 37°C and the capillary pipette was mounted in a chamber on the stage of an inverted microscope. The proximal end of the capillary tube was connected to a PHD2000 Programmable Syringe Pump (Harvard Apparatus). Whole-cell currents were recorded at baseline and during exposure to 10 dynes/cm 2 of SS using an Axopatch 200B integrating amplifier (Molecular Devices), filtered at 2 kHz and digitized at 50 kHz as reported previously (41,42). Clampex 10.4 software (Molecular Devices) was used to generate voltage clamp protocols. Specifically, SK currents, defined as the apamin-sensitive (200 nM) component of whole-cell K ϩ currents, were elicited from a holding potential of Ϫ60 mV to testing potentials of Ϫ40 mV to ϩ150 mV in 10-mV increments. TRPV4 currents, defined as the HC067047-sensitive (200 nM) component of whole-cell currents, were recorded continuously at 30-s intervals from a holding potential of 0 mV to a voltage ramp from Ϫ100 mV to ϩ100 mV over 100 ms (41).

Activation of endothelial ion channels by shear stress SS-induced coronary vasodilation
Video microscopy was employed to measure SS-induced coronary vasodilation of mice (6). Briefly, mouse (C57BL/6J) coronary arteries (1-2 mm in length and 100 -200 m in diameter) were mounted in a vessel chamber filled with Krebs solution (in mM): NaCl 118.3, KCl 4.7, CaCl 2 2.5, MgSO 4 1.2, KH 2 PO 4 1.2, NaHCO 3 25.0, and glucose 11.1, pH 7.4 with NaOH, secured between two borosilicate glass micropipettes with 10-O ophthalmic sutures, and then placed on the stage of an inverted Olympus CK40 microscope (Olympus America) equipped with a Olympus OLY-105 CCD camera and a video micrometer (VIA-100, Boeckeler Instruments). The intraluminal pressure of the mounted coronary arteries filled with Krebs solution was maintained at 80 mmHg using a syringe microinjection pump and a pressure servo controller (Living Systems Instrumentation). After a 1-h equilibration, vessels were subjected to incremental levels of physiological SS (1,5,10,15,20, and 25 dynes/cm 2 ) as described previously (6,44). Incremental levels of shear stress (1,5,10,15,20, and 25 dynes/cm 2 ) were applied to each vessel through the microinjection pump with flow rates calculated according to Equation 1, where Q is the flow rate, D is the vessel diameter, is the shear stress, and is the viscosity of fluid. Vessel diameters were monitored and measured continuously. Flow rates were adjusted to achieve the next level of shear stress based on the diameter reached (6,42).

In situ proximity ligation assay
The PLA assay was performed using the PLA kit according to the manufacturer's instruction. Briefly, freshly isolated BCECs were fixed in coverslips with 4% paraformaldehyde/PBS at room temperature for 20 min, penetrated with 0.2% Triton X-100 for 5 min, and then washed three times with PBS and blocked for 30 min in blocking buffer. After treatment with mouse anti-caveolin-1 antibodies and rabbit anti-KCa2.3 or TRPV4 antibodies (1:250 in PBS with blocking) at 37°C for 2 h, cells were washed three times (5 min each) with PBS. Oligonucleotide-conjugated anti-mouse minus and anti-rabbit plus PLA secondary probes were added with blocking buffer and the cells were incubated in a humidified chamber for 1 h at 37°C. Connector oligonucleotides were hybridized and circularized by ligation for 30 min at 37°C. After thorough washing, the cells were incubated with DNA polymerase for 100 min at 37°C to produce rolling circle amplification products tagged with a red fluorescence probe, when the PLA probes were in close proximity (Ͻ40 nm). The nuclei were counterstained with DAPI, and the PLA signals were visualized at 40ϫ magnifications under Zeiss 510 Meta Confocal Laser Scanning Microscope equipped with DAPI/Texas Red filters and analyzed using Zeiss 510 software.

Sucrose gradient density fractionation
Caveolae targeting of SK and TRPV4 in freshly isolated nonpassaged BCECs was determined by sucrose density gradient centrifugation as described previously (27,45). The cells were homogenized in 500 mM Na 2 CO 3 with 2% protease inhibitor (v/v), and then centrifuged at 5000 rpm at 4°C for 10 min. Two ml of the supernatant were adjusted to 40% sucrose-MES (2-(Nmorpholino)ethanesulfonic acid) by adding 1 ml of 80% sucrose-MES, placed at the bottom of a 12-ml ultracentrifuge tube, layered with a 4-ml discontinuous sucrose gradient (40, 30, and 5%), and centrifuged at 32,000 rpm at 4°C for 20 h. Twelve fractions of 1 ml each were collected and analyzed by immunoblotting. Equal volumes (40 l) of each fraction were used for Western blot analysis. The distribution of TRPV4 was calculated according to the relative densities of the TRPV4 band on the Western blot. The band in each fraction was scanned and the relative density units were added to constitute the denominator (adding up all the densities from each fraction). The contribution of each fraction was calculated as a percentage of the total density.

Co-immunoprecipitation and immunoblotting
Co-IP and immunoblotting analyses were performed as described previously (46,47). In brief, 200 l of the low-buoyant density fraction 5 from each experiment was incubated with rabbit anti-caveolin-1 antibody (1:500, Santa Cruz Biotechnology), anti-SK3 (1:120, Alomone Labs Ltd.), or anti-TRPV4 (1:120, Alomone Labs Ltd.) antibodies at 4°C overnight. Incubation with nonimmune IgG served as the control. The samples were then incubated with 20 l Protein G Plus Agarose (Santa Cruz Biotechnology) at 4°C for 2 h, centrifuged at 1000 rpm, and washed three times with RIPA/protease inhibitor buffer. The immunoprecipitates were collected and eluted from the agarose with 30 l SDS-PAGE loading buffer for further immunoblot analysis.
For immunoblotting, the proteins were boiled with 15 l SDS-PAGE loading buffer at 100°C, resolved by polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and then blotted against rabbit anti-Cav-1, rabbit anti-

Cell surface TRPV4 expression
Cell surface expression of TRAPV4 was determined using the Pierce Cell Surface Protein Isolation Kit (Thermo Fisher Scientific). Briefly, BCECs with or without a 16-h SS were washed by PBS and then incubated with Sulfo-NHS-SS-Biotin reagent at 4°C for 30 min. Cells were washed by TBS and lysed by RIPA buffer. The control and shear-stressed cell lysates containing equal amounts of proteins were incubated with NeutrAvidin Agarose beads at room temperature for 1 h. After washing the beads with lysis buffer thrice, surface biotinylated TRPV4 protein levels were determined by Western blot analysis.

Cone-plate viscometer
A cone-plate viscometer was custom-made based on that described by Malek et al. (28). The methyl methacrylate cone

Activation of endothelial ion channels by shear stress
has an angle of 1°with the bottom of the culture plate and was coupled to a computer-driven motor with precise regulation of SS from 0 to 40 dynes/cm 2 , which allowed us to expose a large number of BCECs to 15 dynes/cm 2 SS overnight. A culture dish containing nonpassaged BCEC (ϳ75% confluent) was exposed to 15 dynes/cm 2 SS in a tissue culture incubator for 16 h at 37°C. A successful response to SS resulted in realignment of cells to the direction of flow.

Intracellular Ca 2؉ measurement
[Ca 2ϩ ] i was measured by the fluorescence Ca 2ϩ indicator fura-2, as reported previously (6). BCECs were seeded on a microscope slide and loaded with 3 M fura-2, AM (Thermo Fisher Scientific) for 30 min at 37°C. The slide was then assembled in a linear SS flow chamber and placed on the stage of an inverted Olympus IX71 microscope (Olympus America) equipped with a Hamamatsu ORCA-R2 CCD camera (Hamamatsu Photonics) and a Sutter LB-LS/17 light source (Sutter Instruments). The cells were subjected to SS (11 dynes/ cm 2 ) with a solution that contained (in mM) NaCl 125.0, KCl 4.5, CaCl 2 2.0, MgCl 2 2.0, HEPES 20.0, and glucose 10.0, pH 7.4. The intracellular Ca 2ϩ fluorescence signals were measured as the ratio of fluorescence intensities at 510 nm from excitations of 340/ 380 nm. The Ca 2ϩ signal (F) was analyzed using MetaFluor software (Molecular Devices) and expressed as a ratio (F/F0), where F0 stands for the baseline fluorescence signal.

NO and PGI 2 measurement
Culture dishes containing nonpassaged BCECs at 75% confluence were exposed to 15 dynes/cm 2 SS overnight using a cone-plate viscometer. The culture medium was collected, and the end products of PGI 2 (6-keto PGF1␣) and NO (NOx) were measured by enzyme immunoassay kits (Cayman Chemical) as reported previously (48).

Chemicals
Unless otherwise mentioned, all chemicals used were obtained from Sigma-Aldrich Company.

Statistical analysis
Data are expressed as mean Ϯ S.E. Statistical analysis was performed using SigmaStat 3.5 software (Systat Software, Inc.). One way analysis of variance (ANOVA) followed by Tukey's test was employed to compare data from multiple groups. Student's t test was used to compare data between two groups, whereas paired t test was used to compare data from the same samples before and after treatment. Statistical significance was defined as p Ͻ 0.05.