G protein-coupled receptor kinase 2 moderates recruitment of THP-1 cells to the endothelium by limiting histamine-invoked Weibel-Palade body exocytosis

Background G protein-coupled receptors (GPCRs) are a major family of signaling molecules, central to the regulation of inflammatory responses. Their activation upon agonist binding is attenuated by GPCR kinases (GRKs), which desensitize the receptors through phosphorylation. G protein-coupled receptor kinase 2(GRK2) down-regulation in leukocytes has been closely linked to the progression of chronic inflammatory disorders such as rheumatoid arthritis and multiple sclerosis. Because leukocytes must interact with the endothelium to infiltrate inflamed tissues, we hypothesized that GRK2 down-regulation in endothelial cells would also be pro-inflammatory. Objectives To determine whether GRK2 down-regulation in endothelial cells is pro-inflammatory. Methods siRNA-mediated ablation of GRK2 in human umbilical vein endothelial cells (HUVECs) was used in analyses of the role of this kinase. Microscopic and biochemical analyses of Weibel-Palade body (WPB) formation and functioning, live cell imaging of calcium concentrations and video analyses of adhesion of monocyte-like THP-1 cells provide clear evidence of GRK2 function in histamine activation of endothelial cells. Results G protein-coupled receptor kinase 2 depletion in HUVECs increases WPB exocytosis and P-selectin-dependent adhesion of THP-1 cells to the endothelial surface upon histamine stimulation, relative to controls. Further, live imaging of intracellular calcium concentrations reveals amplified histamine receptor signaling in GRK2-depleted cells, suggesting GRK2 moderates WPB exocytosis through receptor desensitization. Conclusions G protein-coupled receptor kinase 2 deficiency in endothelial cells results in increased pro-inflammatory signaling and enhanced leukocyte recruitment to activated endothelial cells. The ability of GRK2 to modulate initiation of inflammatory responses in endothelial cells as well as leukocytes now places GRK2 at the apex of control of this finely balanced process.

Summary. Background: G protein-coupled receptors (GP-CRs) are a major family of signaling molecules, central to the regulation of inflammatory responses. Their activation upon agonist binding is attenuated by GPCR kinases (GRKs), which desensitize the receptors through phosphorylation. G protein-coupled receptor kinase 2(GRK2) down-regulation in leukocytes has been closely linked to the progression of chronic inflammatory disorders such as rheumatoid arthritis and multiple sclerosis. Because leukocytes must interact with the endothelium to infiltrate inflamed tissues, we hypothesized that GRK2 down-regulation in endothelial cells would also be pro-inflammatory. Objectives: To determine whether GRK2 down-regulation in endothelial cells is pro-inflammatory. Methods: siRNAmediated ablation of GRK2 in human umbilical vein endothelial cells (HUVECs) was used in analyses of the role of this kinase. Microscopic and biochemical analyses of Weibel-Palade body (WPB) formation and functioning, live cell imaging of calcium concentrations and video analyses of adhesion of monocyte-like THP-1 cells provide clear evidence of GRK2 function in histamine activation of endothelial cells. Results: G protein-coupled receptor kinase 2 depletion in HUVECs increases WPB exocytosis and P-selectin-dependent adhesion of THP-1 cells to the endothelial surface upon histamine stimulation, relative to controls. Further, live imaging of intracellular calcium concentrations reveals amplified histamine receptor signaling in GRK2-depleted cells, suggesting GRK2 moderates WPB exocytosis through receptor desensitization. Conclusions:

Introduction
To initiate an inflammatory response, both leukocytes and endothelial cells need to be activated by hormones and proinflammatory cytokines. The subsequent cell-surface expression of adhesion molecules in both cell types results in the rolling of leukocytes along the endothelial surface, before eventual firm adhesion and extravasation [1].
Many signaling pathways involved in both endothelial and leukocyte activation are initiated by stimulating G protein-coupled receptors (GPCRs) [2]. GPCR signal transduction is attenuated by GPCR kinase (GRK)-mediated phosphorylation of agonist-bound receptor. This promotes b-arrestin binding, which uncouples the receptor from its G proteins and mediates receptor internalization and recycling [3,4]. Disruption of this machinery alters the strength and/or duration of physiological responses to GPCR ligands [5]. Of seven GRK subfamilies, the ubiquitously expressed GRK2 has been most closely linked to inflammatory [2,6] and cardiovascular function [6,7].
The pathologies of both MS and RA are characterized by increased leukocyte infiltration of diseased tissues, probably caused, at least in part, by impaired GRK2-mediated attenuation of chemokine signaling. For example, GRK2 +/À murine T cells show significantly heightened migratory responses towards CCL4. This is concurrent with enhanced calcium signaling and PKB phosphorylation, indicative of impaired CCR5 desensitization [15]. Moreover, loss of GRK2 in the endothelium can enhance cytokine expression, increasing the incidence of macrophage extravasation in endothelial-GRK2 À/À mice [16].
Endothelial activation is mediated by pro-inflammatory and procoagulant factors, delivered to the endothelial cell surface by exocytosing Weibel-Palade bodies (WPBs). These specialized secretory organelles store the multimeric glycoprotein von Willebrand factor (VWF) [17] and are formed at the trans-Golgi network [18][19][20], with the help of an AP-1/clathrin coat [21]. Upon injury or infection, mature organelles, previously anchored to cortical actin [22], fuse with the plasma membrane and release VWF to initiate hemostasis [23,24]. Other WPB cargo such as the leukocyte receptor P-selectin, its cofactor CD63 [25] and pro-inflammatory cytokines are also delivered to the cell surface or released into the circulation. WPBs are thus central to endothelial regulation of inflammation.
As GRK2-deficiency in leukocytes has been closely linked to inflammatory disorders, we determined whether it also affects the pro-inflammatory behaviour of endothelial cells.

Immunofluorescence and WPB quantification
Transfected HUVECs were fixed and stained as described previously [21]. Images were taken using a Leica TCS SPE scanning confocal microscope, a 639 (NA1.3) or 409 (NA1.15) oil immersion lens (NA 1.15) and LAS-AF Software (Leica, Buckinghamshire, UK). Acquisition settings were: 0.5 lm z-stack step size, 1024 9 1024 pixel resolution, 3-4 frame average and 19 zoom. For quantification, the LAS-AF mark-and-find feature and a motorized stage were used to generate 15-25 random fields of view (FoV, 300-400 cells) per treatment condition in each replicate experiment. WPBs were quantified using Image J; background subtraction was performed on the VWF channel using a rolling ball algorithm (radius 2 pixels) and a manual threshold applied. Segmented objects over 0.1 lm 2 were counted and measured for Feret's diameter (defined as the longest diameter across a WPB in 2D projection) using Image J.

Secretion assays
VWF secretion assays have been described previously [21]. Briefly, cells were rinsed and incubated in release medium for 30 min, then release medium plus 100 ng mL À1 phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich, St Louis, MO, USA) or 10 lM histamine for 45 or 30 min, respectively. Medium was collected to sample VWF release and intracellular VWF determined from cell lysates. Secreted VWF is presented as a percentage of the total VWF (media plus lysates). All results are normalized to total lysate protein content, as determined by bicinchoninic assay (Pierce, Rockford, IL, USA). Total VWF measurements were used to compare VWF protein expression in mock and GRK2-depleted cells. To measure unregulated secretion over longer periods, cells were rinsed and incubated in serum-free release medium for 4 h, or in optiMEM reduced serum medium (GIBCO, Paisley, UK) for 7 h.

ssHRP secretion assays
Eight hours post-transfection, cells were rinsed and incubated in phenol red-free HUVEC growth medium (Sigma); 17 h later, medium was collected and cells lysed in 50 mM Tris/Cl pH2 on ice. Samples were processed and analyzed as described previously [29]. Secretion is presented as a proportion of total ssHRP (media plus lysates).

VWF multimer analysis
Samples were concentrated using Vivaspin500 centrifugal filter units (Sartorius, Goettingen, Germany) and run on SDS-agarose gels as described previously [29]. Multimer patterns were analyzed using the Image J plot profile function to measure intensity changes down each lane, normalized against total signal.

Calcium imaging
29 Fluo-4 calcium indicator, containing 25 lm probenecid, was prepared in serum-free release medium as per manufacturer's instructions (Fluo-4 Direct TM Calcium Assay Kit, Molecular Probes, Life Technologies, Paisley, UK). Nucleofected cells seeded onto 1.45-mm glass-bottom imaging dishes (PAA) were rinsed and loaded with 19 Fluo-4 for 30 min at 37°C/5% CO 2 prior to imaging. Movies were acquired using an UltraVIEW VoX spinning disc system (PerkinElmer, Waltham, MA, USA) mounted on an inverted microscope (TiE; Nikon, Surrey, UK) with an EM charge-coupled device camera (512 9 512 pixels; C9100-13; Hamamatsu Photonics, Hertfordshire, UK) and 488-and 561-nm solid-state lasers. Cells were visualized using a 1009 oil immersion lens (NA 1.4), inside a 37°C heat-controlled chamber. Z-stacks of 0.5-lm spacing were acquired using a piezo stage (NanoScanZ; Prior Scientific, Cambridgeshire, UK) every 10 s for 10 min. At time-points 60, 300 and 480 s, 10 mM histamine, 200 lm A21387 ionophore and 50 mM EGTA were added, respectively, to stimulate cells and provide a maximal (fmax) and minimal fluorescent signal. To determine changes in intracellular calcium levels, a 40 9 40 pixel ROI was drawn juxtanuclear in the cytosol for each cell and the mean fluo-4 intensity measured at every time-frame using Velocity software (Per-kinElmer). Measurements were normalized against fmax to eliminate inconsistencies in indicator loading. To align curves for comparison, Fluo-4 intensity at time-point 60 s was subtracted from all values.

THP-1 flow assays
Nucleofected HUVECs were seeded onto gelatin-coated lslides VI 0.4 (ibidi, Munich, Germany). Slides were mounted on the microscope stage of an Axiovert 100 (Carl Zeiss, Welwyn Garden City, UK), maintained at 37°C, and connected to a syringe pump system (Harvard Aparatus, Holliston, MA, USA) to draw fluid through the chamber with a wall shear stress of 0.07 Pa (0.7 dyne/cm 2 ). Cells were rinsed with perfusion medium (HBSS containing Ca +2 and Mg +2 and 0.2% BSA) under flow, then 10 6 THP1 cells mL À1 were perfused across the endothelial surface for 3 min to image steady-state rolling. Next, a 10-min stimulation of HUVECs, using perfusion medium + 10 lM histamine, was followed by a second THP-1 cell perfusion in the absence of secretagogue. The latter was recorded for 5 min to observe monocyte rolling. Movies were taken at 109 objective with a FoV of 784 9 576 pixels (559.78 9 411.26 lm) using a QIMAGING Scientific (QIMAGING Scientific, Surrey, Canada) CMOS Rolera bolt camera, acquiring at a rate of 24 frames/second, and Micro-Manager 1.4.13 software. To quantify firmly adhered monocytes on the endothelial surface, random snapshots were taken in the absence of flow at the end of the movie. In antibody blocking experiments, HUVECs were incubated with 15 lg mL À1 sheep polyclonal antihuman P-selectin (R&D Systems, Minneapolis, MN, USA) or IgG from sheep serum (Sigma-Aldrich) at 37°C for 30 min prior to experimentation. Antibody was present for the remainder of the experiment.
To assess whether other WPB characteristics were altered, recruitment of P-selectin and MyRIP to organelles was also analyzed by immunofluorescence. P-selectin is an integral membrane protein incorporated into WPBs at the trans-Golgi network (TGN) [31], whereas the Rab27A-MyRIP-MyoVA complex is recruited later and predominantly found on mature organelles [32]. Incorporation of these proteins into WPBs is therefore indicative of successful progression through two independent stages of biogenesis. Both proteins were found to localize correctly in GRK2-depleted cells, indicating that cargo selection and maturation is unaffected (Fig. 1D). Furthermore, in both cell populations, 52% of cells showed P-se-lectin recruitment to WPBs, suggesting it is targeted equally efficiently (data not shown).
Disruption of WPB biogenesis can result in the loss of higher molecular weight (HMW) forms of VWF [21]. We therefore determined the multimeric state of intracellular VWF as an additional descriptor of organelle structure. VWF multimerisation was slightly reduced in GRK2depleted cells, suggesting either VWF maturation is reduced or mature protein is being lost from the cell (see Fig. 2A).
Reduction in WPB numbers is not due to reduced VWF expression VWF drives the formation of WPBs [33]. Deficiencies in VWF expression can therefore reduce WPB numbers, as seen in von Willebrand disease [34]. To determine if this is causative of fewer organelles here, VWF protein levels in control and GRK2 knock-down (KD) cells were quan- tified by both ELISA and western blot and found to be comparable (Fig. 2). VWF transcript, however, was consistently, although statistically insignificantly, increased by 24% and 31% (siRNA1 and 2 respectively) in GRK2-depleted cells (Fig. 2C). Loss of GRK2 thus does not reduce WPB numbers by reducing VWF expression.
VWF progression through the early secretory pathway is unaffected by GRK2 depletion We next investigated whether WPB formation is GRK2 dependent. If WPB biogenesis is slowed, VWF is likely to accumulate in pre-Golgi compartments of the secretory pathway, as observed following expression of VWD-causing VWF variants [35]. As VWF passage through the TGN is marked by the furin-mediated cleavage of its propeptide [36], VWF progression through the Golgi can be determined by the ratio of pro-VWF to VWF. As shown in Fig. 2(D), this ratio was unaffected by GRK2 depletion, as was the gross morphology of the TGN (Fig. 2E). GRK2 therefore does not influence WPB numbers by regulating VWF trafficking through the early secretory pathway.
GRK2-deficient HUVECs secrete more VWF in the absence of secretagogue than control cells If VWF expression and WPB formation are normal in GRK2-depleted cells, reductions in organelle number are likely to be the result of increased WPB exocytosis or constitutive VWF secretion. Steady-state VWF release was therefore examined by ELISA. When incubated in reduced-serum medium in the absence of added secreta-gogue, GRK2-depleted cells released almost 60% more VWF than controls (Fig. 3Ai). Furthermore, a consistent (but statistically insignificant) 33% increase in unregulated secretion in the total absence of serum, and hence stimulus, was also seen in KD cells (Fig. 3Aii). GRK2depleted cells thus possess fewer WPBs because they release more VWF under resting conditions. To determine whether constitutive secretion is up-regulated in GRK2deficient cells, the secretion of ssHRP [28], a marker for this pathway, was monitored and found to be unchanged. GRK2 depletion therefore specifically affects VWF release (Fig. 3B).
A GRK2-depleted endothelium is hyper-responsive to histamine stimulation The observed change in VWF release, coupled to the known role of GRK2 in desensitization, suggested that WPB loss might result from enhanced sensitivity to GPCR signaling. To determine whether GRK2 depletion affects endothelial activation, we challenged cells with histamine, a GPCR agonist [37] and pro-inflammatory stimulant [38] of WPB exocytosis. GRK2 KD cells released 78% (45-179%, n = 6) more VWF than controls during a 30-min incubation with 10 lM histamine, consistent with increased sensitivity (Fig. 4A). There was no difference in the multimeric state of the VWF released by GRK2-deficient HUVECs, indicating that similar organelles are secreted in both cell populations (Fig. 4Aii).
We also performed a secretion assay in the presence of PMA, a membrane permeant DAG analogue, which stimulates WPB exocytosis by raising both intracellular calcium and cAMP levels, independent of cell surface receptors. Importantly, there was no difference in regu-  Fig. 3. Unregulated secretion is increased in GRK2 KD cells. (A) Mock and GRK2 KD (siRNA sequence 2) cells were rinsed and incubated with (i) reduced-serum medium (optiMEM) for 7 h or (ii) serum-free medium for 4 h. Media and cell lysates were then collected and assayed for VWF content by ELISA, before normalizing to lysate total protein content as determined by BCA assay. The percentage of total VWF (medium plus lysates) released over 1 h was calculated and is presented here to allow comparison between the two conditions. Error bars represent SEM. Statistics were performed using Student's t-test. (Ai) A 59% increase in VWF release is seen in GRK2 KD cells assayed in Opti-MEM (n = 3, P = 0.01). (Aii) In the absence of serum, a 33% increase in VWF secretion is seen (n = 4, P = 0.3). (B) HUVECs were transfected with luciferase-or GRK2-targeting siRNA plus ssHRP cDNA. Secreted ssHRP was collected over 17 h in phenol red-free medium before lysing cells in Tris-Cl pH2. The ssHRP content of media and lysates was determined by kinetic ELISA. Secreted ssHRP is expressed as a percentage of total ssHRP present in cells at the beginning of the assay (medium plus lysates) and is 84% under both conditions (82-88%, n = 3). Error bars represent SEM. lated secretion between control and KD cells in this instance, suggesting that the organelles of siRNAtreated cells are not generally secretion super-competent (Fig. 4B).
To confirm that the enhanced histamine-stimulated secretion observed in GRK2-depleted cells was due to impaired GPCR desensitization, we next investigated the amplitude of downstream signaling events. Intracellular calcium concentrations before and during histamine stimulation were monitored by live-imaging cells loaded with Fluo-4 indicator. As shown in Fig. 5, Ca 2+ influx in response to histamine was augmented in GRK2-depleted cells relative to controls. This was not due to general changes in receptor abundance, as HRH1 mRNA, the only histamine receptor expressed in HUVECs [39], was equivalent in GRK2 KD and control cells (Fig. 5B).

Endothelial GRK2 is anti-inflammatory
To establish the functional relevance of these changes, we assayed monocyte-like THP-1 cell adhesion to HUVEC monolayers. Control and GRK2-depleted HUVECs were stimulated with histamine before being perfused with 10 6 THP-1 cells mL À1 under flow. The number of firmly adherent cells after 5 min was found to be increased almost 3-fold in GRK2-depleted cells (Fig. 6B). This increase was dependent on P-selectin, because, in the presence of 15 lg mL À1 function-blocking P-selectin antibody, THP-1 adhesion was reduced to control levels in GRK2 KD cells (Fig. 6B). There was no significant difference in adhesion between control and GRK2-depleted cells in the absence of secretagogue (Fig. 6B).
To verify that these observations result from increased WPB exocytosis, and not changes in P-selectin expression, P-selectin protein levels in mock and KD cell lysates were examined by SDS-PAGE and found to be comparable (Fig. 6C). Furthermore, P-selectin targeting to WPBs was not affected by loss of GRK2 (Fig. 1D). Increased THP-1 cell adhesion to GRK2-depleted HUVECs therefore results from increased delivery of P-selectin to the plasma membrane upon stimulation.

Discussion
Extravasation of circulating leukocytes and their subsequent migration into afflicted tissue during inflammation must be tightly regulated, because excessive recruitment results in inflammatory disease. Leukocyte GRK2 activity is known to limit leukocyte migration along chemotactic gradients and thus prevent aberrant tissue infiltration [9,12,13,15,40]. Here we show for the first time that GRK2 also regulates endothelial activation and leukocyte recruitment in vitro. Together these activities have the potential to limit both early and late events in the initiation of inflammation and thus prevent the harmful accumulation of leukocytes inside the body.
A key pro-inflammatory stimulant of endothelial cells is histamine [38], which triggers calcium-mediated WPB exocytosis by binding to HRH1 [39,41], a GPCR phosphorylated by GRK2 [37]. Consistent with a failure in HRH1 desensitization, we report that histamine-invoked calcium influx is amplified in GRK2-deficient HUVECs, as is subsequent VWF release. Similar effects of GRK2 depletion on calcium signaling have been reported upon CCR5 stimulation in activated T cells, suggesting a general mechanism for regulation of inflammatory signal transduction [15]. In addition to GRK2, HUVECs have been reported to express GRK5 and 6 [42]. To the best of our knowledge, there is no reported interaction between these kinases and HRH1 that may complicate interpretation of the results presented. GRK5 and 6 do, however, desensitize other receptors that are capable of stimulating WPB exocytosis, such as PAR-1 [42], and thus may contribute to the regulation of other hemostatic and inflammatory pathways in a similar manner to GRK2.
Histamine signaling through HRH1 has previously been shown to induce P-selectin-mediated leukocyte rolling in post-capillary venules [39,43]. Consistent with this, we see a 3-fold increase in the number of THP-1 cells adhering to GRK2-depleted HUVECs following enhanced receptor activation. These leukocyte-endothelial interactions rely on the regulated translocation of adhesion molecules and receptors to the cell surface. In the case of endothelial P-selectin, this is achieved through WPB exocytosis. The importance of P-selectin in mediating tethering between endothelial cells and leukocytes is well established [44]. In P-selectin-deficient mice, both initial rolling, as seen by intra-vital microscopy, and subsequent neutrophil recruitment to inflamed sites are severely impaired [45]. Similarly, antibodies against P-selectin glycoprotein ligand-1 (PSGL-1), the leukocyte counterreceptor for P-selectin, block rolling of human polymorphonuclear cells in rat mesenteric venules [46]. Here we indicate that the converse is also true; enhanced delivery of P-selectin to the endothelial surface can promote excessive accumulation of adherent leukocytes. Consistent with our data, increased cell-surface P-selectin has been shown to correlate with the development of inflammatory infiltrates in a number of pathologies. In murine atherosclerotic lesions, histochemical staining of endothelial P-selectin is strongest at sites of active macrophage infiltration [47]. Moreover, VEGF-induced P-selectin up-regulation leads to psoriasis and contact dermatitis [48], whereas enhanced surface translocation of P-selectin in pancreatic capillaries, through WPB exocytosis, vastly contributes to the progression of severe acute necrotizing pancreatitis [49]. Endothelial P-selectin expression is also increased in the inflamed synovial tissue of patients with rheumatoid arthritis [50], where it promotes monocytemicrovasculature interactions [51]. Paradoxically, loss of P-selectin is reported to enhance progression of murine collagen-induced arthritis [52]; the contribution of adhesion molecules to inflammatory responses is therefore complex. It is unknown whether endothelial GRK2 is down-regulated in any of these pathologies; however, it is tempting to speculate that augmentation of endothelial activation would serve to exacerbate the already enhanced chemotactic responses of GRK2-deficient leukocytes.
Recently, endothelial-targeted deletion of GRK2 in mice revealed that, even in the absence of pro-inflammatory mediators, loss of GRK2 in the endothelium triggers macrophage infiltration [16]. This is attributed to up-regulation of cytokine expression in response to reactive oxygen species (ROS), generated by GRK2-depleted mitochondria. Here we provide a more direct mechanism by which loss of GRK2 could promote leukocyte adherence to the endothelial wall, and thus extravasation, through impaired inflammatory receptor desensitization and enhanced WPB exocytosis. This could occur downstream of the systemic cytokine signaling induced by loss of GRK2 itself, or upon development of inflammatory disease. How these mice respond to infection or induction of chronic inflammatory conditions remains to be tested. HUVECs transfected with luciferase or GRK2 siRNA were lysed and assayed for P selectin and GRK2 content by SDS-PAGE. As a control, blots were also probed for actin.
The storage of both P-selectin and VWF in the same secretory compartment ensures coordinated delivery of both proteins to the cell surface, implying they function in the same processes. Although THP-1 accumulation in GRK2-depleted cells was P-selectin dependent here, the increased secretion of VWF from these cells may also affect inflammation. VWF is itself capable of interacting with PSGL-1 via its A1 domain, an interaction enhanced by the presence of b2-integrins [53]. The involvement of the latter, plus observations of reduced rolling in P-selectin-deficient mice [45], suggests these interactions are more important in the latter stages of leukocyte recruitment and adhesion. The combined action of increased P-selectin and VWF secretion is therefore consistent with the strong impact of GRK2 depletion on firm adhesion, described here, as opposed to just rolling. We speculate that in vivo, the inflammatory effects of increased VWF secretion could be yet further enhanced through the recruitment of large numbers of activated platelets, which themselves induce Pselectin-dependent leukocyte rolling and WPB exocytosis [54]. Platelets also up-regulate expression of additional adhesion molecules, such as E-selectin and VCAM-1 [55], and further enhance leukocyte activation [56].
Delivery of a bolus of adhesion molecules to the endothelial membrane requires the formation of functionally competent secretory organelles. We find that GRK2 depletion does not significantly affect WPB morphology or cargo recruitment, including incorporation of P-selectin. Interestingly, loss of GRK2 does, however, result in a 30% reduction in WPB numbers. This may be explained by the observed increase in unregulated VWF secretion, which is most likely attributable to basal exocytosis of WPBs as described by Giblin et al. [57]. Interestingly, this increase in VWF release is not accompanied by a change in steady-state levels of VWF protein, although as WPBs contain approximately 50% of all intracellular VWF [58], a 30% fall in organelle number is only expected to result in a relatively small reduction in total protein. As the relationship between levels of VWF and numbers of WPBs is complex, with both initial formation and subsequent pre-exocytic stages of WPB biogenesis offering opportunities to adjust this ratio [24], it is currently difficult to determine why a fall in protein is not observed.
The simplest explanation for the effects of GRK2 depletion on unregulated VWF secretion is a general failure in GPCR desensitization. The observed difference in VWF release between GRK2-depleted cells assayed in serum-free and reduced-serum media does suggest that unidentified extracellular agonists may play such a role. Unregulated secretion is, however, still enhanced by 30% in the absence of serum. This could result from autocrine or paracrine signaling; HUVEC confluency is known to affect WPB numbers [59]. Alternatively, the recent observation that GRK2 depletion increases mitochondrial production of ROS [16], known stimulants of WPB exocytosis [60], may promote low-level endothelial-autonomous stimulation of WPB exocytosis.
Reduced organelle number had no inhibitory effect on histamine-evoked VWF secretion. On the basis that, at most, only 25% of total intracellular VWF was released from GRK2-deficient cells upon histamine stimulation, we suggest that organelle numbers are not limiting under these conditions. Whether WPB number is relevant under conditions such as chronic stimulation, remains to be determined. Moreover, in vivo, endothelial cells are unlikely to be confronted by a single stimulus following injury or infection. Angiotensin, vasopressin and adrenaline all bind to receptors that are under GRK2 regulation [61][62][63] and capable of stimulating WPB exocytosis [64][65][66]. Indeed, VWF release is also enhanced in response to adrenaline in GRK-depleted HUVECs (data not shown). It may be that the combined activities of these secretagogues, enhanced by the absence of receptor desensitization, would exhaust WPBs in GRK-depleted cells quicker than in control cells.
In conclusion, our data are consistent with a model in which GRK2 limits endothelial activation at steady-state and during an inflammatory response by desensitizing GPCRs, including the histamine receptor, to the presence of agonist. Together with previously published data, this suggests that GRK2 activity could be required in both endothelial cells and leukocytes to limit excess cellular infiltration of inflamed tissues. The ability to modulate an inflammatory response in at least two of the cell types involved potentially allows for GRK2 to mount a coordinated control of this finely balanced process.
Addendum N. L. Stevenson performed research, carried out data interpretation and wrote the paper. B. Martin-Martin performed research. J. Freeman performed research. J. Kriston-Vizi performed research and contributed to design of the project. R. Ketteler contributed to design of the project. D. F. Cutler contributed to design of the project and interpretation of data, and wrote the paper.