vascular endothelial cells ( ecs ) are 
 constantly subjected to shear stress caused by the flow of blood . they are able to convert these 
 mechanical stimuli into relevant biological signals by a process that is known 
 as mechanotransduction . the best - known elements in the 
 early stages of this response are the cell - anchoring integrins   and certain membrane - associated 
 receptors , such as the vascular endothelial growth factor receptor 2 ( vegfr2 ) [ 3 , 4 ] and g - protein coupled receptors . after the initial activation of 
 these molecules , the biological signal is transmitted into the cell by activation 
 of major signal transduction pathways , such as mitogen - activated protein kinase 
 ( mapk ) pathways , the protein kinase b ( pkb / akt ) pathway , and the endothelial 
 nitric oxide synthase ( enos ) signaling route . these events lead to a functional 
 response of the cell , influencing rate of apoptosis and proliferation [ 1 , 6 ] , sensitivity to inflammation , and cytoskeletal remodeling . studies have shown that 50-nanometer , 
 omega - shaped membrane invaginations , known as caveolae , are linked to 
 mechanotransduction . the majority of membrane - associated proteins that are 
 phosphorylated in response to shear stress localize to these domains [ 9 , 10 ] . also , as ecs are subjected to shear 
 stress , the density of caveolae in the cell membrane increases , modulating the 
 activation of signaling pathways [ 11 , 12 ] . moreover , mice that lack a 
 structural protein of the caveolar domain , caveolin-1 , have an abnormal 
 vascular response when shear stress is altered . in order to gain more mechanistic insight 
 into the exact role of caveolae in the ec response to shear stress , in vitro studies caveolae were disrupted by cholesterol extraction [ 9 , 14 , 15 ] , and in one study caveolar 
 functioning was inhibited by introducing blocking antibodies to caveolin-1 . these studies have shown that interfering 
 with caveolar function causes an impaired response to shear stress , as 
 characterized by a lowered activation of mapks [ 9 , 14 , 16 ] , akt , and enos . an important molecular biological 
 tool to study the role of proteins in cellular processes is rna interference 
 ( rnai ) . by transfecting cells with short interfering rna ( sirna ) molecules with 
 a sequence that is complementary to the mrna of the protein of interest , the tool is very 
 specific and using it to lower expression levels of caveolin-1 could be useful 
 in confirming the results obtained by the more crude method of cholesterol 
 extraction . in this study , we use rnai in human 
 umbilical vein endothelial cells ( huvecs ) to confirm the essential role of 
 caveolin-1 in ec mechanotransduction . we also show that lowering caveolin-1 levels leads to 
 impaired vegfr2 signaling , but not integrin signaling , in these cells . this 
 suggests that the role of caveolin-1 
  in mechanotransduction lies in coupling vegfr2 
 activation to downstream signaling . caveolin-1 sirna was bought from qiagen ( west sussex , ireland ) 
 ( cat . si00299635 ) ; negative control sirna was obtained from invitrogen ( calif , usa ) ( stealth rnai negative control with medium gc content ) . antibodies for immunodetection were from the following companies : caveolin-1 ( sigma , mo , usa ; c4490 ) , akt ( abcam ( cambridge , uk ) ; ab28422 ) , phospho - akt ( abcam , ab27773 ) , erk1 ( abcam , ab9363 ) , phospho - erk ( santa cruz , calif , usa ; sc-7976 ) , gapdh ( abcam , ab9485 ) , goat anti - rabbit igg - alexa 633 ( molecular probes , ore , usa ; a21071 ) , goat anti - rabbit igg - hrp ( sigma , a0545 ) , and mouse anti - goat igg - hrp ( zymed , calif , usa ; 81 - 1620 ) . all products for cell culturing were from lonza ( breda , the netherlands ) except for the partially purified fibronectin , which was obtained as a coproduct during purification of human factor viii at sanquin , amsterdam , the netherlands , and which was used at 2 mg / ml in phosphate buffered saline ( pbs ) to coat surfaces for cell culture . huvecs were isolated from umbilical 
 cords by the method of jaffe et al . , using trypsin solution ( 0.05% ( w / v ) 
 trypsin , 0.02% ( w / v ) edta in pbs ) . the obtained endothelial cells were cultured 
 in fibronectin - coated culture flasks in endothelial growth medium-2 ( egm-2 ) , 
 containing 2% fetal bovine serum , until they reached confluency . when 
 confluent , cells were detached from the surface using trypsin solution and 
 diluted 1 : 3 in a fresh fibronectin - coated culture flask . cells were kept in a 
 humidified incubator ( 37c , 5%   co2 ) up to passage 8 , after which they were discarded . the day before transfection , huvecs were seeded in a 6-well plate , 30010 cells per well in egm-2 . the 
 following day , cells were washed once with optimem medium ( gibco , okla , usa ) and then overlaid with 
 800 l optimem . 15 l of 20 m sirna solution were mixed with 145 l of 37c optimem medium and left at 
 room temperature for 15 minutes . 8 l of oligofectamine ( invitrogen ) were mixed 
 with 32 l optimem , and after 5 minutes the oligofectamine mixture was added to 
 the tube with sirna . complexes were allowed to form for 15 minutes , and they 
 were added to the cells . after 4 hours in the incubator , 500 l egm-2 with 
 three times the normal amount of bovine serum were added to the wells . after 
 overnight incubation , the cells were washed with pbs and incubated in normal 
 egm-2 for 6 hours . for shear stress experiments , four 
 wells of sirna - transfected cells were trypsinized and replated on a 
 fibronectin - coated glass plate of 40 cm . the cells were left to 
 adhere for two hours in egm-2 , after which the medium was replaced by endothelial 
 basal medium-2 ( ebm-2 ) to starve the cells overnight . the next day , 48 hours 
 after transfection , cells were subjected to shear stress in a custom - built 
 parallel plate flow chamber . the chamber consisted of two parallel glass plates , spaced 0.6 mm apart by 
 glass spacers , held together by a stainless steel housing . the chamber was 
 connected to a glass reservoir containing 200 ml shear medium ( medium 199 with 
 100 units / ml penicillin and 100 g / ml streptomycin ) and to a peristaltic pump 
 ( watson - marlow , rotterdam , the netherlands ) . the different parts of the setup 
 were connected to each other in a closed circuit using silicone tubing ( versitec 
 silicone , 5 mm inner diameter , rubber bv , the netherlands ) , and the entire 
 setup was put in an incubator to maintain proper culturing conditions . medium 
 was then pumped through the flow chamber for 10 or 30 minutes at a rate of 200 ml 
 per minute . this yields a theoretical estimate for the shear stress of approximately 
 15 dyne / cm . after the treatment , the flow chamber was disassembled ; the cells were washed once with pbs and scraped in 100 l ice - cold lysis buffer ( 1% ( w / v ) triton x-100/pbs with added protease inhibitors ( 4-(2-aminoethyl)benzenesulfonyl fluoride , pepstatin a , e-64 , bestatin , leupeptin , and aprotinin ) and phosphatase inhibitors ( microcystin lr , cantharidin , ( )-p - bromotetramisole , sodium vanadate , sodium molybdate , sodium tartrate , and imidazole ) ) . then , the lysate was incubated on ice for 15 minutes and centrifuged at 12000  g for 10 
 minutes . the supernatant was transferred to a fresh tube , and the protein 
 concentration was determined using a bicinchonic acid assay ( pierce , etten - leur , the netherlands ) . the resulting sample was boiled for 5 minutes and then 
 stored at   20c until use . for vegf stimulation experiments , 
 sirna transfected cells were incubated overnight in growth medium and then 
 starved for 6 hours with ebm-2 . subsequently , 48 hours after transfection , the 
 cells were washed once with pbs , followed by addition of ebm-2 with 200 ng / ml vegf165 . cells were put in the incubator for 10 minutes , after which a lysate was 
 prepared as described above . the following day , 48 hours after transfection , cells were 
 trypsinized , spun down , and resuspended in 2 ml ebm-2 with 2% ( w / v ) bovine 
 serum albumin ( bsa ) . the cells were kept in suspension in the incubator under 
 constant , light agitation for 30 minutes . then , 1 ml of the cell suspension was 
 plated on a fibronectin - coated surface , while the remaining cells were spun 
 down , washed once with pbs , and then resuspended in lysis buffer . the plated 
 cells were left to adhere for 20 minutes , after which a lysate was prepared as 
 described above . samples were subjected to sodium 
 dodecyl sulphate ( sds ) poly(acrylamide ) gel electrophoresis , western blotting , 
 and immunodetection according to common protocols . shortly , samples were 
 separated by size on a 10% ( w / v ) poly(acrylamide ) gel , and the protein band 
 pattern was transferred to a poly(vinylidene difluoride ) membrane , using a 
 bio - rad mini - protean 3 system . the membrane was blocked with 1% ( w / v ) nonfat 
 dry milk in 25 mm tris , 150 mm nacl , 0.05% ( v / v ) tween-20 , ph 8.3 ( tbs - t ) . the primary antibodies were applied to the membranes in blocking buffer , 
 overnight at 4c . after washing the membrane with tbs - t four times for 20 
 seconds and one time for 15 minutes , the secondary antibody was applied to the 
 membrane in blocking buffer for one hour at room temperature . the same washing 
 regime was repeated , and the membrane was overlayed with supersignal west femto 
 substrate ( pierce ) . after three minutes of incubation , the chemiluminescent 
 signal was detected with a kodak image station with ccd camera . cells were seeded at a density of 150  10 cells per well on 
 fibronectin - coated glass coverslips in a 12-well plate . they were transfected with sirna as 
 described earlier , but with half the reagents per well . after transfection , the 
 cells were incubated overnight in egm-2 , then washed with pbs , and fixed with 
 4% ( w / v ) formaldehyde / pbs for 15 minutes at room temperature . the coverslips 
 were then covered with permeabilization buffer ( pbs with 1 mg / ml bsa and 0,1% 
 ( w / v ) primary antibodies were diluted in 
 permeabilization buffer and then incubated on the coverslips for 1 hour at 
 37c . the coverslips were washed three times for 5 minutes with pbs and were 
 subsequently covered with the secondary antibody in a mild permeabilization 
 buffer ( pbs with 1 mg / ml bsa and 0.05% ( w / v ) triton x-100 ) . after incubating for 1 hour at 37c , coverslips were washed and a 100 ng / ml solution of 4,6-diamidino-2-phenylindole 
 ( dapi ) was applied for 5 minutes to stain nuclei . after three more washes with 
 pbs , coverslips were mounted on microscope slides using mowiol ( calbiochem , darmstadt , germany ) and stored at 4c in the dark until 
 they were imaged with a zeiss lsm 510 confocal microscope . an estimate of the 
 average staining intensity was performed by dividing the total signal in a 
 field by the number of nuclei , as determined by using imagej image analysis 
 software . the intensity of the specific 
 signal was normalized to total protein content , as assessed by the loading 
 control in the same lane . in order to compare these signal intensities between 
 different experiments , the ratios between the intensity level of a sample and 
 the total intensity of all samples in that experiment were determined . the 
 averages of these normalized ratios were plotted , with the error bars 
 representing standard deviation . differences between these means were tested 
 for statistical significance by performing an unpaired , two - tailed student 's t - test . differences were considered to be statistically significant at p - values 
 smaller than .05 . in order to investigate 
 mechanotransduction in our flow chamber system , huvecs were subjected to 
 physiologically relevant shear stresses for 10 minutes and 30 minutes . after these 
 treatments , cell lysates were tested for phosphorylated akt and erk1/2 by 
 immunoblotting . as shown in figure 1 , 10 minutes of shear stress led to 
 significantly enhanced phosphorylation of both akt and erk1/2 . after 30 minutes 
 of shear stress , phosphorylation levels had dropped to values comparable to the 
 static situation . therefore , we decided to focus on the early timepoint for our 
 studies into the role of caveolin-1 in the ec mechanotransduction . when huvecs were treated with 
 caveolin-1 sirna , the expression of caveolin-1 decreased significantly to less 
 than one - third of the caveolin-1 level in untreated cells , as shown by 
 immunoblotting ( see figures 2(a ) and 2(b ) ) . when cells were transfected with 
 negative control sirna , no statistically significant decrease in caveolin-1 
 expression was detected , showing the specificity of the sirna treatment . moreover , 
 expression of akt and erk1/2 was not affected by the transfection procedure or 
 the lowering of caveolin-1 levels ( see figure 2(a ) ) . the downregulation of 
 caveolin-1 was confirmed by using immunocytofluorescence ( see figure 2(c ) ) . using the same microscope settings , staining intensity was approximately four 
 times higher in untreated cells than in caveolin-1 sirna - treated cells . the 
 remaining caveolin-1 in the sirna - treated cells was located mostly in the 
 perinuclear area , with a lack of intense membrane staining like in the 
 untreated and control rna treated cells . huvecs with normal and lowered levels 
 of caveolin-1 were subjected to physiological levels of shear stress in a 
 parallel plate flow chamber . after 10 minutes of shear stress , phosphorylation 
 status of the important mechanotransducing molecules akt and erk1/2 was 
 determined by phospho - specific immunoblotting . total expression levels of these 
 proteins were not affected by sirna treatment , as determined by immunoblotting 
 ( see figure 2(a ) ) . after subjecting huvecs to shear stress , phosphorylation 
 status of akt and erk1/2 increased approximately five to ten times ( see figure 3 ) . we found that the activation of both signal transduction pathways was 
 significantly lower in caveolin-1 sirna - treated cells than in cells treated 
 with negative control sirna . no significant differences were found among the activation levels of control sirna - treated cells and untreated cells . in order to uncover the mechanism for 
 the ineffective mechanotransduction in cells with low caveolin-1 levels we chose activation 
 of integrins and vegfr2 , because these proteins are well known to be essential 
 for mechanotransduction . in order to assess integrin - mediated signal 
 transduction , cells were kept in suspension for 30 minutes and then plated for 
 20 minutes on a fibronectin - coated surface . when checking the phosphorylation 
 status of akt and erk1/2 by phospho - specific immunoblotting , no significant 
 differences could be detected between the activation of signal transduction 
 pathways in negative control sirna - treated cells and in cells with low caveolin-1 levels ( see figures 4(a ) and 4(b ) ) . activation of vegfr2 was accomplished by stimulating starved cells with 200 ng / ml vegf165 for 10 minutes . in this case , signal transduction was found to be significantly lower in cells that had been treated with caveolin-1 sirna than in cells with normal levels of caveolin-1 ( see figures 4(c ) and 4(d ) ) . our study shows that lowering the 
 expression level of caveolin-1 in human ecs by rnai inhibits 
 mechanotransduction in response to shear stress . thus , we confirm the essential 
 role of caveolae in the ec response to shear stress which has also been 
 suggested by research groups that interfered with caveolar function using 
 methods like cholesterol extraction   and antibody blocking . we show that the activation of both 
 akt and erk1/2 is affected by the lowering of caveolin-1 . in both cases , akt is well known 
 for its role in cell survival and resistance to apoptosis . indeed , activation 
 of akt in response to shear stress has been shown to suppress apoptosis in ecs [ 15 , 19 ] . based on the findings in this 
 study , one would expect that ecs with low caveolin-1 levels are more sensitive 
 to apoptosis when subjected to shear stress than untreated cells . this would be 
 an interesting and counterintuitive result , because lack of caveolin-1 
 generally renders cells less sensitive to apoptosis . the exact mechanisms of this 
 decreased apoptosis sensitivity have not yet been elucidated , although the akt 
 pathway is definitely involved . a number of important membrane receptors 
 in ecs , such as activin receptor - like kinase 1 ( alk-1 ) , vegfr2 , and epithelial growth factor 
 receptor ( egfr ) , have been shown to associate with 
 caveolin-1 . moreover , therefore , loss of the association 
 between these receptors and caveolin-1 by downregulation of the latter may be a 
 reason for changes in akt activation and apoptosis sensitivity . given the 
 importance of ec apoptosis in the onset and development of atherosclerosis , future studies should be performed 
 to provide more insight in this functional implication of the substantially 
 lowered akt activation in cells with less caveolin-1 . another well - known downstream effect 
 of akt activation is increased enos activity . based on our finding that lowered 
 levels of caveolin-1 inhibit activation of akt , it can be expected that enos 
 activation and the resulting effects on blood vessels , such as vasodilatation , 
 are also impaired . ex vivo studies 
 have indeed shown that enos activation and vasodilatation in response to fluid 
 flow are lower in arteries of caveolin-1 knockout mice than in blood vessels of 
 wild - type mice . the dramatically lowered activation 
 of erk1/2 in response to shear stress in caveolin-1 sirna - treated ecs that we 
 describe in this study will also have functional implications . activation of erk1/2 
 has an important role in modulating gene expression in response to shear stress . two well - described examples show that erk1/2 activation is essential for the 
 shear stress - induced increase in the expression of matrix metalloprotease-9   and enos . the absence of erk1/2 activation in 
 our system will surely have an impact on these shear stress - induced changes in 
 expression . when attempting to identify the 
 mechanism by which caveolin-1 contributes to mechanotransduction , we first 
 decided to investigate the signal transduction in response to integrin binding 
 in these cells . integrin - mediated signaling is an essential process in 
 mechanotransduction [ 2 , 31 ] . moreover , there have been multiple 
 reports of the involvement of caveolin-1 in this signaling pathway [ 3234 ] . despite these reports , we were 
 unable to detect any changes in activation of akt or erk1/2 between caveolin-1 sirna - treated 
 cells and untreated cells . it could be that in our system the remaining 
 caveolin-1 is still sufficient to properly induce these signaling events . our 
 system differs from the ones used in the aforementioned reports by cell type , 
 caveolin-1 levels , and the chosen output parameters to determine efficient 
 signaling . based on our experiments , we can conclude that it is unlikely that 
 the inefficient mechanotransduction as seen in our system is due to impaired 
 integrin signaling . we decided also to investigate signal 
 transduction in response to vegf in caveolin-1 sirna - treated ecs . ligand - independent activation of vegfr2 is another essential event in 
 mechanotransduction , and stimulation with vegf can serve 
 as a way to specifically activate and monitor this part of the signaling 
 network . the role of caveolin-1 in vegf - induced signal transduction is still 
 ambiguous and may depend on cell type and culture conditions . in some cell 
 systems , downregulation of caveolin-1 leads to hyperactivation of signal 
 transduction pathways . in addition , other studies that 
 focus on the role of caveolin-1 in vegf - initiated signaling have shown that both an increase and a decrease of the levels of caveolin-1 in ecs have a negative impact on downstream signal transduction [ 22 , 36 ] . the direct association between caveolin-1 and vegfr2 inhibits activation of the latter , but on the other hand , the association also seems necessary for proper initiation of downstream signaling after growth factor receptor activation . moreover , caveolin-1 knock - out mice have impaired vasculogenic 
 potential , due to impaired vegf signaling . also in our system , lowering the 
 level of caveolin-1 has a negative impact on signal transduction when ecs are 
 stimulated with vegf . this suggests that the lack of mechanotransduction in ecs 
 with low caveolin-1 levels is due to the important role that caveolin-1 plays 
 in coupling vegfr2 activation to downstream signaling events . based on the results presented in 
 this article , it can be concluded that caveolin-1 is needed for activation of 
 akt and erk1/2 in ecs that are subjected to shear stress . our results suggest 
 that the impaired activation of these pathways in ecs with low levels of 
 caveolin-1 is due to inefficient vegfr2 signaling . the role that caveolin-1 plays in the biology of ecs and 
 the impact it has on the physiology of the vasculature in vivo are becoming more and more clear . because of the 
 importance of caveolin-1 in endothelial signaling , it is a potential target for 
 clinical applications , for example , in tumor angiogenesis and atherosclerosis . 
 therefore , the role of caveolin-1 in the ec shear stress response is definitely 
 worth studying in more detail .
vascular endothelial cells have an extensive response to physiological levels of shear stress . 
 there is evidence that the protein caveolin-1 is involved in the early phase of this response . in this study , caveolin-1 was downregulated in human endothelial cells by rnai . when these cells were subjected to a shear stress of 15 dyn / cm2 for 10 minutes , activation of akt and erk1/2 was significantly lower than in control cells . 
 moreover , activation of akt and erk1/2 in response to vascular endothelial growth factor was significantly lower in cells with low levels of caveolin-1 . 
 however , activation of integrin - mediated signaling during cell adhesion onto fibronectin was not hampered by lowered caveolin-1 levels . in conclusion , caveolin-1 is an essential component in the response of endothelial cells to shear stress . 
 furthermore , the results suggest that the role of caveolin-1 in this process lies in facilitating efficient vegfr2-mediated signaling .