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Glucose-induced endothelial heparanase secretion requires cortical and stress actin reorganization

Fang Wang, Ying Wang, Min Suk Kim, Prasanth Puthanveetil, Sanjoy Ghosh, Dan S. Luciani, James D. Johnson, Ashraf Abrahani, Brian Rodrigues
DOI: http://dx.doi.org/10.1093/cvr/cvq051 127-136 First published online: 17 February 2010

Abstract

Aims Heparanase, which specifically cleaves carbohydrate chains of heparan sulfate, has been implicated in the pathology of diabetes-associated complications. Using high glucose (HG) to replicate hyperglycaemia observed following diabetes, the present study was designed to determine the mechanism by which HG initiates endothelial heparanase secretion.

Method and results To examine the effect of HG on endothelial heparanase, bovine coronary artery endothelial cells were incubated with 25 mM glucose. Strategies using different agonists and antagonists were used to determine the mechanism behind HG-induced heparanase secretion. In endothelial cells, heparanase colocalized with lysosomes predominately around the nucleus, and HG caused its dispersion towards the plasma membrane for subsequent secretion. ATP release, purinergic receptor activation, cortical actin disassembly, and stress actin formation were essential for this HG-induced heparanase secretion. With HG, phosphorylation of filamin likely contributed to the cortical actin disassembly, whereas Ca2+/calmodulin-dependent protein kinase II and p38 mitogen-activated protein kinase /heat shock protein 25 phosphorylation mediated stress actin formation. The endothelial secreted heparanase in response to HG demonstrated endoglucuronidase activity, cleaved heparan sulfate, and released attached proteins like lipoprotein lipase and basic fibroblast growth factor.

Conclusion Our results suggest that HG is a potent stimulator of endothelial heparanase secretion. These data may assist in devising new therapeutic strategies to prevent or delay the cardiovascular complications associated with diabetes.

  • Diabetes
  • High glucose
  • Endothelial cell
  • Cytoskeleton
  • Heparan sulfate

1. Introduction

Heparan sulfate proteoglycans are ubiquitous macromolecules present on cell membranes and extracellular matrix, and consist of a core protein to which several linear heparan sulfate side-chains are covalently linked.1,2 They serve not only as structural proteins, but also as receptors; heparan sulfate can bind almost a hundred different proteins [growth factors, chemokines, cytokines, coagulation factors, and enzymes such as lipoprotein lipase (LPL)].3,4 This binding function provides the cell with a rapidly accessible reservoir, precluding the need for de novo synthesis when the requirement for a particular protein is increased.

Heparanase, cloned in 1999, is an endoglucuronidase that specifically cleaves carbohydrate chains of heparan sulfate.5 The role of heparanase in physiology includes its impact on bone formation, vascularization, hair growth, and wound healing.5 Its contribution to pathophysiology is also well documented. For example, in cancer progression, degradation of heparan sulfate chains is associated with disruption of the extracellular matrix and basement membrane; loss of this physical barrier facilitates tumour cell propagation and angiogenesis.6 In atherosclerosis-prone arteries, intense staining of heparanase is observed in apoE-null mice.7 In recent studies, heparanase has also been implicated in the pathology of diabetes. Elevated levels of heparanase have been detected in serum and urine of patients suffering from diabetic nephropathy.8 The inference is that heparanase disrupts the permselective property of the glomerular basement membrane, leading to urinary protein excretion.8 Confirmation of this property of heparanase was established in transgenic mice overexpressing mammalian heparanase; these mice had increased levels of urinary protein and creatinine.9

Heparanase is synthesized as a latent 65 kDa enzyme that undergoes secretion followed by reuptake into the cells. After undergoing proteolytic cleavage (primarily by cathepsin-L in lysosomes), a 50 kDa polypeptide is formed that is ∼200-fold more active than the 65 kDa type.5,10 Within the acidic compartment of the lysosome, active heparanase is stored in a stable form (half-life ∼30 h), and can assist in the turnover of heparan sulfate side chains.11 Mobilization by demand can also occur, where the enzyme is either translocated to the nucleus to affect gene transcription, or secreted to degrade cell surface heparan sulfate.10 An intriguing non-enzymatic function of latent heparanase has also been identified, and embraces promotion of cell adhesion and migration.6 We have previously reported that high glucose (HG) caused endothelial heparanase secretion by mechanisms that are yet to be identified.12 Using HG to replicate hyperglycaemia observed following diabetes, the present study was designed to determine the mechanism by which HG initiates this enzyme secretion. Our data suggest that actin reorganization, which encompasses cortical actin disassembly and stress actin formation, mediates HG-induced secretion of endothelial heparanase. Appreciating this mechanism by which endothelial cells manage and release heparanase may assist in devising new therapeutic strategies to prevent or delay the cardiovascular complications associated with diabetes.

2. Methods

An expanded version of the Supplementary material online, materials and methods together with figures is available at Cardiovascular Research online.

2.1 Cell culture

The current study adheres to the guide for the care and use of laboratory animals published by the US National Institutes of Health and the University of British Columbia. Bovine coronary artery endothelial cells (bCAECs, Clonetics) were cultured in endothelial growth medium (EGM) supplemented with EGM-MV BulletKit (Lonza) at 37°C in a 5% CO2 humidified incubator. Where indicated, bCAECs were also co-cultured with adult rat cardiomyocytes. These cells were prepared in medium 199 from hearts isolated from male Wistar rats obtained from Charles River Laboratories.

2.2 Western blot and immunoprecipitation

Western blot was carried out as described previously.12 Membranes were incubated with anti-heparanase mAb130 (that recognizes both 50 and 65 kDa heparanase forms; only the active 50 kDa heparanase data are presented), anti-phospho Ca2+/calmodulin-dependent protein kinase II (CaMKII) (Thr286), anti-phospho p38 MAPK (Thr180/Tyr182), anti-phospho Hsp25 (Ser86), anti-phospho filamin 1 (Ser2151) and anti-filamin 1 antibodies, and subsequently treated with secondary antibodies. Endothelial cells were lysed in lysis buffer and immunoprecipitated using anti-P2Y2 antibody for 3 h at 4°C. The immunocomplex was pulled down with protein A/G-sepharose overnight, and then heated for 5 min with sample loading dye at 95°C. The immunocomplex was immunoblotted for filamin and reaction products were visualized using an ECL® detection kit, and quantified by densitometry.

2.3 Heparanase activity

Following separation of medium from the cells, heparanase activity was determined in the culture medium using a HTRF heparanase assay kit.

2.4 Determination of heparan sulfate-bound proteins

LPL activity was assayed by measuring the hydrolysis of a [3H] triolein substrate emulsion. An ELISA kit was used to determine basic fibroblast growth factor (bFGF).

2.5 Immunofluorescence

Cells were fixed for 10 min with 4% paraformaldehyde in phosphate buffered saline (PBS), permeabilized with 0.1% Triton X-100 in PBS for 3 min, treated with PBS containing 1% goat serum for 1 h, and finally rinsed with PBS. Cells were incubated with the indicated antibodies (anti-heparanase mAb130, rabbit polyclonal P2Y2, mouse monoclonal filamin 1, anti-heparan sulphate 10E4 epitope). Following washes with PBS (3×), cells were incubated with secondary Alex633 (red) or Alex488 (green) conjugated goat anti-mouse or anti-rabbit antibodies. DAPI was used to stain nuclei. Where indicated, Alexa Fluor 488 Phalloidin was used to stain actin filaments, and LysoTracker to identify lysosomes. Slides were visualized using a Zeiss Pascal confocal microscope.

2.6 Extracellular ATP determination

ATP released into the extracellular medium was measured using a bioluminescent assay kit (Sigma).

2.7 Estimation of endothelial intracellular free Ca2+

For the measurement of [Ca2+]i, endothelial cells grown on glass coverslips were loaded with 1 µM of the acetoxymethylester form of the Ca2+ fluorescent dye Fura-2 in culture medium for 25 min. The coverslips were then mounted in a chamber and put on a temperature-controlled (37°C) stage of the Zeiss Axiovert 200 M inverted microscope (Carl Zeiss, Thornwood, NY, USA). Fura-2 was excited at 340 and 380 nm, and results were expressed as the ratio of the fluorescence emission intensity (F340/F380).

2.8 Isolation of membrane and cytosolic fractions

Isolation of plasma membrane and cytosolic fractions were determined using a previously described method.13

2.9 Treatments

To examine the effect of HG on endothelial heparanase, bCAECs were incubated with 25 mM glucose in DMEM at the indicated times (0–30 min). When co-culturing, 25 mM glucose in DMEM was placed in both the upper (endothelial cells) and lower (cardiomyocytes) chambers.

Mannitol [Mnt (20 mM) in 5 mM glucose] or 5 ng/mL TNF-α was also added to the culture medium, and served as osmolarity or positive controls, respectively. Various antagonists like suramin (100 µM, P2 receptor antagonist), jasplakinolide (1 µM, stabilizes actin filaments), cytochalasin D (0.5 µM, actin-depolymerizing agent), KN93 (10 µM, CaMKII inhibitor), and SB202190 (20 µM, p38 MAPK inhibitor) were used where indicated. 2-Methylthio ATP (MeSATP) was used as an ATP analog that acts as a P2Y receptor agonist.

2.10 Statistical analysis

Values are means ± SEM. Wherever appropriate, one-way ANOVA followed by the Tukey test was used to determine differences between group mean values. The level of statistical significance was set at P < 0.05.

3. Results

3.1 High glucose induces endothelial lysosomal heparanase secretion

To simulate diabetes-induced hyperglycaemia, endothelial cells were incubated with 25 mM glucose. Heparanase mRNA was not affected by HG, up to 2 h of treatment (data not shown). Glucose time-dependently lowered endothelial intracellular heparanase, with approximately 60% of the cellular enzyme released after 30 min of incubation (Figure 1A). This time-dependent decrease in intracellular heparanase was strongly associated to increased appearance of heparanase activity and protein in the incubation medium (Figure 1B). The osmolarity control mannitol had no effect on either intracellular (Figure 1A, inset) or medium (Figure 1B, inset) heparanase, whereas the positive control, TNF-α, had effects similar to HG; it lowered intracellular heparanase (Figure 1A, inset), and increased medium heparanase activity (Figure 1B, inset). Confirmation of the effect of HG and TNF-α on heparanase secretion was done using immunofluroscence in single (Figure 1C) and multiple cells (see Supplementary material online, Figure S1). In endothelial cells, heparanase colocalized with lysosomes. Interestingly, both HG and TNF-α caused the intracellular heparanase located predominately around the nucleus to disperse towards the plasma membrane.

Figure 1

High glucose induces endothelial lysosomal heparanase secretion. bCAECs (0.5 × 106 cells) were grown to 80–90% confluence and incubated with high glucose (HG, 25 mM) over a period of 30 min. Following separation of medium from the cells, heparanase was determined in both cell lysates (A) and medium (B) at the indicated times. The insets depict the influence of mannitol [Mnt (20 mM) in 5 mM glucose] and TNF-α (5 ng/mL) on heparanase in cell lysates (A) and medium (B) after a 30 min incubation. Results are means ± SEM of three separate experiments, and are expressed as a ratio to 0 min or to control (CON, 5 mM glucose). In a different set of bCAECs, lysosomes were first labelled with LysoTracker (green) for 3 h, before being treated with glucose (25 mM) or TNF-α (5 ng/mL) for 30 min. Cells were then fixed, permeabilized, and incubated with anti-heparanase mAb 130 (red) and DAPI (blue), and examined under a confocal microscope (C). The merged image of heparanase, lysosomes, and nucleus is described in the fourth panel from left (C). Bar = 20 µm. Data are from a representative experiment done twice. *Significantly different from 0 min or control; #significantly different from all other groups, P < 0.05.

3.2 Extracellular ATP mediates the effect of high glucose to induce heparanase secretion

In various cell types, intracellular ATP can be released into the extracellular space.14 bCAECs exposed to glucose (5–25 mM) released ATP into the medium, an effect that was concentration-dependent (Figure 2A, inset). Comparing 5–25 mM glucose, the release with both concentrations was rapid, with a maximum effect observed within 2 min. However, at this time, 25 mM glucose induced a more robust release of ATP compared with 5 mM glucose (Figure 2A). With prolongation of the incubation time, medium ATP decreased, and the difference in medium ATP observed with 5 and 25 mM glucose was lost. In HEK293 cells, extracellular nucleotides have been implicated in inducing heparanase secretion through purinergic receptors (P2Y receptor).15 Interestingly, the HG-induced endothelial heparanase secretion was inhibited by suramin (a nonspecific P2 receptor antagonist) (Figure 2B, inset). Another strategy used MeSATP, an ATP analog that can act as a P2Y receptor agonist. As shown in see Supplementary material online, Figure S2, MeSATP stimulated endothelial heparanase secretion in a concentration-dependent manner, with the maximum effect seen at 100 µM. Using this concentration of MeSATP in a different experiment, a time-dependent lowering of endothelial heparanase (Figure 2C) and an augmentation of medium heparanase activity (Figure 2C, inset) was observed.

Figure 2

Extracellular ATP mediates the effect of high glucose to induce heparanase secretion. Endothelial cells were incubated in 5–25 mM glucose for 2 min (A, inset), or 5 and 25 mM glucose over a period of 10 min (A). Following separation of medium from cells, ATP concentration in the media was measured. Cells were also pre-treated in the absence or presence of Suramin (100 µM, P2 receptor antagonist) for 30 min, followed by incubation with glucose (25 mM) for another 30 min. Heparanase protein remaining in the cells (B) or secreted into the incubation medium (B, inset) was then examined. The effects of MeSATP (100 µM, an ATP analog that acts as a P2Y receptor agonist) on endothelial intracellular (C) and medium heparanase (C, inset) was determined over a period of 30 min. Results are means ± SEM of three separate experiments, and are expressed as ratio to 0 min or control. *Significantly different from 0 min; +significantly different from 5 mM glucose; #significantly different from all other groups, P < 0.05.

3.3 Glucose-induced endothelial heparanase secretion is dependent on cytoskeleton reorganization

We have previously shown that endothelial actin cytoskeleton plays an important role in glucose-induced heparanase secretion.12 In this study, we more closely examined the time-dependent changes in endothelial actin cytoskeleton in response to HG using immunofluroscence in single (Figure 3) and multiple cells (see Supplementary material online, Figure S3). In untreated cells, actin filaments presented as continuous fluorescent rings, predominantly in the outer portion of the cells (cortical actin). Interestingly, exposure of cells to 25 mM glucose caused discontinuation of this cortical actin ring within 5 min, and after 30 min in HG, no evidence of continuous cortical actin remained. Instead, cells now demonstrated a robust increase in actin formation stretching across the cell body (stress actin). Using cytochalasin D (actin-depolymerizing agent, Supplementary material online, Figure S4A) or jasplakinolide (that stabilizes actin filaments, Supplementary material online, Figure S4B), we were able to prevent the glucose-induced endothelial heparanase secretion.

Figure 3

Glucose-induced endothelial heparanase secretion is dependent on cytoskeleton reorganization. Following treatment of bCAECs with glucose (25 mM) over a period of 30 min, cells were fixed, permeabilized, and double stained with Alexa Fluor 488 Phalloidin for filamentous actin (green) and DAPI for nucleus (blue) at the indicated times. Bar = 20 µm. The image is from a representative experiment done three times.

3.4 Ca2+/calmodulin-dependent protein kinase II (CAMKII) and p38 MAPK/Hsp25 phosphorylation are augmented in endothelial cells exposed to high glucose

CaMKII-mediated activation of p38 MAPK/Hsp has been suggested to play an important role in H2O2-induced stress actin formation in bovine aortic endothelial cells.16 Measurement of intracellular calcium revealed that HG was able to provoke a rapid elevation of [Ca2+]i within 5 min; this effect was transient with [Ca2+]i rapidly returning to baseline values (Figure 4A). Pre-treatment of endothelial cells with the P2 receptor antagonist suramin for 30 min did not affect [Ca2+]i (data not shown). However, in response to subsequent exposure to 25 mM glucose, suramin completely abolished the calcium response seen with HG (Figure 4A). MeSATP also caused a rapid but transient increase in [Ca2+]i. However, compared with HG, peak [Ca2+]i after administration of MeSATP was almost three-fold higher, and demonstrated a small but sustained rise in [Ca2+]i after the transient spike (Figure 4A, inset). Associated with the increase in [Ca2+]i, measurement of CaMKII demonstrated an increase in Thr286 phosphorylation at 20 min following glucose incubation, with a subsequent return to baseline at 30 min (Figure 4B). Unlike CaMKII phosphorylation, phosphorylation of p38 MAPK (Figure 4C) and Hsp25 (Figure 4D) were elevated by 20 min of glucose incubation, and remained high for the duration of the exposure to HG.

Figure 4

High glucose stimulates Ca2+/calmodulin-dependent protein kinase II (CaMKII) and p38 MAPK/Hsp25 phosphorylation. bCAECs were pre-treated in the absence or presence of suramin (100 µM) for 30 min, followed by stimulation with 25 mM glucose. The cytosolic Ca2+ changes over time were measured as the F340/F380 ratio using Fura-2 (A). The inset depicts cytosolic Ca2+ changes in response to MeSATP (100 µM; A). Results are expressed as an average response of 15–25 endothelial cells from four different endothelial cell preparations. In a separate experiment, bCAECs were exposed to 25 mM glucose for a period of 30 min. At the indicated times, protein was extracted to determine phosphorylation of CaMKII (B), p38 MAPK (C), and Hsp25 (D) using western blot. Results are means ± SEM of three separate experiments, and are expressed as ratio to 0 min. *Significantly different from 0 min; P < 0.05.

3.5 Inhibiting CAMKII or p38 MAPK regulated stress actin formation prevents glucose-induced endothelial heparanase secretion

To examine the relationship between CaMKII and p38 MAPK activation and stress actin formation, we used the specific inhibitors KN93 and SB202190. As depicted in Figure 5A, HG induced cortical actin disassembly and stress actin formation. Inhibiting CaMKII with KN93 reduced phosphorylation of p38 MAPK/Hsp25 (Figure 5B) and prevented the effect of HG on stress actin formation (Figure 5A). With inhibition of p38 MAPK phosphorylation using SB202190, Hsp25 phosphorylation was reduced (Figure 5B), and the stress actin formation observed with HG appeared discontinuous (Figure 5A). More importantly, the glucose-induced endothelial heparanase secretion was abolished by both KN93 and SB202190 (Figure 5C, inset). Our data suggest that in response to HG, stress actin is an important mediator of endothelial heparanase secretion.

Figure 5

Inhibiting CaMKII or p38 MAPK regulated stress actin formation prevents glucose-induced endothelial heparanase secretion. Following a 1 h pre-treatment of bCAECs with KN93 (KN; 10 µM) or SB202190 (SB; 20 µM), specific inhibitors of CaMKII or p38 MAPK respectively, cells were treated with glucose (25 mM) for 30 min. A control37 and a high glucose (HG) groups were also included. Cells were fixed, permeabilized, and double stained with Alexa Fluor 488 Phalloidin (a probe for filamentous actin; green) and DAPI (blue) (A). Bar = 20 µm. The image is from a representative experiment done twice. Using a similar protocol in a different set of cells, cellular proteins were extracted to determine phosphorylation of CaMKII, p38 MAPK and Hsp25 (B) using Western blot. Intracellular heparanase protein (C) and medium heparanase activity (C, inset) were also determined. The representative Western blot and densitometric results illustrated are means ± SEM from three separate experiments. #Significantly different from all groups; +Significantly different from HG group; P < 0.05.

3.6 Colocalization of filamin with the P2Y2 receptor is disrupted by high glucose bringing about filamin redistribution

Filamin attaches actin filaments to the plasma membrane through its binding to a number of plasma membrane proteins (including the P2Y2 receptor), and its phosphorylation disrupts cortical actin.17,18 Using immunofluorescence, we confirmed the plasma membrane colocalization of P2Y2 receptor (green) and filamin (red) in untreated endothelial cells (Figure 6A, merge). In the presence of HG, dissociation between the two proteins was observed at 30 min (Figure 6A). Interestingly, at this time, filamin located at the plasma membrane decreased, and much of the protein was observed in the cytosol (Figure 6A). Using immunoprecipitation, we confirmed a decreased association between filamin and the P2Y2 receptor after 30 min of HG (Figure 6B). In addition, the membrane to cytosolic transfer of filamin in HG was verified using cell fractionation and western blot (Figure 6C, inset). Coupled to this translocation, HG also time-dependently increased the phosphorylation of filamin (Figure 6C).

Figure 6

Colocalization of filamin with the P2Y2 receptor is disrupted by high glucose bringing about filamin redistribution. bCAECs treated with 25 mM glucose (for 0 and 30 min) were fixed, permeabilized, and stained for P2Y2 receptor (green), filamin (red) and nucleus (blue), and examined under a confocal microscope (A). The merged image of P2Y2 receptor, filamin, and nucleus is described in the fourth panel from left (A). Bar = 20 µm. The image is from a representative experiment done three times. Using a similar protocol in a different set of bCAECs, cells were lysed, and subjected to immunoprecipitation (IP) using anti-P2Y2 receptor antibody, and immunoblotted with anti-filamin and anti-P2Y2 receptor antibodies (B). To determine filamin redistribution, endothelial cells were exposed to glucose (25 mM) over a period of 30 min. At the indicated times, total protein was extracted to determine phosphorylation of filamin (C). Membrane and cytosolic fractions was also isolated to examine filamin distribution using western blot (C, inset). The representative western blot and densitometric results illustrated are the means ± SEM from three separate experiments. *Significantly different from 0 min; P < 0.05.

3.7 Endothelial secreted heparanase cleaves cardiomyocyte heparan sulfate to release attached proteins

The cardiomyocyte cell surface is rich in heparan sulfate side chains that contain many attached proteins such as LPL and bFGF.19,20 To test the endoglucuronidase activity of endothelial heparanase to specifically cleave the carbohydrate chains of heparan sulfate, endothelial cells were co-cultured with cardiomyocytes. Initially, we determined that HG time-dependently lowered endothelial intracellular heparanase in the insert, with maximum release observed after 60 min of incubation (Figure 7A). As heparanase secretion is polarized, with preferential secretion towards the basolateral rather than the apical side of endothelial cells,3 we also measured heparanase activity in the medium from the lower chamber. Medium heparanase activity increased at 30 min, and reached a maximum at 60 min of HG incubation (Figure 7A, inset). To allow for maximum ability of heparanase to cleave heparan sulfate, cardiac cells were co-cultured with or without endothelial cells in HG for 4h, and cardiomyocytes then imaged for heparan sulfate staining. The increase in heparanase activity in the lower chamber (in the cardiomyocyte + EC group) was associated with a robust decrease in immunofluorescent staining for heparan sulfate (Figure 7B). Given the ability of heparan sulfate to bind proteins, we predicted that loss of cardiomyocyte heparan sulfate would increase the appearance of heparan sulfate-bound proteins such as LPL and bFGF in the culture medium. Indeed, co-culture of endothelial cells with cardiomyocytes in HG increased medium LPL activity (Figure 7C) and bFGF protein in the lower chamber (Figure 7D).

Figure 7

Endothelial secreted heparanase cleaves cardiomyocyte heparan sulfate to release attached proteins. Primary rat cardiomyocytes were plated in a 6-well plate as described. A cell culture insert (Falcon, diameter 23.1 mm) grown with or without confluent endothelial cells4 was placed above the cardiomyocytes. Cells were incubated in 25 mM glucose over a period of 0–2 h. At the times indicated, endothelial cells on the insert were lysed and heparanase protein (A) and medium heparanase activity (A, inset) detected. To test the endoglucuronidase activity of endothelial heparanase to specifically cleave the carbohydrate chains of cardiomyocyte heparan sulfate, co-culture (with or without EC) was maintained for 4 h. Cardiomyocytes were then fixed, and stained for heparan sulfate (red) (B). Bar = 20 µm. Data are from a representative experiment done twice. Medium was also collected from the lower chambers to measure LPL activity (C) and bFGF (D). Results are means ± SEM of three separate experiments. *Significantly different from 0 min; #significantly different from all groups; P < 0.05.

4. Discussion

Following diabetes, endothelial heparanase has been reported to manage cardiac metabolism by transferring LPL from the cardiomyocyte surface to the vascular lumen.12 Diabetes can also lead to arterial structural and mechanical changes that are related to upregulation of endothelial heparanase.7,21,22 Previously, we examined endothelial heparanase regulation following diabetes and reported that HG had a dose-dependent effect in releasing this enzyme.12 The present study describes a novel signal pathway by which HG induces cortical and stress actin reorganization to facilitate endothelial heparanase exocytosis.

Cellular heparanase is stored in lysosomes with predominant perinuclear localization.15,23 Exposure of bCAECs to HG time-dependently (within 30 min) released heparanase. Thus, for heparanase to undergo secretion, movement of lysosomes to the cell surface would be a prerequisite. Indeed, immunofluorescent images of endothelial cells revealed that heparanase colocalized with lysosomes, in close proximity to the nucleus. More importantly, for the first time, we demonstrate that in response to HG, there is a repositioning of lysosomal heparanase away from the nucleus and loss of total staining for endothelial heparanase and lysosomes. It should be noted that although heparanase gene expression is augmented in HEK 293 cells exposed to HG for 24 h,8 we were unable to detect any change in gene expression up to 2 h with HG. Overall, our data suggest that in response to stimulation by HG, mechanisms that regulate lysosomal exocytosis are also likely to control endothelial heparanase secretion.

In various cell types including endothelial cells, cytoplasmic ATP can be released by shear stress, hypoxia, or agents such as thrombin.2426 When released, ATP is an important mediator of vascular tone and blood coagulation by regulating endothelial nitric oxide and von Willebrand factor secretion, respectively.27,28 We observed that bCAECs exposed to 5 or 25 mM glucose rapidly released ATP into the medium, with 25 mM glucose having a more robust effect. Extracellular ATP, through a P2Y G-protein-coupled receptor signal cascade, is known to initiate intracellular heparanase secretion.15 P2Y is the predominant purinergic receptor subtype in endothelial cells.29,30 Using the P2 receptor antagonist suramin, we prevented the effect of HG to bring about heparanase secretion. As MeSATP, a more potent P2Y agonist than ATP, also had a profound effect in releasing endothelial heparanase, our data provide strong evidence that ATP release and subsequent purinergic receptor activation are essential for mediating the effect of HG on endothelial heparanase release.

Actin cytoskeleton is a dynamic network, and its reorganization from a distribution predominantly in the outer portion of the cells (cortical actin) into fibres that stretch across the cell body (stress actin), is a characteristic change of endothelial cells in response to inflammatory agents.31,32 For example, a 30 min exposure to thrombin significantly increases calf pulmonary artery endothelial cell permeability to albumin, which was associated with the loss of cortical and increases in stress actin.31 Recent studies have also specified a dual role of actin cytoskeleton in controlling vesicle transport and exocytosis.33 It was suggested that cortical actin serves as a barrier, and its transient depolymerization is necessary for vesicle secretion.34 Related to stress actin, its formation as a track for vesicle translocation towards the plasma membrane would also be essential for vesicular secretion. In this study, immunofluorescent images of endothelial cells demonstrated cortical actin disassembly on exposure to HG, with subsequent stress actin formation. As agents that disrupted actin dynamics prevented the HG-induced depletion of intracellular heparanase, our data suggest that both cortical actin disassembly and stress actin formation are indispensable for HG-induced heparanase secretion.

In pancreatic beta cells, CaMKII contributes to calcium-regulated exocytosis of insulin secretory granules.35 In our study, albeit with different kinetics, HG and MeSATP provoked an increase in [Ca2+]i. As suramin impeded this effect of HG on [Ca2+]i, our data suggest that ATP is a key element that facilitates HG-induced increases in endothelial [Ca2+]i. Related to this increase in [Ca2+]i, there was an associated augmentation in phosphorylation of CaMKII that was not sustainable and declined to control levels at 30 min even though heparanase was continuously being secreted. We considered the possibility that the early activation of CaMKII may have turned on other downstream signals. Downstream targets of CaMKII include p38 MAPK/Hsp25, and there was coincident activation of this pathway following HG. Hsp25 is known to inhibit actin polymerization, and its phosphorylation results in a decline of this inhibitory function.36 In this setting, actin monomers are released from the phosphorylated Hsp25 to self-associate to form actin filaments. Interestingly, CaMKII inhibition with KN93 or blocking of p38 MAPK with SB202190 prevented stress actin formation, and abolished HG-induced endothelial heparanase secretion. These data suggest that in response to HG, CaMKII and p38 MAPK, through their control of Hsp25 and stress actin, act in unison to facilitate heparanase secretion from endothelial cells.

Filamin is a 280 kDa protein that contains an N-terminal actin-binding domain (that assists in actin filament cross linking), and a C-terminal protein binding end (that connects the actin network to plasma membrane bound proteins such as the insulin and P2Y2 receptors).18,3739 In this way, filamin plays a critical role in cortical actin organization. We established a strong association between filamin and P2Y2 receptors in endothelial cells. More importantly, incubation with HG reduced this association, facilitating filamin translocation to the cytosol. In endothelial cells exposed to thrombin, filamin phosphorylation is required for its translocation from the cell periphery to cytosol.38 Our data also indicate that HG induces a robust phosphorylation of filamin with subsequent membrane to cytosol relocation, and is associated with disassembly of the endothelial cortical actin network and endothelial heparanase secretion.

Co-culturing of endothelial cells and cardiomyocytes is an appropriate means by which the endoglucuronidase activity of endothelial heparanase can be tested. This is because in endothelial cells, heparanase exocytosis is polarized, with preferential secretion towards the basolateral rather than the apical side.3,37 Using this co-culture preparation, we were successful in depleting endothelial intracellular heparanase. More importantly, we observed that in response to HG and heparanase secretion, cardiomyocyte surface heparan sulfate was cleaved, with an associated increase in LPL activity and bFGF protein in the lower chamber.

In summary, our results suggest that HG stimulates ATP release and purinergic receptor activation. One downstream cascade of this event is increased in [Ca2+]i, stimulation of CaMKII, phosphorylation of p38 MAPK/Hsp25, and eventual formation of stress actin. Another outcome is detachment of filamin from the P2Y receptor, resulting in cortical actin disassembly. Together, these processes contribute to robust stimulation of endothelial heparanase secretion. These data may help in our understanding of the complications associated with diabetes.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Conflict of interest: none declared.

Funding

This work was supported by operating grants from the Canadian Diabetes Association and the Canadian Institutes of Health Research. F.W., P.P., and M.S.K. are the recipients of Doctoral Student Research Awards from the Canadian Diabetes Association. M.S.K. also received a Doctoral Student Research Award from the Heart and Stroke Foundation of Canada.

References

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