while macroautophagy , herein simply termed autophagy , plays an indispensible role in the intracellular degradation of proteins and organelles , there is growing evidence that also implicates this process in protein secretion .
this link includes a role for autophagy in regulating unconventional secretion of acb1 in yeast   and interleukin-1 ( il-1 ) in mammalian cells .
for instance , the constitutive secretion of il-6 and il-8 from senescent cells appears to involve the formation of a specialized cellular domain termed the tor - autophagy spatial coupling compartment ( tascc ) .
this compartment is directly adjacent to the trans golgi network and is enriched for both signaling proteins such as mtor , as well as for autophagic vacuoles . finally , there are a growing number of studies suggesting a connection between components of the autophagy machinery and the regulated secretion of intracellular lysosomes or granules . for instance ,
deficiency in specific essential autophagy proteins appear to impair regulated secretion from the intestinal paneth cell , bone marrow derived mast cells , pancreatic -cells , melanocytes , osteoclasts and vestibular epithelial cells .
exocytosis from endothelial cells represents one of the first lines of defense following vascular injury .
specific secretory granules within the endothelial cell known as weibel - palade bodies ( wpbs ) contain numerous biologically active molecules , although von willebrand factor ( vwf ) is by far the most abundant .
indeed , vwf is required for formation of wpbs , and heterologous expression of vwf in non - endothelial cells can induce the formation of wpb - like structures .
once secreted , vwf multimers can assemble into long strings that can become tethered to the underlying connective tissue while simultaneously ensnaring circulating platelets .
impaired production , processing or secretion of vwf results in the clinical syndrome known as von willebrand disease , recognized as the most common inherited bleeding disorder .
the production of wpbs is a complex process involving dimerization and disulfide bond formation in the endoplasmic reticulum followed by furin - dependent cleavage in the golgi complex .
the vwf multimers that form can then be folded , in a ph - sensitive fashion , into tubular structures . while structural mutations in vwf can lead to defects in multimer formation or tubulation , relatively little
is known about what other cellular processes regulate the number , ph or secretory potential of wpbs . here
, we demonstrate that autophagy plays a critical role in the biology of wpbs and regulates the in vitro and in vivo release of vwf .
while there is a growing understanding of the molecular and biological role of autophagy , the role this process plays within the vasculature is poorly understood . in an effort to better understand how autophagy might contribute to vessel homeostasis , we analyzed electron micrographs of primary human umbilical vein endothelial cells ( huvecs ) .
both wpbs and autophagosomes have distinctive appearance by electron microscopy , with the former exhibiting a classical cigar shaped appearance and the latter being an intracellular organelle often filled with diverse cellular contents surrounded by a unique double membrane structure .
surprisingly , we noted that within endothelial cells , wpbs and autophagosomes were often found in close proximity .
moreover , we noted multiple instances in which a wpb appeared to be very near and actively fusing with an autophagosome ( fig . 1a and supplemental fig .
examination of the contents of autophagosomes also revealed the presence of what appeared to be wholly or partially intact wpbs , clearly identifiable by their unique shape and internal striations running parallel to their long axis ( fig .
1b ) . to further pursue these observations we performed additional immunogold labeling coupled with electron microscopy analysis to assess the distribution of vwf within endothelial cells .
these studies revealed that as expected , vwf was present at high abundance within mature wpbs ( fig
this immunogold labeled vwf signal appeared to be composed of both free protein , as well as , what appeared to be wholly or partially intact wpbs ( fig . 1d and supplemental fig .
1c - e ) . analyzing 30 random section containing several hundred autophagosomes revealed that 17 % of the autophagosomes had no detectable vwf signal ( 48/279 ) , 42% ( 117/279 ) of the autophagosomes had a moderate amount of vwf ( 1 - 10 immunogold particles ) and 41% ( 114/279 ) of the autophagosomes had a robust signal for vwf ( > 10 immunogold particles per autophagosome ) .
the electron microscopic observations suggested that autophagosomes , and by extension autophagy , might somehow modulate vwf levels or secretion .
autophagosome formation requires that the cytosolic form of lc3 ( lc3-i ) be conjugated in an atg5 and atg7 dependent fashion to form the lipidated lc3-ii species .
based on these past observations , we next sought to analyze the distribution of vwf and lc3 ( atg8 ) using super resolution structured illumination microscopy .
these images revealed the expected cigar shaped wpb morphology and the classical spherical punctae of lipidated lc3-ii ( fig .
1e ) . within endothelial cells , super resolution microscopy revealed that endogenous vwf and lc3 were clearly separable .
nonetheless , at any given time , a substantial fraction of wpbs were closely associated with a lc3 signal ( 37/224 wpbs or 16.7% ) with a smaller percentage of wpbs ( 7/224 or 3.2% ) appearing to be surrounded by a structure consistent with a lc3-coated autophagosome ( fig . 1e and supplemental movie 1 ) .
a separate analysis using confocal microscopy came to a similar conclusion , namely that a substantial fraction ( approximately 30% ) of intracellular lc3 and vwf spots had a centroid to centroid distance of less than 1.5 m ( supplemental fig .
finally , using two color confocal live cell imaging , we could capture in real time , the apparent direct interaction of a lc3-gfp autophagosome with a vwf - mcherry positive wpb ( fig .
nonetheless , additional dual - color ( lc3-mcherry and vwf - gfp ) or single color ( lc3-gfp ) tirf imaging suggested these interactions were rare and that under normal circumstances , autophagosomes do not appear to directly fuse with the plasma membrane ( supplemental movie 2&3 ) . to begin to address whether autophagy modulates vwf biology
, we performed lentiviral shrna mediated knockdown of atg7 in human endothelial cells . in atg7 knockdown cells , western blot analysis confirmed a significant reduction in atg7 expression ( fig .
assessment of autophagic flux often relies on the simultaneous measurements of the level of expression of the multidomain , signaling adaptor protein p62/a170/sqstm1 , hereafter referred to simply as p62 , along with the ratio of lc3-ii to lc3-i .
a rise in p62 levels and a fall in lc3-ii are consistent with impaired autophagic flux .
as expected , knockdown of the essential autophagy gene atg7 had the expected effects on these well characterized markers of autophagic flux ( fig .
we then asked whether knockdown of atg7 altered the stimulated release of vwf . using an elisa - based method of the culture medium , we detected an increase in vwf secretion following stimulation with histamine or vegf ( fig .
we saw similar effects on vwf secretion using an alternative and independent method for knockdown of atg7 that employed sirna ( supplemental fig .
the reduction in the stimulated release of vwf was further confirmed by direct western blot analysis of the endothelial cell supernatant ( fig .
2c , d ) . a recent report has suggested that disruption of autophagy might alter intracellular calcium signaling .
given the central role of calcium in stimulated exocytosis from the endothelium , we wondered whether such a mechanism might explain our observations . using a calcium sensitive fluorometric probe
, we could not however detect significant differences in ligand - induced calcium transients between control and atg7 knockdown cells ( fig .
similarly , other proximal signaling events such as extracellular regulated kinase ( erk ) activation appeared to be unaffected by atg7 knockdown ( supplemental fig .
4 ) . we next sought to establish whether other genetic or pharmacological inhibitors of autophagy had similar effects .
knockdown of the essential autophagy gene atg5 alone or in combination with atg7 also inhibited vwf release ( fig .
following histamine stimulation , the combined knockdown of atg5 and atg7 appeared slightly more effective than single knockdown of either atg7 or atg5 in blocking vwf secretion ( fig .
incubation of human endothelial cells with chloroquine or bafilomycin , two known inhibitors of autophagic flux , also resulted in a marked inhibition of ligand - stimulated vwf secretion ( fig .
in contrast , knockdown of atg5 or atg7 did not appear to alter ligand - stimulated release of endothelin-1 from endothelial cells ( supplemental fig .
5b ) . in addition , while knockdown of atg6 ( beclin 1 ) was quite effective in reducing atg6 expression ( fig 2i ) , it had only a very modest effect on markers of autophagic flux ( supplemental fig .
we next sought to attempt to get a better understanding of why knockdown of an essential autophagy gene such as atg7 might inhibit the secretion of vwf . following synthesis , vwf undergoes a series of processing events in the er and golgi that eventually lead to maturation of the protein and incorporation into wpbs . using a quantitative elisa - based method we determined that the total intracellular level of vwf protein was not significantly different between control and atg7 knockdown endothelial cells ( fig .
similar results were observed following knockdown of atg5 or combined atg5 and atg7 knockdown ( supplemental fig .
we next sought to analyze whether differences might exist in the processing , maturation and secretion of vwf .
the processing of vwf involves a furin - dependent proteoltyic cleavage within the trans - golgi network with the pro - vwf precursor distinguishable from mature vwf based on electrophoretic mobility .
we observed that the steady state ratio of pro - vwf to mature vwf was significantly altered in atg7 or atg5 knockdown cell ( fig .
consistent with a defect in vwf processing , we also noted a reduction in the number of mature wpbs observed in atg7 knockdown cells ( fig .
we saw similar reduction in wpbs in endothelial cells with either atg5 knockdown or following pharmacological inhibition of autophagy using chloroquine or bafilomycin ( supplemental fig .
in contrast , knockdown of atg6 did not alter the number of mature wpbs ( supplemental fig .
these results suggest that one explanation for our observation with atg5 or atg7 knockdown is that these strategies , while not altering overall vwf expression , appear to inhibit the processing and maturation of vwf into mature wpbs .
the formation of wpbs is believed to occur at the trans - golgi network ( tgn ) and autophagy is known to be critical for the homeostatic maintenance of both the er and golgi . furthermore , the ph of wpbs is known to be acidic and this low ph is essential for the shape of the wpbs , as well as vwf tubulation and function . using previously described methods , we were able to establish that wpbs within atg7 knockdown cells exhibited a higher ph than wpbs found in control cells ( fig . 4a and supplemental fig .
consistent with previous results , when assessed by confocal microscopy , this higher ph in atg7 knockdown cells resulted in rounder and less rod - shaped wpbs ( fig .
this also translated into a statistically significant reduction in the length of wpbs found in atg7 knockdown cells ( fig .
similar observations were evident by electron microscopy where we observed that knockdown of atg7 resulted in wpbs that were shorter , rounder and had less distinct tubulation ( fig .
whether in addition to a processing defect , there was also an effect of atg7 on the secretion of mature wpbs , we employed a previously described system using live cell imaging of the secretion of a vwf - gfp chimeric fusion protein .
expression of this tagged vwf results in assembly of wpbs containing the co - expressed fluorescently labeled vwf protein ( supplemental fig .
consistent with the alterations in processing described above , the number of gfp - positive wpbs was reduced in cells where atg7 was knockdown ( supplemental fig .
9b ) and the wpbs that formed , had an altered , more rounded morphology ( supplemental movies 4 , 5 ) .
using this system , in conjunction with total internal reflection fluorescence ( tirf ) microscopy , we could monitor individual secretion events of gfp - positive wpbs that localized near the plasma membrane ( fig .
our analysis demonstrated that knockdown of atg7 resulted in reduced wpb secretion after histamine stimulation ( fig .
4 g ) . in an effort to extend these observations to an in vivo situation
, we conditionally deleted atg7 within endothelial cells by crossing mice harboring atg7 floxed alleles with transgenic mice expressing the previously described ve - cadherin cre recombinase .
parental mice both of which bore the genotype atg7 ; ve - cadherin cre were crossed to generate offspring that were either atg7 ; ve - cadherin cre ( hereafter denoted as atg7 ) or atg7 ; ve - cadherin cre ( hereafter denoted as control ) .
given the relatively small contribution of endothelial cells to overall organ mass , we did not observe a significant reduction in atg7 expression in the tissues and organs isolated from atg7 mice ( supplemental fig .
primary cultures of mouse endothelial cells isolated from atg7 mice exhibited the expected reduction in atg7 expression ( fig .
the residual expression of atg7 in these cells may represent a combination of incomplete deletion with ve - cadherin cre , as well as potentially some degree of contamination of non - endothelial cells in these primary cultures .
nonetheless , this level of atg7 expression was sufficient to induce a reduction in autophagic flux as evidenced by the lc3-i / ii ratio and p62 levels ( fig .
similar to our observations employing knockdown of atg7 in human endothelial cells , we did however observe a reduction in the number of mature wpbs in endothelial cells derived from atg7 mice ( fig 5b ) . while conditional deletion of essential autophagy genes in tissues such as brain led to marked alterations in tissue homeostasis and early mortality , atg7 mice exhibited no obvious vascular phenotypes .
similarly , atg7 mice exhibited no increase in mortality over the first year of life ( unpublished observations ) .
assessment of vascular density demonstrated that atg7 mice were indistinguishable from control animals ( fig .
5c ) . in addition , while autophagy has been implicated in angiogenesis , we saw no consistent or reproducible abnormalities in atg7 mice when post - natal retinal angiogenesis was assessed ( fig .
furthermore , the endothelium and underlying architecture of large vessels were similar in atg7 and control mice ( fig .
we also observed no reduction in the level of vwf expression in atg7 mice either by immunohistochemical ( fig .
we next asked whether atg7 mice exhibited defects in vwf secretion . in control mice ,
6a , fold increase : 1.5 + / 0.04 , n=27 mice , p<0.001 pre versus post ) .
this increase in vwf in response to subcutaneous epinephrine injection is similar in magnitude to previous reports .
in contrast , atg7 mice failed to respond in a significant fashion to an epinephrine challenge ( fig .
6a , fold increase : 1.17 + / 0.05 , n=13 mice , p = ns pre versus post ) .
this lack of stimulated epinephrine release was also evident in mice where atg5 had been conditionally deleted again using ve - cadherin cre .
similar to atg7 mice , atg5 mice also failed to respond to epinephrine ( fig .
6a , fold increase : 1.13 + / 0.07 , n=9 mice , p = ns pre versus post ) .
neither atg5 mice nor atg7 mice had a significant reduction in basal vwf ( fig .
similarly , basal levels of circulating p - selectin levels were not altered between control and atg7 mice ( supplemental fig .
12 ) . to therefore further assess the role of autophagy in regulating basal vwf levels , we analyzed mice having a complete , whole body knockout of atg7 .
previous studies demonstrated that these total body atg7 knockout mice rarely survive for more than 48 hours after birth .
when we analyzed atg7 mice shortly after delivery , we did note a reduction in plasma vwf when compared to wild type littermates , perhaps reflective of a more complete knockout in this model then can be achieved using conditional approaches ( fig .
we next sought to further characterize the physiological importance of atg7 in vwf processing in vivo .
consistent with the observed in vitro deficit in vwf processing , we noted that on non - reducing gels , plasma vwf analyzed from atg7 mice contained diminished levels of the biologically active , higher molecular weight multimers ( fig .
consistent with this reduction in high molecular weight vwf , coupled with the decrement in stimulated vwf release ( fig .
6a ) , we noted a significant increase in measured bleeding time when control mice were compared to atg7 mice ( fig .
such differences occurred in the absence of any measurable differences in various hemostatic parameters ( supplemental fig .
furthermore , we did not see any obvious abnormalities in platelet morphology or platelet vwf levels that could have potentially contributed to the observed alterations in the measured bleeding time ( supplemental fig .
14 ) . given that one of the many effects for chloroquine is inhibition of autophagy and since this agent is approved for human usage , we next randomized mice to saline or chloroquine ( cq ) injections .
after 9 days , basal plasma vwf levels in cq treated and vehicle treated mice were similar ( supplemental fig .
nonetheless , direct assessment of hemostasis revealed that similar to atg7 mice , pharmacological inhibition of autophagic flux resulted in a significant prolongation of bleeding time ( fig . 6e and supplemental fig .
while there is a growing understanding of the molecular and biological role of autophagy , the role this process plays within the vasculature is poorly understood . in an effort to better understand how autophagy might contribute to vessel homeostasis , we analyzed electron micrographs of primary human umbilical vein endothelial cells ( huvecs ) .
both wpbs and autophagosomes have distinctive appearance by electron microscopy , with the former exhibiting a classical cigar shaped appearance and the latter being an intracellular organelle often filled with diverse cellular contents surrounded by a unique double membrane structure .
surprisingly , we noted that within endothelial cells , wpbs and autophagosomes were often found in close proximity .
moreover , we noted multiple instances in which a wpb appeared to be very near and actively fusing with an autophagosome ( fig . 1a and supplemental fig .
examination of the contents of autophagosomes also revealed the presence of what appeared to be wholly or partially intact wpbs , clearly identifiable by their unique shape and internal striations running parallel to their long axis ( fig .
1b ) . to further pursue these observations we performed additional immunogold labeling coupled with electron microscopy analysis to assess the distribution of vwf within endothelial cells .
these studies revealed that as expected , vwf was present at high abundance within mature wpbs ( fig
this immunogold labeled vwf signal appeared to be composed of both free protein , as well as , what appeared to be wholly or partially intact wpbs ( fig . 1d and supplemental fig .
1c - e ) . analyzing 30 random section containing several hundred autophagosomes revealed that 17 % of the autophagosomes had no detectable vwf signal ( 48/279 ) , 42% ( 117/279 ) of the autophagosomes had a moderate amount of vwf ( 1 - 10 immunogold particles ) and 41% ( 114/279 ) of the autophagosomes had a robust signal for vwf ( > 10 immunogold particles per autophagosome ) .
the electron microscopic observations suggested that autophagosomes , and by extension autophagy , might somehow modulate vwf levels or secretion .
autophagosome formation requires that the cytosolic form of lc3 ( lc3-i ) be conjugated in an atg5 and atg7 dependent fashion to form the lipidated lc3-ii species .
based on these past observations , we next sought to analyze the distribution of vwf and lc3 ( atg8 ) using super resolution structured illumination microscopy .
these images revealed the expected cigar shaped wpb morphology and the classical spherical punctae of lipidated lc3-ii ( fig .
1e ) . within endothelial cells , super resolution microscopy revealed that endogenous vwf and lc3 were clearly separable .
nonetheless , at any given time , a substantial fraction of wpbs were closely associated with a lc3 signal ( 37/224 wpbs or 16.7% ) with a smaller percentage of wpbs ( 7/224 or 3.2% ) appearing to be surrounded by a structure consistent with a lc3-coated autophagosome ( fig . 1e and supplemental movie 1 ) .
a separate analysis using confocal microscopy came to a similar conclusion , namely that a substantial fraction ( approximately 30% ) of intracellular lc3 and vwf spots had a centroid to centroid distance of less than 1.5 m ( supplemental fig .
finally , using two color confocal live cell imaging , we could capture in real time , the apparent direct interaction of a lc3-gfp autophagosome with a vwf - mcherry positive wpb ( fig .
nonetheless , additional dual - color ( lc3-mcherry and vwf - gfp ) or single color ( lc3-gfp ) tirf imaging suggested these interactions were rare and that under normal circumstances , autophagosomes do not appear to directly fuse with the plasma membrane ( supplemental movie 2&3 ) .
to begin to address whether autophagy modulates vwf biology , we performed lentiviral shrna mediated knockdown of atg7 in human endothelial cells . in atg7 knockdown cells , western blot analysis confirmed a significant reduction in atg7 expression ( fig .
assessment of autophagic flux often relies on the simultaneous measurements of the level of expression of the multidomain , signaling adaptor protein p62/a170/sqstm1 , hereafter referred to simply as p62 , along with the ratio of lc3-ii to lc3-i . a rise in p62 levels and a fall in lc3-ii are consistent with impaired autophagic flux . as expected , knockdown of the essential autophagy gene atg7 had the expected effects on these well characterized markers of autophagic flux ( fig .
we then asked whether knockdown of atg7 altered the stimulated release of vwf . using an elisa - based method of the culture medium
, we detected an increase in vwf secretion following stimulation with histamine or vegf ( fig .
we saw similar effects on vwf secretion using an alternative and independent method for knockdown of atg7 that employed sirna ( supplemental fig .
the reduction in the stimulated release of vwf was further confirmed by direct western blot analysis of the endothelial cell supernatant ( fig .
2c , d ) . a recent report has suggested that disruption of autophagy might alter intracellular calcium signaling .
given the central role of calcium in stimulated exocytosis from the endothelium , we wondered whether such a mechanism might explain our observations . using a calcium sensitive fluorometric probe
, we could not however detect significant differences in ligand - induced calcium transients between control and atg7 knockdown cells ( fig .
similarly , other proximal signaling events such as extracellular regulated kinase ( erk ) activation appeared to be unaffected by atg7 knockdown ( supplemental fig .
4 ) . we next sought to establish whether other genetic or pharmacological inhibitors of autophagy had similar effects .
knockdown of the essential autophagy gene atg5 alone or in combination with atg7 also inhibited vwf release ( fig .
following histamine stimulation , the combined knockdown of atg5 and atg7 appeared slightly more effective than single knockdown of either atg7 or atg5 in blocking vwf secretion ( fig .
incubation of human endothelial cells with chloroquine or bafilomycin , two known inhibitors of autophagic flux , also resulted in a marked inhibition of ligand - stimulated vwf secretion ( fig .
in contrast , knockdown of atg5 or atg7 did not appear to alter ligand - stimulated release of endothelin-1 from endothelial cells ( supplemental fig .
5b ) . in addition , while knockdown of atg6 ( beclin 1 ) was quite effective in reducing atg6 expression ( fig 2i ) , it had only a very modest effect on markers of autophagic flux ( supplemental fig .
we next sought to attempt to get a better understanding of why knockdown of an essential autophagy gene such as atg7 might inhibit the secretion of vwf .
following synthesis , vwf undergoes a series of processing events in the er and golgi that eventually lead to maturation of the protein and incorporation into wpbs . using a quantitative elisa - based method we determined that the total intracellular level of vwf protein was not significantly different between control and atg7 knockdown endothelial cells ( fig .
similar results were observed following knockdown of atg5 or combined atg5 and atg7 knockdown ( supplemental fig .
we next sought to analyze whether differences might exist in the processing , maturation and secretion of vwf .
the processing of vwf involves a furin - dependent proteoltyic cleavage within the trans - golgi network with the pro - vwf precursor distinguishable from mature vwf based on electrophoretic mobility .
we observed that the steady state ratio of pro - vwf to mature vwf was significantly altered in atg7 or atg5 knockdown cell ( fig .
consistent with a defect in vwf processing , we also noted a reduction in the number of mature wpbs observed in atg7 knockdown cells ( fig .
we saw similar reduction in wpbs in endothelial cells with either atg5 knockdown or following pharmacological inhibition of autophagy using chloroquine or bafilomycin ( supplemental fig .
in contrast , knockdown of atg6 did not alter the number of mature wpbs ( supplemental fig .
these results suggest that one explanation for our observation with atg5 or atg7 knockdown is that these strategies , while not altering overall vwf expression , appear to inhibit the processing and maturation of vwf into mature wpbs .
the formation of wpbs is believed to occur at the trans - golgi network ( tgn ) and autophagy is known to be critical for the homeostatic maintenance of both the er and golgi .
furthermore , the ph of wpbs is known to be acidic and this low ph is essential for the shape of the wpbs , as well as vwf tubulation and function .
using previously described methods , we were able to establish that wpbs within atg7 knockdown cells exhibited a higher ph than wpbs found in control cells ( fig . 4a and supplemental fig .
consistent with previous results , when assessed by confocal microscopy , this higher ph in atg7 knockdown cells resulted in rounder and less rod - shaped wpbs ( fig .
this also translated into a statistically significant reduction in the length of wpbs found in atg7 knockdown cells ( fig .
similar observations were evident by electron microscopy where we observed that knockdown of atg7 resulted in wpbs that were shorter , rounder and had less distinct tubulation ( fig .
4e ) . to further assess whether in addition to a processing defect , there was also an effect of atg7 on the secretion of mature wpbs , we employed a previously described system using live cell imaging of the secretion of a vwf - gfp chimeric fusion protein .
expression of this tagged vwf results in assembly of wpbs containing the co - expressed fluorescently labeled vwf protein ( supplemental fig .
consistent with the alterations in processing described above , the number of gfp - positive wpbs was reduced in cells where atg7 was knockdown ( supplemental fig .
9b ) and the wpbs that formed , had an altered , more rounded morphology ( supplemental movies 4 , 5 ) . using this system , in conjunction with total internal reflection fluorescence ( tirf ) microscopy
, we could monitor individual secretion events of gfp - positive wpbs that localized near the plasma membrane ( fig .
our analysis demonstrated that knockdown of atg7 resulted in reduced wpb secretion after histamine stimulation ( fig .
in an effort to extend these observations to an in vivo situation , we conditionally deleted atg7 within endothelial cells by crossing mice harboring atg7 floxed alleles with transgenic mice expressing the previously described ve - cadherin cre recombinase .
parental mice both of which bore the genotype atg7 ; ve - cadherin cre were crossed to generate offspring that were either atg7 ; ve - cadherin cre ( hereafter denoted as atg7 ) or atg7 ; ve - cadherin cre ( hereafter denoted as control ) . given the relatively small contribution of endothelial cells to overall organ mass , we did not observe a significant reduction in atg7 expression in the tissues and organs isolated from atg7 mice ( supplemental fig .
in contrast , primary cultures of mouse endothelial cells isolated from atg7 mice exhibited the expected reduction in atg7 expression ( fig .
the residual expression of atg7 in these cells may represent a combination of incomplete deletion with ve - cadherin cre , as well as potentially some degree of contamination of non - endothelial cells in these primary cultures .
nonetheless , this level of atg7 expression was sufficient to induce a reduction in autophagic flux as evidenced by the lc3-i / ii ratio and p62 levels ( fig .
similar to our observations employing knockdown of atg7 in human endothelial cells , we did however observe a reduction in the number of mature wpbs in endothelial cells derived from atg7 mice ( fig 5b ) . while conditional deletion of essential autophagy genes in tissues such as brain led to marked alterations in tissue homeostasis and early mortality , atg7 mice exhibited no obvious vascular phenotypes .
similarly , atg7 mice exhibited no increase in mortality over the first year of life ( unpublished observations ) .
assessment of vascular density demonstrated that atg7 mice were indistinguishable from control animals ( fig .
5c ) . in addition , while autophagy has been implicated in angiogenesis , we saw no consistent or reproducible abnormalities in atg7 mice when post - natal retinal angiogenesis was assessed ( fig .
, the endothelium and underlying architecture of large vessels were similar in atg7 and control mice ( fig .
we also observed no reduction in the level of vwf expression in atg7 mice either by immunohistochemical ( fig .
we next asked whether atg7 mice exhibited defects in vwf secretion . in control mice , injection of epinephrine led to a significant rise in plasma vwf levels ( fig .
6a , fold increase : 1.5 + / 0.04 , n=27 mice , p<0.001 pre versus post ) .
this increase in vwf in response to subcutaneous epinephrine injection is similar in magnitude to previous reports .
in contrast , atg7 mice failed to respond in a significant fashion to an epinephrine challenge ( fig .
6a , fold increase : 1.17 + / 0.05 , n=13 mice , p = ns pre versus post ) .
this lack of stimulated epinephrine release was also evident in mice where atg5 had been conditionally deleted again using ve - cadherin cre .
similar to atg7 mice , atg5 mice also failed to respond to epinephrine ( fig .
6a , fold increase : 1.13 + / 0.07 , n=9 mice , p = ns pre versus post ) .
neither atg5 mice nor atg7 mice had a significant reduction in basal vwf ( fig .
basal levels of circulating p - selectin levels were not altered between control and atg7 mice ( supplemental fig .
to therefore further assess the role of autophagy in regulating basal vwf levels , we analyzed mice having a complete , whole body knockout of atg7 .
previous studies demonstrated that these total body atg7 knockout mice rarely survive for more than 48 hours after birth .
when we analyzed atg7 mice shortly after delivery , we did note a reduction in plasma vwf when compared to wild type littermates , perhaps reflective of a more complete knockout in this model then can be achieved using conditional approaches ( fig .
we next sought to further characterize the physiological importance of atg7 in vwf processing in vivo .
consistent with the observed in vitro deficit in vwf processing , we noted that on non - reducing gels , plasma vwf analyzed from atg7 mice contained diminished levels of the biologically active , higher molecular weight multimers ( fig .
consistent with this reduction in high molecular weight vwf , coupled with the decrement in stimulated vwf release ( fig .
6a ) , we noted a significant increase in measured bleeding time when control mice were compared to atg7 mice ( fig .
such differences occurred in the absence of any measurable differences in various hemostatic parameters ( supplemental fig .
furthermore , we did not see any obvious abnormalities in platelet morphology or platelet vwf levels that could have potentially contributed to the observed alterations in the measured bleeding time ( supplemental fig .
14 ) . given that one of the many effects for chloroquine is inhibition of autophagy and since this agent is approved for human usage , we next randomized mice to saline or chloroquine ( cq ) injections .
after 9 days , basal plasma vwf levels in cq treated and vehicle treated mice were similar ( supplemental fig .
nonetheless , direct assessment of hemostasis revealed that similar to atg7 mice , pharmacological inhibition of autophagic flux resulted in a significant prolongation of bleeding time ( fig . 6e and supplemental fig .
our data suggests that knockdown or deletion of atg5 or atg7 results in the impaired in vitro and in vivo stimulated secretion of vwf .
interestingly , we and others have demonstrated that conditional deletion of atg7 within the pancreatic -cell leads to impaired secretion of insulin .
similarly , impaired granular exocytosis is observed following disruption of autophagy within mast cells , the intestinal paneth cell and osteoclasts .
thus , our current results , in conjunction with these past observations , suggest that in addition to its known role in intracellular recycling , autophagy plays a fundamental role in the regulated secretion that occurs in certain specialized cell types .
it is currently unclear in the case of mast cells , paneth cells or osteoclasts exactly how autophagy regulates granule secretion .
our data suggests that autophagosomes and wpbs can directly interact and that vwf protein and wpb remnants appear to be found within autophagosomes .
recent evidence suggests that under certain conditions ( e.g. starvation ) , autophagic vacuoles can actually be directly secreted by endothelial cells .
consistent with our microscopy observations , proteomic analysis of these secreted endothelial autophagic vacuoles reveals they contain vwf .
nonetheless , although our data suggests an interaction between autophagosomes and wpbs , this interaction is relatively rare when assessed by electron microscopy , super resolution microscopy or dual - color tirf imaging .
one intriguing possibility is that the observed interaction represents a mechanism to remove damaged or dysfunctional wpbs , in a similar fashion to how mitophagy is thought to remove damaged mitochondria .
how autophagosomes could potentially distinguish a normal from a damaged wpb is unclear , although in this regard , the basis of mitophagic selectivity also remains unresolved .
finally , it is interesting to note that our observations may have important therapeutic implications .
this agent has many effects on cells since it can suppress the acidification of late endosomes , mvbs and lysosomes .
chloroquine is widely given for the treatment and prevention of malaria as well as for treatment of certain classes of autoimmune diseases .
interestingly , one known side effect of the drug is an increased risk for bleeding and the longer acting derivative hydroxychloroquine has been shown to be effective in humans as a prophylaxis against thromboembolic complications following surgery .
our data provides support to the notion that other agents that similarly target autophagic flux might also modulate vwf secretion . as such
, targeted pharmacological inhibition of the autophagic pathway may provide a novel approach to reduce thrombus formation .
 endothelial secretion of von willebrand factor ( vwf ) from intracellular organelles known as weibel - palade bodies ( wpbs ) is required for platelet adhesion to the injured vessel wall . here 
 , we demonstrate that wpbs are in some cases found near or within autophagosomes and that endothelial autophagosomes contain abundant vwf protein . 
 pharmacological inhibitors of autophagy , or knockdown of the essential autophagy genes atg5 or atg7 , inhibits the in vitro secretion of vwf . 
 furthermore , while mice with an endothelial specific deletion of atg7 have normal vessel architecture and capillary density , these animals exhibit impaired epinephrine - stimulated vwf release , reduced levels of high molecular weight vwf multimers and a corresponding elevation of their bleeding times . 
 endothelial deletion of atg5 or pharmacological inhibition of autophagic flux results in a similar in vivo alteration of hemostasis . 
 thus , autophagy regulates endothelial vwf secretion and transient pharmacological inhibition of autophagic flux may be a useful strategy to prevent thrombotic events . 
Introduction
Results
Proximity of endothelial WPBs to autophagosomes
Autophagy regulates 
Autophagy is required for VWF protein maturation
Autophagy regulates in vivo VWF release
Discussion
Supplementary Material