Thrombosis

Storage of Tissue-Type Plasminogen Activator in Weibel-Palade Bodies of Human Endothelial Cells

Corinne Rosnoblet, Ulrich M. Vischer, Robert D. Gerard, Jean-Claude Irminger, Philippe A. Halban, Egbert K. O. Kruithof
https://doi.org/10.1161/01.ATV.19.7.1796
Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1796-1803
Originally published July 1, 1999

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Abstract

Abstract—Tissue-type plasminogen activator (t-PA) is acutely released by endothelial cells. Although its endothelial storage compartment is still not well defined, t-PA release is often accompanied by release of von Willebrand factor (vWf), a protein stored in Weibel-Palade bodies. We investigated, therefore, whether t-PA is stored in these secretory organelles. Under basal culture conditions, a minority of human umbilical vein endothelial cells (HUVEC) exhibited immunofluorescent staining for t-PA, which was observed only in Weibel-Palade bodies. To increase t-PA expression, HUVEC were infected with a t-PA recombinant adenovirus (AdCMVt-PA). Overexpressed t-PA was detected in Weibel-Palade bodies and acutely released together with endogenous vWf by thrombin or calcium ionophore stimulation. In contrast, plasminogen activator inhibitor type 1 and urokinase were not detected in Weibel-Palade bodies after adenovirus-mediated overexpression. Infection of HUVEC with proinsulin recombinant adenovirus resulted in the storage of insulin in Weibel-Palade bodies, indicating that these organelles can also store nonendothelial proteins that show regulated secretion. Infection of AtT-20 pituitary cells, a cell type with regulated secretion, with AdCMVt-PA resulted in the localization of t-PA in adrenocorticotropic hormone–containing granules, indicating that t-PA can be diverted to secretory granules independently of vWf. Coinfection of AtT-20 cells with AdCMVt-PA and proinsulin recombinant adenovirus resulted in the colocalization of t-PA and insulin in the same granules. Taken together, these results suggest that HUVEC have protein sorting mechanisms similar to those of other regulated secretory cells. Although the results did not exclude an alternative storage site for t-PA in HUVEC, they established that t-PA can be stored in Weibel-Palade bodies. This finding may explain the acute coordinate secretion of t-PA and vWf.

  • Received October 16, 1998.
  • Accepted December 22, 1998.

Tissue-type plasminogen activator (t-PA) is a key enzyme for the removal of incipient thrombi in the vascular system, and recombinant t-PA is now widely used for the thrombolytic therapy of myocardial infarction. Endothelial cells (EC) are the main source for plasma t-PA.1 2 3 4 Although a variety of stimuli such as venous occlusion, exercise, or injection of vasoactive substances are known to acutely increase plasma levels of t-PA, the precise mechanisms responsible for this increase are poorly defined. They may involve an acute release of t-PA from EC, variations in the rate of production of t-PA by EC, or changes in the clearance rate.5 6

Several in vivo and ex vivo studies give clear evidence for the occurrence of acute release of t-PA. Experimental induction of disseminated intravascular coagulation in chimpanzees or baboons results in a 50-fold increase in t-PA plasma levels within a few minutes.7 8 The rapidity and the magnitude of the increase in the t-PA concentration, as well as the rapid return to normal levels, are consistent only with a massive release of t-PA from storage pools. The findings that injection of vasoactive agents such as thrombin or calcium ionophore leads to an acute increase of t-PA in isolated vascular systems are also consistent with an endothelial t-PA storage pool.9 10 11

Acute release of t-PA in vivo or in vitro is often accompanied by the release of von Willebrand factor (vWf), an adhesive glycoprotein involved in primary hemostasis. Plasma vWf originates mainly from EC, which store the protein in specific rod-like secretory granules known as Weibel-Palade bodies.12 Concomitant release of t-PA and vWf has been observed in vivo after injection of 1-desamino-8-d-arginine vasopressin (DDAVP)13 or after experimental disseminated intravascular coagulation,7 8 14 ex vivo in isolated rat hindlegs10 11 and in vitro in human umbilical vein endothelial cells (HUVEC).15 Furthermore, patients with severe von Willebrand’s disease are deficient in acute t-PA release.13 16 17 Taken together, these data suggest that t-PA and vWf are released either from the same storage granules or from distinct granules responsive to common stimuli. Support for the existence of distinct granules is provided by studies that have observed a discrepancy in the release of t-PA and vWf. In the rat hindleg model, adenosine diphosphate stimulated the release of t-PA but not of vWf.18 In cultured HUVEC, the thrombin-induced release of t-PA and vWf revealed subtle differences in calcium requirement and pertussis toxin sensitivity.19 Furthermore, cell fractionation and immunofluorescence suggest that t-PA and vWf are stored in distinct granules.20 However, the heterogeneity in t-PA expression by EC3 does not allow firm conclusions to be drawn as to whether t-PA and vWf have distinct cellular localization(s).

The key role of t-PA in protecting the vascular system from thrombotic occlusions makes it important to better understand the mechanisms of t-PA storage and release. The present immunofluorescence study was undertaken to investigate the intracellular sorting of t-PA in HUVEC. Because endogenous t-PA expression is very low in HUVEC, we enhanced t-PA expression by adenovirus-mediated t-PA gene transfer. This approach was chosen because primary human EC are known to be resistant to other transfection methods. We also investigated the localization of t-PA in AtT-20 cells, a model cell system that is used to study the sorting of proteins into the constitutive or the regulated secretory pathway.21 A further aim was to determine whether Weibel-Palade bodies can store non-EC proteins known to follow the regulated pathway of secretion. Our observations indicate that both t-PA and other regulated proteins can be sorted to Weibel-Palade bodies.

Methods

Materials

Unless stated otherwise, all biochemical reagents and chemicals used in this study were from Sigma or Fluka and of the highest grade available. The following antibodies were used: monoclonal anti-human t-PA antibody PAM-3 (directed against the protease domain) was from Biopool; monoclonal anti-human t-PA antibody ESP-4 (kringle 2 domain) was from American Diagnostica; monoclonal anti-human u-PA antibodies 7C7, 2L3, and 4D1E822 23 were a gift from Dr Paul de Clerck (Institute for Pharmacological Sciences, University of Leuvan, Leuvan, Belgium); rabbit anti-human vWf antibodies were from Dako and immunopurified on immobilized vWf before use; rabbit anti-human adrenocorticotropic hormone (ACTH) antibodies and monoclonal anti-human insulin (used for insulin and vWf double label experiments) and anti-rat Golgi 58K protein antibodies were from Sigma; monoclonal anti-human Lamp-2 H4B4 used as a lysosomal marker was from the Developmental Studies Hybridoma Bank; monoclonal 6C4 antibody used as a marker for late endosomes24 was a gift from Dr Toshihide Kobayashi and Jean Gruenberg, (University of Geneva, Geneva, Switzerland); monoclonal anti-human PAI-1 antibody was from Monozyme; guinea pig anti-human insulin antibodies (used for insulin and t-PA double label experiments) were a gift from Dr Paolo Meda, (University of Geneva Medical School, Geneva, Switzerland); rhodamine-conjugated goat anti-mouse antibodies were from Jackson (these antibodies were passed over immobilized rabbit antibodies to remove cross-reactivity); fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibodies were from Organon Teknika Cappel; FITC-conjugated sheep anti-guinea pig antibodies were from Biosys.

Cell Culture

Human umbilical vein endothelial cells (HUVEC) were isolated by collagenase digestion25 and grown at 37°C in a humidified atmosphere containing 5% CO2. Briefly, the umbilical vein was washed with Krebs-Ringer–bicarbonate buffer (KRBH, 120 mmol/L NaCl, 4.75 mmol/L KCl, 1.2 mmol/L KH2PO4, 0.6 mmol/L MgSO4, 1.2 mmol/L CaCl2, 25 mmol/L NaHCO3, and 25 mmol/L HEPES, pH 7.4) and incubated for 10 minutes with 1 mg/mL collagenase (CLS, type 1, Worthington Biochemical). Cells were collected by flushing the vein with 50 mL RPMI 1640 supplemented with 10% FBS (Life Technologies) and then grown in RPMI 1640 containing 90 μg/mL heparin (Boehringer Ingelheim), 15 μg/mL endothelial cell growth supplement (Upstate Biotechnology), 10 mmol/L HEPES, 100 U/mL penicillin, and 100 μg/mL streptomycin and supplemented with 10% FBS. Cells were passaged by trypsin/EDTA (Biochrom KG, Berlin, Germany) treatment at a split ratio of 1/3 and used at passage 1 or 2. Tissue culture dishes (Falcon Becton Dickinson Labware, Lincoln Parc, NJ), 24-well plates (Costar, Cambridge, MA) and glass coverslips were coated with 0.1% gelatin. Cultured cells were identified as endothelial by the presence of vWf antigen and labeling of Weibel-Palade bodies using indirect immunofluorescence.

AtT-20 mouse pituitary tumor-derived cells were grown in Dulbecco’s modified Eagle’s medium containing 4,5 g/L glucose (DMEM, Life Technologies) and supplemented with 10% FBS. AtT-20 cells were passaged by trypsin/EDTA treatment at a split ratio of 1:10.

Cell Infection With Recombinant Adenoviruses

The following recombinant adenoviruses have been previously described: t-PA recombinant adenovirus (AdCMVt-PA),26 PAI-1 recombinant adenovirus (AdCMVPAI-1),27 and proinsulin recombinant adenovirus.28 Recombinant u-PA adenovirus was prepared as follows: a plasmid containing the human pro-urokinase cDNA, pu-PA, was made available by William Bennett of Genentech Inc, San Francisco, CA. The uPA cDNA fragment from HindIII to SspI, containing 75 bp of 5′ untranslated sequence and 600 bp of 3′ untranslated sequence was inserted into the adenovirus construction plasmid, pACskCMV2. This plasmid contains the left end of adenovirus type 5 including the origin of replication and packaging sequences (nt no. 1-454), an SV40 ori/hGH terminator, a polylinker to facilitate cDNA insertion, the CMV promoter, and flanking adenovirus sequences (nt no. 3334-5779) serving as the target for homologous recombination. It was kindly provided by Joseph Alcorn of UT Southwestern Medical Center (Dallas, Texas). The resulting plasmid, pACskCMVu-PA, was cotransfected with ClaI digested Ad5dL309 into 293 cells using standard methods29 to yield AdCMVu-PA. The recombinant adenoviruses were propagated on a monolayer of 293 cells (CRL 1573, ATCC) and titrated by plaque assay.29

HUVEC or AtT-20 cells were grown to 50% confluency in 24-well plates or on 12-mm diameter glass coverslips. They were then infected for 1 hour at 37°C in 300 μL RPMI 1640/10% FBS or DMEM/10% FBS with t-PA recombinant adenovirus (AdCMVt-PA), with u-PA recombinant adenovirus (AdCMVu-PA), with PAI-1 recombinant adenovirus (AdCMVPAI-1), or with proinsulin recombinant adenovirus at a titer of 108 pfu/mL. These virus titers were chosen to obtain positive staining for the recombinant proteins in 30% to 50% of the cells. After infection, the cells were washed and incubated for 48 hours at 37°C before fixation or secretion studies.

Indirect Immunofluorescence Staining

HUVEC or AtT-20 cells grown on glass coverslips were fixed for 20 minutes in 4% freshly depolymerized paraformaldehyde in phosphate buffered saline (PBS: 10 mmol/L NaH2PO4/Na2HPO4, 136 mmol/L NaCl, and 4 mmol/L KCl, pH 7.4), washed with PBS, quenched for 20 minutes with 0.27% NH4Cl/0.38% glycine in PBS, pH 7.4 and permeabilized for 30 minutes with 0.1% saponin/0.1% BSA in PBS, pH 7.4. For insulin staining, HUVEC were fixed for 30 minutes in Bouin solution (75% saturated picric acid solution, 7.4% formaldehyde, and 5% acetic acid) and permeabilized by progressive 3 minutes ethanol dehydration (30%, 50%, 70%, and 90% ethanol in water) and rehydration (70%, 50%, 30%, and 0% ethanol in water).

For single label analyses, cells were incubated sequentially with the primary antibody and the fluorescent second antibody. For double label analyses, cells were incubated sequentially with a mixture of primary antibodies and a mixture of fluorescent second antibodies. Antibodies were diluted in 0.1% BSA in PBS, pH 7.4. Monoclonal antibodies for primary staining were used at 10 μg/mL for detection of t-PA, u-PA, and PAI-1, 5 μg/mL for late endosomal membranes and at dilutions of 1/5000 for lysosomal membranes, 1/50 for insulin, and 1/100 for Golgi; polyclonal antibodies for primary staining were used at 2 μg/mL for detection of vWf, 1/100 for ACTH, and 1/200 for insulin. Rhodamine-conjugated goat anti-mouse and FITC-conjugated goat anti-rabbit antibodies were used at a dilution 1/100. FITC-conjugated sheep anti-guinea pig antibodies was used at a dilution of 1/400. All incubations were performed for 1 hour at room temperature. After washing, coverslips were mounted in polyvinyl alcohol. Pictures were taken with a Zeiss Axiophot (Carl Zeiss) photomicroscope equipped with epi-illumination and specific filters for fluorescein and rhodamine using a plan apochromate×63/1.40 objective and Tmax black and white film (Eastman Kodak).

Negative control experiments were performed by omitting the primary antibodies or by using an irrelevant primary antibody of the same species or IgG subclass (for monoclonal antibodies). For double label analyses, we verified that the FITC fluorescence gave no signal in the rhodamine channel and conversely. We also confirmed the absence of cross-reactivity between mouse antibodies and FITC-conjugated goat anti-rabbit antibodies as well as between rabbit antibodies and rhodamine-conjugated goat anti-mouse antibodies.

Secretion Studies

Forty-eight hours after infection with recombinant AdCMVt-PA, HUVEC grown on 24-well plates were washed 3× with KRBH/BSA 0.1%, pH 7.4, preincubated for 30 minutes in 1 mL KRBH/BSA, and then incubated again for 30 minutes in 0.5 mL fresh KRBH/BSA alone, KRBH/BSA containing 1 NIH U/mL of human thrombin (Sigma), or KRBH/BSA containing 2 μmol/L calcium ionophore A23187 (Sigma). The cell supernatants were centrifuged and kept at −20°C until determination of t-PA and vWf antigen concentrations.

Antigen Determinations

Human t-PA antigen was determined by ELISA (Imulyse t-PA, Biopool). vWf was measured by ELISA as described elsewhere.14 Data are presented as means±SEM. The significance of differences was calculated using the Student t test.

Results

Localization of t-PA in Endothelial Cells

To study the intracellular localization of t-PA in HUVEC grown under basal culture conditions, we performed double label indirect immunofluorescence analysis for t-PA and vWf. All data shown in this article concerning the immunolocalization of t-PA were obtained using monoclonal antibody ESP-4 (similar data were obtained using monoclonal antibody PAM-3). A minority of the cells (<2%) were weakly but distinctly stained by anti–t-PA antibodies. These antibodies stained elongated organelles spread throughout the cytoplasm and positive for vWf, which identifies them as Weibel-Palade bodies (Figure 1⇓). This pattern of staining was observed in HUVEC originating from different umbilical cords and using either monoclonal antibody. The presence of cells positive for vWf but negative for t-PA provided an intra-image negative control that ruled out the possibility of interference from the bright vWf/FITC signal into the rhodamine channel and the possibility of cross-reactivity between the rabbit anti-vWf and the rhodamine-conjugated goat anti-mouse antibodies.

Figure 1.
Figure 1.

Localization of tissue-type plasminogen activator (t-PA) and von Willebrand factor (vWf) in human umbilical vein endothelial cells cultured under basal conditions. Cells were processed for double-label indirect immunofluorescence. The elongated structures stained with anti-t-PA antibody (upper panel) were identified as Weibel-Palade bodies by co-staining for vWf (lower panel). Arrows point to Weibel-Palade bodies that are positive for both t-PA and vWf. Note that only 1 cell in the field expressed t-PA, whereas the other cells were negative for t-PA. The same field is shown in the upper and lower panel. Scale bar=20 μm.

We compared the staining pattern for t-PA with that for plasminogen activator inhibitor-1 (PAI-1), the principal inhibitor of t-PA that is produced in high amounts by HUVEC and is constitutively secreted by these cells. Strong PAI-1 staining was observed in all cells (Figure 2⇓, left panel). It was mainly restricted to a distinct perinuclear region that was similar to that labeled with an antibody against protein 58 K, a membrane protein of the Golgi apparatus (data not shown). No staining of elongated organelles was observed using anti–PAI-1 antibodies.

Figure 2.
Figure 2.

Localization of endogenous plasminogen activator inhibitor type 1 (PAI-1), endosomes and lysosomes in human umbilical vein endothelial cells. Cells were processed for single-label indirect immunofluorescence using monoclonal antibodies specific for PAI-1 (left panel), for late endosomal (middle panel) or lysosomal membranes (right panel). PAI-1 staining was mainly restricted to the Golgi apparatus. The antibodies specific for late endosomes and lysosomes stained round vesicles spread over the perinuclear area of the cells which were clearly different from Weibel-Palade bodies. Scale bar=20 μm.

Many cell types are able to internalize and degrade t-PA. To test whether endocytosis of t-PA by some of the cells could have contributed to the immunofluorescence signal for t-PA, we compared the morphology of late endosomes (labeled with antibody 6C4, Figure 2⇑, middle panel) and lysosomes (labeled with anti–Lamp-2 antibody, Figure 2⇑, right panel) with that of the t-PA–labeled organelles. The morphology of the numerous endocytotic/lysosomal structures stained in all cells by these antibodies was clearly different from that of the t-PA–positive organelles.

Localization of Overexpressed t-PA in Endothelial Cells

The results presented above for t-PA can be interpreted in 2 ways: 1) there is heterogeneity in the intracellular sorting of t-PA with only a minority of EC able to target t-PA to Weibel-Palade bodies, or 2) there is a heterogeneity in t-PA expression levels, with only a minority of cells expressing sufficient t-PA to be detected by our technique. To increase t-PA expression in HUVEC we used a recombinant adenovirus vector expressing human t-PA (AdCMVt-PA). A multiplicity of infection was chosen such that approximately 30% to 50% of the cells over-expressed t-PA to preserve some noninfected cells as negative controls in the immediate vicinity of cells that overexpressed t-PA. Double label immunofluorescence experiments for t-PA and vWf were then performed. In infected cells, the staining pattern for overexpressed t-PA, namely elongated organelles and a bright perinuclear pattern, was identical to that for vWf (Figure 3⇓). No staining for t-PA was detected in areas devoid of vWf. The perinuclear staining for vWf was identical to that previously described30 and similar to the pattern seen in studies using antibodies specific for the rough endoplasmic reticulum.31 There were no endosome or lysosome-type structures labeled with anti–t-PA antibody in AdCMVt-PA–infected HUVEC. This suggested that endocytosis of overexpressed t-PA did not contribute to the immunofluorescence signal. The presence of cells positive for vWf but negative for t-PA again provided the intra-image negative control. At higher AdCMVt-PA titers, almost all HUVEC expressed high amounts of t-PA in Weibel-Palade bodies (data not shown).

Figure 3.
Figure 3.

Localization of t-PA and vWf in human umbilical vein endothelial cells after adenovirus-mediated t-PA gene transfer. Forty-eight hours after infection, cells were processed for double-label indirect immunofluorescence analysis. Staining for overexpressed t-PA (upper panel) colocalized with staining for vWf (lower panel) both in the perinuclear area and in Weibel-Palade bodies. Arrows point to Weibel-Palade bodies that are positive for both t-PA and vWf. Note that viral titers were chosen such that some cells were not infected and could serve as negative controls. The same field is shown in the upper and lower panel. Scale bar=20 μm.

To verify whether overexpressed t-PA follows the regulated pathway of secretion, we measured t-PA release in AdCMVt-PA infected HUVEC after stimulation with known secretagogues. Treatment with thrombin or the calcium ionophore A23187 led to an acute increase in release of t-PA (3- to 4-fold) and of vWf (6-fold) as compared with untreated HUVEC (Table 1⇓).

View this table:
Table 1.

Acute Release of Overexpressed Tissue-Type Plasminogen Activator (t-PA) and von Willebrand Factor (vWf) by Thrombin and Calcium Ionophore A23187 in Human Umbilical Vein Endothelial Cells

Localization of Overexpressed PAI-1 and u-PA in Endothelial Cells

To analyze whether adenovirus-mediated overexpression of a protein would artifactually result in a detectable signal in Weibel-Palade bodies, we infected HUVEC with recombinant adenoviruses that encode PAI-1 or u-PA under control of the same promoter (cytomegalovirus [CMV]) as that used for the t-PA recombinant adenovirus. We then performed double immunofluorescence analysis for vWf and either PAI-1 or u-PA.

The staining pattern for overexpressed PAI-1 was similar to that for endogenous PAI-1 and markedly different from that for vWf (Figure 4⇓). We observed strong staining for PAI-1 in the perinuclear region corresponding the Golgi apparatus and diffuse staining throughout the cell. As observed in uninfected HUVEC, the anti–PAI-1 antibodies did not label Weibel-Palade bodies.

Figure 4.
Figure 4.

Localization of PAI-1 and vWf in human umbilical vein endothelial cells after adenovirus-mediated PAI-1 gene transfer. Forty-eight hours after infection, cells were processed for double-label indirect immunofluorescence analysis. Staining for overexpressed PAI-1 (upper panel) was particularly strong in the Golgi apparatus, whereas no staining was detected in Weibel-Palade bodies (positive for vWf; lower panel). The arrows point to the Golgi apparatus of cells that are positive for both PAI-1 and vWf. The same field is shown in the upper and lower panel. Scale bar=20 μm.

HUVEC cultured under basal conditions exhibited a weak immunofluorescence signal for u-PA (data not shown). Infection of these cells with AdCMVu-PA led to a marked increase in u-PA expression. The staining pattern for overexpressed u-PA, corresponding to the Golgi apparatus and to focal contact points was distinct from that for vWf (Figure 5⇓). The anti–u-PA antibodies did not label Weibel-Palade bodies.

Figure 5.
Figure 5.

Localization of urokinase (u-PA) and vWf in human umbilical vein endothelial cells after adenovirus-mediated u-PA gene transfer. Forty-eight hours after infection, cells were processed for double-label indirect immunofluorescence analysis. Staining for overexpressed u-PA (upper panel) was particularly strong in the Golgi apparatus and at focal contact points, whereas no staining was detected in Weibel-Palade bodies (positive for vWf; lower panel). The same field is shown in the upper and lower panel. Scale bar=20 μm.

Expression of t-PA in AtT-20 Cells

To test whether the sorting of t-PA into regulated secretory granules requires the presence of vWf, we infected AtT-20 cells with AdCMVt-PA. Double label immunofluorescence analysis showed a strong signal for t-PA in approximately 50% of the cells, whereas no t-PA was detected in uninfected cells. t-PA was localized in round storage granules containing ACTH (Figure 6⇓), an endogenous protein with regulated secretion. Staining was particularly strong at the tips of the extended processes of these cells. The presence of cells positive for ACTH but negative for t-PA provided an intra-image negative control.

Figure 6.
Figure 6.

Localization of t-PA and adrenocorticotropic hormone (ACTH) in AtT-20 cells after adenovirus-mediated t-PA gene transfer. Forty-eight hours after infection the cells were processed for double label indirect immunofluorescence analysis. Staining for t-PA (upper panel) colocalized with staining for endogenous ACTH (lower panel) in granules that were primarily clustered at the tips of the extended processes of the cells. Arrows point to granules that are positive for both t-PA and ACTH. Note that viral titers were chosen such that some cells were not infected and could serve as negative controls. The same field is shown in the upper and lower panel. Scale bar=20 μm.

To test whether AtT-20 cells can be infected by more than 1 recombinant adenovirus and then express and store different proteins in the same organelle, these cells were concomitantly infected with both AdCMVt-PA and proinsulin recombinant adenoviruses. The AtT-20 cells were then analyzed by double label immunofluorescence for t-PA and insulin (Figure 7⇓). AtT-20 cells simultaneously infected by both t-PA and proinsulin recombinant adenoviruses efficiently expressed these proteins. Furthermore, staining for t-PA and insulin was observed within the same granules.

Figure 7.
Figure 7.

Localization of t-PA and insulin in AtT-20 cells after adenovirus-mediated t-PA and proinsulin gene transfer. Forty-eight hour after infection, cells were processed for double-label indirect immunofluorescence analysis. Staining for t-PA (upper panel) colocalized with staining for insulin (lower panel) in round granules. Arrows point to granules that are positive for both t-PA and insulin. The same field is shown in the upper and lower panels. Scale bar=20 μm.

Expression of Insulin in Endothelial Cells

To determine whether Weibel-Palade bodies are able to store non-EC proteins with known regulated secretion, we infected HUVEC with proinsulin-recombinant adenovirus. Double-label immunofluorescence analysis showed that adenovirus-mediated proinsulin gene transfer in HUVEC resulted in the targeting of insulin in Weibel-Palade bodies (Figure 8⇓).

Figure 8.
Figure 8.

Localization of insulin and vWf in human umbilical vein endothelial cells after adenovirus-mediated proinsulin gene transfer. Forty-eight hours after infection, cells were processed for double-label indirect immunofluorescence analysis. Staining for insulin (upper panel) colocalized with staining vWf (lower panel) in Weibel-Palade bodies. Arrows point to Weibel-Palade bodies that are positive for both insulin and vWf. The same field is shown in the upper and in the lower panel. Scale bar=20 μm.

Discussion

The regulated release of t-PA enables EC to acutely generate a strong, local fibrinolytic response. Indeed, in chimpanzees and baboons a thrombogenic stimulus leads to a massive increase in plasma concentrations of t-PA resulting in the rapid appearance of fibrin degradation products.7 8 From these results it appears that the regulated release of t-PA by EC is a major defense mechanism against thrombosis. However, other proteins stored by EC have a prothrombotic action. Thus, exocytosis from Weibel-Palade bodies leads to exposure of P-selectin at the cell surface, which stimulates leukocyte adhesion and release of vWf that contributes to platelet aggregation.12 Many factors—such as thrombin, fibrin, bradykinin, histamine, platelet activating factor, DDAVP, and the calcium ionophore A23187—are known to induce the release of vWf from EC in vitro and in vivo.5–11,14,15,19,32 A majority of these factors also induce the acute release of t-PA. These observations suggest that t-PA and vWf are stored either in the same granules or in distinct granules that respond to similar stimuli.

Our data provide strong evidence that EC are able to sort t-PA into Weibel-Palade bodies. In HUVEC cultured under basal conditions, only a small percentage of the cells had sufficient t-PA to be detected by immunofluorescence analysis. In these t-PA positive cells, t-PA was detected in Weibel-Palade bodies. To increase the expression of t-PA in HUVEC, we used an adenoviral vector containing the t-PA cDNA under control of the strong CMV promoter (AdCMVt-PA).26 Thrombin and the calcium ionophore A23187 induced the parallel acute release of vWf and of overexpressed t-PA, indicating that both proteins were released from preformed storage compartments. The secretion data obtained in our model of adenovirus-mediated t-PA overexpression are in agreement with previous findings in HUVEC cultivated under basal conditions and using the same secretagogues in which acute t-PA release could be observed using an ultra-sensitive t-PA antigen assay.15 19 By immunofluorescence analysis, we observed a clear localization of overexpressed t-PA in the Weibel-Palade bodies of AdCMVt-PA infected cells. At high titers of AdCMVt-PA, almost all HUVEC expressed t-PA in Weibel-Palade bodies. This showed that the ability to store t-PA in Weibel-Palade bodies is not restricted to a small subpopulation of the cells. Rather, it suggests that the presence of a t-PA signal in the Weibel-Palade bodies of some HUVEC cultured under basal conditions is due to differences in t-PA expression levels among cells.

The targeting of overexpressed t-PA to Weibel-Palade bodies was unlikely to be an artifact of overexpression because 1) a qualitatively similar staining pattern was observed in HUVEC cultured under basal conditions; 2) we observed no staining of Weibel-Palade bodies using anti–PAI-1 antibody, even though PAI-1 is endogenously expressed by HUVEC at much higher levels than t-PA after AdCMVt-PA treatment; and 3) no PAI-1 or u-PA could be detected in Weibel-Palade bodies after adenovirus-mediated overexpression of these proteins. This implies that protein over-expression by itself is not sufficient to result in protein sorting into Weibel-Palade bodies.

Our finding on the localization of t-PA in Weibel-Palade bodies is in contradiction to several recent studies that have suggested distinct storage compartments for t-PA and vWf. In 1 study using butyrate to increase t-PA expression, subtle differences in calcium requirement and pertussis toxin sensitivity were observed between t-PA and vWf release.19 However, in our hands, treatment of HUVEC with butyrate increased t-PA expression in only a fraction of the cells devoid of vWf (Rosnoblet et al, unpublished observations, 1996), and it cannot be excluded that butyrate has modified the secretory response of these cells. Another study reported that the density of t-PA storage granules was the same as that of Weibel-Palade bodies when analyzed in Nycodenz gradients but different in sucrose gradients.20 Furthermore, an immunoelectron microscopy analysis of HUVEC and of murine capillary EC detected t-PA in small electron dense granules distinct from Weibel-Palade bodies.20 To what extent the heterogeneity of t-PA expression in EC in culture (this study) and in vivo33 34 may explain these differences remains to be established. Also, our data do not rule out the possibility that t-PA is stored in 2 compartments, ie, small dense granules and Weibel-Palade bodies. Storage in Weibel-Palade bodies may be more prominent in organs/sites where t-PA is expressed in high amounts or at times of increased synthesis such as inflammation.8

The sorting of t-PA into Weibel-Palade bodies may be an intrinsic property of t-PA or be due to a noncovalent interaction with vWf. The relevance of this question is strengthened by the recent observation that transfected vWf can function as a targeting chaperone that diverts transfected coagulation factor VIII from a constitutive to a regulated secretory pathway in AtT-20 cells.35 Sorting of endogenous or transfected t-PA to the secretory granules of PC12 cells36 37 suggests that t-PA storage can occur independently of an interaction with vWf. In the present study, we expressed t-PA in the ACTH-producing AtT-20 cells, a model cell system that expresses neither t-PA nor vWf and that has been widely used to study the targeting of exogenous proteins toward constitutive or regulated secretory pathways. t-PA was found in round granules in colocalization with endogenous ACTH. This confirms that targeting of t-PA to the regulated pathway is not dependent on an interaction with vWf and strongly suggests that t-PA contains its own cis-acting sorting signal.

Coinfection of AtT-20 cells with adenoviral vectors expressing t-PA and proinsulin resulted in storage of the 2 proteins within the same granules. Although ACTH, t-PA, and proinsulin share no obvious structural homology, they probably each contain a signal that is appropriately recognized by the sorting machinery of the pituitary cells. The ability of different adenoviral vectors to infect the same cell is in keeping with our previous findings38 and suggests possibilities to study the elements that play a critical role in the process of storage and secretion. Indeed, complementation experiments in model cell systems may be achieved more easily by using simultaneous infection with different recombinant adenoviruses than by performing successive stable transfection experiments.

Weibel-Palade bodies are regulated secretory granules not only for vWf but also for EC proteins like vasoactive peptides39 and t-PA (this report). In addition, transfection of EC with coagulation factor VIII has demonstrated that factor VIII can be stored in Weibel-Palade bodies in a vWf-dependent fashion.35 The ability of Weibel-Palade bodies to function as storage granules for non-EC proteins with regulated secretion is illustrated by our finding that insulin is found in these organelles after adenovirus-mediated proinsulin gene transfer. This suggests that the mechanisms of sorting of proteins to Weibel-Palade bodies are not fundamentally different from those in other cells possessing regulated secretory mechanisms.

In conclusion, the present study demonstrates that EC are able to target t-PA to Weibel-Palade bodies. This finding may explain the acute coordinated secretion of t-PA and vWf observed in numerous in vitro and in vivo studies. The storage of t-PA within secretory granules after adenovirus-mediated t-PA gene transfer in AtT-20 cells that do not express vWf suggests that t-PA contains its own sorting signal. Adenovirus-mediated expression of insulin in HUVEC and storage of this protein in Weibel-Palade bodies suggests similar storage mechanisms for endothelial and neuroendocrine cells.

Acknowledgments

We gratefully thank Katherine Meyer and Nicole Aebischer for their technical help. We are grateful to Dr Marie-Luce Bochaton-Piallat and Prof Giulio Gabbiani for assistance with the immunofluorescence technique and for access to the fluorescent microscope. This work was supported by grants from the Swiss National Fund for Scientific Research to E.K.O.K. (31.050645.97), to U.M.V. (32.052667.97), and to P.A.H. (31.50811.97), from the Office Fédéral de l’Education et de la Science (95.0155/CT96-1438 and 95.0654/CT96-0023) and from the Foundation for Research on Atherosclerosis and Thrombosis. E.K.O.K. is a recipient of a personal grant from the Association-Vaud-Genève and U.M.V. is a recipient of a SCORE subsidy from the Swiss National Fund for Scientific Research.

References

  1. ↵
    Kristensen P, Larsson LI, Nielsen LS, Grøndahl-Hansen J, Andreasen PA, Danø K. Human endothelial cells contain one type of plasminogen activator. FEBS Lett. 1984;168:33–37.
    OpenUrlCrossRefPubMed
  2. ↵
    Larsson A, Astedt B. Immunohistochemical localisation of tissue plasminogen activator and urokinase in the vessel wall. J Clin Pathol. 1985;38:140–145.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    Levin EG, del Zoppo GJ. Localization of tissue plasminogen activator in the endothelium of a limited number of vessels. Am J Pathol. 1994;144:855–861.
    OpenUrlPubMed
  4. ↵
    Lupu F, Heim DA, Bachmann F, Hurni M, Kakkar VV, Kruithof EKO. Plasminogen activator expression in human atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 1995;15:1444–1455.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Emeis JJ. Regulation of the acute release of tissue-type plasminogen activator from the endothelium by coagulation activation products. Ann N Y Acad Sci. 1992;667:249–258.
    OpenUrlCrossRefPubMed
  6. ↵
    Chandler WL, Levy WC, Stratton JR. The circulatory regulation of tPA and uPA secretion, clearance and inhibition during exercise and during the infusion of isoproterenol and phenylephrine. Circulation. 1995;92:2984–2994.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Giles AR, Nesheim ME, Herring SW, Hoogendoorn H, Stump DC, Heldebrant CM. The fibrinolytic potential of the normal primate following the generation of thrombin in vivo. Thromb Haemost. 1990;63:476–481.
    OpenUrlPubMed
  8. ↵
    Kruithof EKO, Mestries JC, Gascon MP, Ythier A. The coagulation and fibrinolytic responses of baboons after in vivo thrombin generation: effect of interleukin 6. Thromb Haemost. 1997;77:905–910.
    OpenUrlPubMed
  9. ↵
    Klöcking HP, Markwardt F. Pharmacological stimulation of t-PA release. Pharmazie. 1994;49:227–230.
    OpenUrlPubMed
  10. ↵
    Tranquille N, Emeis JJ. The simultaneous acute release of tissue-type plasminogen activator and von Willebrand factor in the perfused rat hindleg region. Thromb Haemost. 1990;63:454–458.
    OpenUrlPubMed
  11. ↵
    Tranquille N, Emeis JJ. On the role of calcium in the acute release of tissue-type plasminogen activator and von Willebrand factor from the rat perfused hindleg region. Thromb Haemost. 1991;66:479–483.
    OpenUrlPubMed
  12. ↵
    Wagner DD. The Weibel Palade body: the storage granule for von Willebrand factor and P-selectin. Thromb Haemost. 1993;70:105–110.
    OpenUrlPubMed
  13. ↵
    Nilsson IM, Vilhardt H, Holmberg L, Astedt B. Association between factor VIII related antigen and plasminogen activator. Acta Med Scand. 1982;211:105–112.
    OpenUrlPubMed
  14. ↵
    Vischer UM, Ingerslev J, Wollheim CB, Mestries JC, Tsakiris DA, Haefeli WE, Kruithof EKO. Acute von Willebrand factor secretion from the endothelium in vivo: assessment through plasma propeptide (vWf:AgII) levels. Thromb Haemost. 1997;77:387–393.
    OpenUrlPubMed
  15. ↵
    van den Eijnden-Schrauwen Y, KooistraT, de Vries REM, Emeis JJ. Studies on the acute release of tissue-type plasminogen activator from human endothelial cells in vitro and in rats in vivo: evidence for a dynamic storage pool. Blood. 1995;85:3510–3517.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Casonato A, Sartori MT, Pontara E, Zucchetto A, Dannhauser D, Patrassi G, Girolami A. Impaired release of tissue plasminogen activator (t-PA) following DDAVP infusion in von Willebrand’s disease with low platelet von Willebrand factor content. Blood Coagul Fibrinolysis. 1992;3:149–153.
    OpenUrlPubMed
  17. ↵
    Wieczorek I, Ludlam CA, MacGregor IR. Study of the endothelial t-PA and vWf in normal subjects and in von Willebrand’s disease. Blood Coagul Fibrinolysis. 1994;5:329–334.
    OpenUrlPubMed
  18. ↵
    Smalley DM, Fitzgerald JE, O’Rourke J. Adenosine diphosphate stimulates the endothelial release of tissue-type plasminogen activator but not von Willebrand factor from isolated-perfused rat hind limbs. Thromb Haemost. 1993;70:1043–1048.
    OpenUrlPubMed
  19. ↵
    van den Eijnden-Schrauwen Y, Atsma DE, Lupu F, de Vries REM, Kooistra T, Emeis JJ. Involvement of calcium and G proteins in the acute release of tissue-type plasminogen activator and von Willebrand factor from cultured human endothelial cells. Arterioscler Thromb Vasc Biol. 1997;17:2177–2187.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    Emeis JJ, van den Eijnden-Schrauwen Y, van den Hoogen CM, de Priester W, Wesmuckett A, Lupu F. An endothelial storage granule for tissue-type plasminogen activator. J Cell Biol. 1997;139:245–256.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Moore HPH, Kelly RB. Secretory protein targeting in a pituitary cell line: differential transport of foreign secretory proteins to distinct secretory pathways. J Cell Biol. 1985;101:1773–1781.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    DeClerck PJ, Lijnen R, Verstreken M, Moreau H, Collen D. A monoclonal antibody specific for two-chain urokinase-type plasminogen activator. Application to the study of clot lysis with single-chain urokinase-type plasminogen activator in plasma. Blood. 1990;75:1794–1800.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    DeClerck PJ, Van Keer L, Verstreken M, Collen D. An enzyme-linked immunosorbent assay for urokinase-type plasminogen activator (u-PA) and mutants and chimeras containing the serine protease domain of u-PA. Thromb Haemostas. 1992;67:95–100.
    OpenUrlPubMed
  24. ↵
    Kobayashi T, Stang E, Fang KS, de Moerloose P, Parton RG, Gruenberg J. A lipid associated with the antiphospholipid syndrome regulates endosome structure and function. Nature. 1998;392:193–197.
    OpenUrlCrossRefPubMed
  25. ↵
    Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest. 1973;52:2745–2756.
  26. ↵
    Carmeliet P, Stassen JM, van Vlaenderen I, Meidell RS, Collen D, Gerard RD. Adenovirus-mediated transfer of tissue-type plasminogen activator augments thrombolysis in tissue-type plasminogen activator-deficient and plasminogen activator inhibitor-1-overexpressing mice. Blood. 1997;90:1527–1534.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    Carmeliet P, Moons L, Lijnen R, Janssens S, Lupu F, Collen D, Gerard RD. Inhibitory role of plasminogen activator inhibitor-1 in arterial wound healing and neointima formation: a gene targeting and gene transfer study in mice. Circulation. 1997;96:3180–3191.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Irminger JC, Meyer K, Halban P. Proinsulin processing in the rat insulinoma cell line INS after overexpression of the endoproteases PC2 or PC3 by recombinant adenovirus. Biochem J. 1996;320:11–15.
  29. ↵
    Gerard RD, Meidell RS. Adenovirus vectors. In: Hames BD, Glover D, eds. DNA Cloning-A Practical Approach: Mammalian Systems. Oxford, UK: Oxford University Press; 1995:285–306.
  30. ↵
    Wagner DD, Olmsted JB, Marder VJ. Immunolocalization of von Willebrand protein in Weibel-Palade bodies of human endothelial cells. J Cell Biol. 1982;95:355–360.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Louvard D, Reggio H, Warren G. Antibodies to the Golgi complex and the rough endoplasmic reticulum. J Cell Biol. 1982;92:92–107.
    OpenUrlAbstract/FREE Full Text
  32. Ribes JA, Francis CW, Wagner DD. Fibrin induces release of von Willebrand factor from endothelial cells. J Clin Invest. 1987;79:117–123.
  33. ↵
    Levin EG, Santell L, Osborn KG. The expression of endothelial tissue plasminogen activator in vivo: a function defined by vessel size and anatomic location. J Cell Sci. 1997;110:139–148.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Yamamoto K, de Waard V, Fearns C, Loskutoff DJ. Tissue distribution and regulation of murine von Willebrand factor gene expression In vivo. Blood. 1998;92:2791–2801.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Rosenberg JB, Foster PA, Kaufman RJ, Vokac EA, Moussalli M, Kroner PA, Montgomery RR. Intracellular trafficking of factor VIII to von Willebrand factor storage granules. J Clin Invest. 1998;101:613–624.
    OpenUrlCrossRefPubMed
  36. ↵
    Harrison TM, Chidgey MAJ, Uff S. Novel markers for constitutive secretion used to show that tissue plasminogen activator is sorted to the regulated pathway in transfected PC12 cells. Cell Biol Int. 1996;20:293–300.
    OpenUrlCrossRefPubMed
  37. ↵
    Parmer RJ, Mahata M, Mahata S, Sebald MT, O’Connor DT, Miles LA. Tissue plasminogen activator (t-PA) is targeted to the regulated secretory pathway. Catecholamine storage vesicles as a reservoir for the rapid release of t-PA. J Biol Chem. 1997;272:1976–1982.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    Kaufmann JE, Irminger JC, Mungall J, Halban PA. Proinsulin conversion in GH3 cells after coexpression of human proinsulin with the endoproteases PC2 and/or PC3. Diabetes. 1997;46:978–982.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    Ozaka T, Doi Y, Kayashima K, Fujimoto S. Weibel-Palade bodies as a storage site of calcitonin gene-related peptide and endothelin-1 in blood vessels of the rat carotid body. Anat Rec. 1997;247:388–394.
    OpenUrlCrossRefPubMed
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  • Storage of Tissue-Type Plasminogen Activator in Weibel-Palade Bodies of Human Endothelial Cells
    Corinne Rosnoblet, Ulrich M. Vischer, Robert D. Gerard, Jean-Claude Irminger, Philippe A. Halban and Egbert K. O. Kruithof
    Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1796-1803, originally published July 1, 1999
    https://doi.org/10.1161/01.ATV.19.7.1796
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