Endothelial APC/PAR1 distinctly regulates cytokine-induced pro-inflammatory VCAM-1 expression

Introduction: Dysfunction of the endothelium impairs its’ protective role and promotes inflammation and progression of vascular diseases. Activated Protein C (APC) elicits endothelial cytoprotective responses including barrier stabilization, anti-inflammatory and anti-apoptotic responses through the activation of the G protein-coupled receptor (GPCR) protease-activated receptor-1 (PAR1) and is a promising therapeutic. Despite recent advancements in developing new Activated protein C variants with clinical potential, the mechanism by which APC/PAR1 promotes different cytoprotective responses remains unclear and is important to understand to advance Activated protein C and new targets as future therapeutics. Here we examined the mechanisms by which APC/PAR1 attenuates cytokine-induced pro-inflammatory vascular cell adhesion molecule (VCAM-1) expression, a key mediator of endothelial inflammatory responses. Methods: Quantitative multiplexed mass spectrometry analysis of Activated protein C treated endothelial cells, endothelial cell transcriptomics database (EndoDB) online repository queries, biochemical measurements of protein expression, quantitative real-time polymerase chain reaction (RT-qPCR) measurement of mRNA transcript abundance, pharmacological inhibitors and siRNA transfections of human cultured endothelial cells. Results: Here we report that Activated Protein C modulates phosphorylation of tumor necrosis factor (TNF)-α signaling pathway components and attenuates of TNF-α induced VCAM-1 expression independent of mRNA stability. Unexpectedly, we found a critical role for the G protein-coupled receptor co-receptor sphingosine-1 phosphate receptor-1 (S1PR1) and the G protein receptor kinase-2 (GRK2) in mediating APC/PAR1 anti-inflammatory responses in endothelial cells. Discussion: This study provides new knowledge of the mechanisms by which different APC/PAR1 cytoprotective responses are mediated through discrete β-arrestin-2-driven signaling pathways modulated by specific G protein-coupled receptor co-receptors and GRKs.


Introduction
Endothelial dysfunction, a hallmark of inflammation, is linked to the pathogenesis of vascular diseases and results in barrier disruption, inflammation and apoptosis (Pober and Sessa, 2007).There are currently limited treatment options for improving endothelial dysfunction resulting in high morbidity and mortality (Dombrovskiy et al., 2007;Goldenberg et al., 2011).Activated Protein C (APC), a natural anti-coagulant protease, is a promising therapeutic and exhibits multiple pharmacological benefits in preclinical studies of injury and inflammation (Kerschen et al., 2007;Griffin et al., 2015;Griffin et al., 2018).In endothelial cells, APC elicits several cytoprotective responses including barrier stabilization, anti-inflammatory and antiapoptotic activities (Riewald and Ruf, 2005;Mosnier et al., 2007;Molinar-Inglis et al., 2021).Protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR), is the central mediator of APC cellular signaling in endothelial cells.
Activation of PAR1 occurs through irreversible proteolytic cleavage of its' N-terminus, generating a new N-terminal tethered ligand sequence that binds intramolecularly to the receptor to trigger transmembrane signaling.The classic mechanism of PAR1 activation established for the coagulant protease thrombin occurs through cleavage at the arginine (R)-41 site within the N-terminus (Vu et al., 1991).However, APC bound to endothelial protein C receptor activates PAR1 via cleavage at an N-terminal R-46 site, generating a distinct tethered ligand that drives endothelial cytoprotection (Mosnier et al., 2012;Sinha et al., 2018).In addition, APC/PAR1 localization to caveolae, caveolin-1 enriched microdomains, abundant in endothelial cells is required for cytoprotective signaling (Bae et al., 2008;Russo et al., 2009;Birch et al., 2021;Molinar-Inglis et al., 2021).We discovered that APC/PAR1 signals primarily via βarrestin-2 rather than heterotrimeric G proteins to promote endothelial barrier stabilization (Soh and Trejo, 2011) and protection against cytokine-induced apoptosis (Molinar-Inglis et al., 2021).β-arrestin-2 has also been implicated in mediating APC/PAR1 endothelial antiinflammatory responses in vitro and in vivo (Roy et al., 2016;Kanki et al., 2019).However, there remains a limited understanding of the diverse mechanisms by which APC-activation of PAR1 promotes endothelial cytoprotection.
In the present study, we examined the mechanisms by which APC modulates tumor necrosis factor-α (TNF-α) cytokine induced inflammatory responses in human cultured endothelial cells.We show that APC stimulation alters phosphorylation of several TNF-α signal pathway components and attenuates of TNF-α induced vascular cell adhesion molecule-1 (VCAM-1) expression independent of mRNA stability.Unexpectedly, we found a role for S1PR1 and GRK2 in APC/PAR1 mediated suppression of cytokine-induced VCAM-1 expression.This study reveals that different APC/PAR1 cytoprotective responses are mediated by discrete β-arrestin-2-driven signaling pathways modulated by specific co-receptors and GRKs.
2 Materials and methods

Endothelial cell database (EndoDB) analysis
To explore the effect of TNF-α on adhesion molecule and GRK mRNA expression in endothelial cells, we examine the online publicly available endothelial cell database (EndoDB) (https:// endotheliomics.shinyapps.io/endodb/)(Khan et al., 2019), which contains normalized and curated RNA-seq datasets collected from the Gene Expression Omnibus and ArrayExpress repositories.We examined four independent studies from HUVEC (#1, E-GEOD-8166_P2; #2, E-SGRP-3; #3, E-GEOD-2639 and #4, E-GEOD-9055) and one from aorta derived endothelial cells (#5, E-MEXP-3540).Data were analyzed for changes in mRNA transcript levels in control cells and TNF-α treated cells for the adhesion molecules VCAM1, ICAM1 and SELE, as well as for the GRKs (GRK2, GRK3, GRK5, GRK6).The relative mRNA levels, including replicates, of each of the 5 independent studies were analyzed by box-and-whisker plot with median and range from minimum-to-maximum.

mRNA stability assay
Endothelial EA. hy926 cells were seeded in 6-well plates at 3.2 × 10 5 cells per well, cultured and serum-starved and pretreated with 20 nM APC for 3 h, followed by incubation with TNF-α (10 ng/mL) for 24 h 37 °C.Cells were then washed with PBS and then left untreated (0 h) or treated with 10 μg/mL Actinomycin D for 2, 4, 5 and 6 h at 37 °C.RNA was extracted and VCAM1 mRNA transcripts quantified by RT-qPCR using a TaqMan Gene Expression Probe (VCAM1, #Hs01003372_m1 ThermoFisher) as described below.

Bioluminescence resonance energy transfer (BRET) assay
HEK293 cells were purchased from ATCC and maintained at 37 °C in 5% CO 2 in Dulbecco's Modified Eagle Medium (DMEM) (Gibco #10-013-CV) supplemented with 10% fetal bovine serum (FBS) (Gibco #10437-028).HEK293 cells were seeded at 2.5 × 10 5 cells per well of a 12-well plate, grown overnight, and then transfected with BRET donor RLuc-β-arr2 (100 ng) together with BRET acceptor PAR1-YFP (400 ng) and the APC co-receptor EPCR Halo (200 ng), with polyethylenimine (PEI) diluted in Opti-MEM.The next day, cells were pooled and re-seeded into poly-D-lysine coated 96-well plate at 3 × 10 4 cells per well and grown overnight.The following day, cells were washed with PBS and starved for 1 h using a 1:1 equal mixture of DMEM no phenol red (Gibco, #31053-028) combined with PBS.After starvation, cells were preincubated with 5 µM of RLuc substrate Coelenterazine H (Biotium, #10111-1) for 5 min followed by the addition of 20 nM APC (Prolytix, HCAPC-0080) and BRET measurements were taken at 37 °C over time.All BRET measurements were performed with a Berthold TriStar LB941 multimode plate reader running MicroWIN 2010 software (Berthold Biotechnologies) using two filter settings: 480 nm for Rluc and 530 nm for YFP.The BRET signal was calculated as the emission at 530 nm divided by the emission at 480 nm.The BRET signals were normalized to basal BRET ratios and expressed as the percent over basal.

Quantitative phospho-proteomics mass spectrometry analysis
APC-induced changes in phosphorylation of TNF-α signaling pathway components were determined from three biological replicates using tandem mass tag 10-plex quantitative phosphoproteomic analysis as we previously described (Lin et al., 2020).Briefly, endothelial EA. hy926 cells were grown, serum starved overnight, and stimulated with 20 nM APC for 0, 15 or 30 min and processed as described (Lin et al., 2020).Temporal changes in phosphorylation of TNF-α pathway components are displayed as heat maps, where each row is colored by relative abundance from minimum (blue) and maximum (red) intensity of the phosphopeptide abundance or not detected (N.D.) colored as gray.The data are also displayed as floating bar graphs of the average and minimum-to-maximum of the individual replicates (not including N.D.).

Model and prediction analysis
Adobe Illustrator and Photoshop were used to create figures.The cartoons were created with BioRender.com.The "TNF Pathway" was adapted from BioRender.com(2023) https://app.biorender.com/biorender-templates.

Statistical analysis
Data were analyzed using Prism software (version 9.4.1;GraphPad Software).Statistical analysis was determined by performing one-way analysis of variance (ANOVA) with multiple comparisons.

APC attenuates TNF-α-induced VCAM-1 protein expression but not mRNA stability
Next, we examined TNF-α-induced VCAM-1 and ICAM-1 adhesion molecule protein expression in endothelial EA. hy926 cells by immunoblot.TNF-α induced a significant increase in VCAM-1 protein expression that remained elevated after 6 h of TNF-α stimulation (Figure 3A).Similarly, a significant increase in ICAM-1 protein expression in endothelial cells was also observed following TNF-α treatment (Figure 3A).To determine how APC/ PAR1 modulates TNF-α inflammatory responses, endothelial cells were pretreated with or without APC and then stimulated with TNF-α.In APC pretreated cells, TNF-α-stimulated VCAM-1 protein expression was significantly inhibited (Figure 3B), indicating that APC attenuates cytokine-induced VCAM-1 expression.However, APC pretreatment failed to modulate ICAM-1 expression in HUVEC-derived EA. hy926 cells detected by immunoblot (Figure 3C) and was not further evaluated.We next examined if APC's capacity to attenuate TNF-α-induced VCAM-1 expression was related to VCAM1 mRNA stability.In these experiments, endothelial cells were pretreated with APC, stimulated with TNF-α and then incubated with actinomycin D to block gene transcription and the abundance of VCAM1 mRNA measured over time using RT-qPCR.In the absence of APC pretreatment, an approximately 50% loss of VCAM1 mRNA was detected after actinomycin D-mediated blockade of gene transcription (Figure 3D).A similar 50% loss of VCAM1 mRNA was observed in endothelial cells pretreated with APC and actinomycin D (Figure 3D).We further examined the effect of APC pretreatment on TNF-α expression by RT-qPCR and observed a negligible effect (Figure 3E), whereas TNF-α induced a significant increase in TNF-α mRNA transcripts.These data suggest that APC's capacity to modulate VCAM-1 expression does not occur at the level of VCAM mRNA stability or modulation of TNF-α mRNA expression.
In addition to PAR1, PAR3 has been shown to function as a coreceptor for APC-mediated barrier protection but its role in antiinflammatory responses is not known (Burnier and Mosnier, 2013).To determine if PAR3 contributes to APC-mediated protection against TNF-α induced inflammatory responses we used siRNA to deplete cells of PAR3 expression, since there are no effective antagonists.We first optimized the efficiency and specificity of siRNA-targeted PAR1 and PAR3 mRNA degradation in endothelial cells using RT-qPCR.Endothelial cells transfected with the PAR1-specific siRNA caused significant degradation of PAR1 mRNA transcripts but not PAR3 mRNA transcripts measured by RT-qPCR (Figure 4B).Similarly, the PAR3-specific siRNA caused a significant loss of PAR3 mRNA transcripts but not PAR1 mRNA abundance in transfected endothelial cells (Figure 4C).We next examined the contribution of PAR1 and PAR3 to APC-mediated protection against TNF-α-induced VCAM-1 protein expression utilizing the PAR1 and PAR3 specific siRNAs.As expected, APC pretreatment reduced TNF-αstimulated VCAM-1 protein expression in control non-specific siRNA transfected endothelial cells (Figure 4D).In contrast to control cells, in endothelial cells depleted of PAR1 by siRNA, APC was not able to reduce TNF-α stimulated VCAM-1 expression (Figure 4D).Interestingly, in endothelial cells diminished of PAR3 expression by siRNA, APC retained the capacity to inhibit TNF-α induced VCAM-1 expression (Figure 4D).These data suggest that PAR1 and not PAR3 is the critical mediator of APC-protection against TNF-α-induced VCAM-1 expression.

APC/PAR1 protection against TNF-α induced VCAM-1 expression requires GRK2 and not GRK5
APC/PAR1 cellular signaling is mediated primarily by the βarrestin-2 isoform, which drives most if not all aspects of endothelial cytoprotection (Soh and Trejo, 2011;Roy et al., 2016;Kanki et al., 2019;Molinar-Inglis et al., 2021).GRKs are known to mediate phosphorylation of activated GPCRs and facilitate the recruitment of β-arrestins.To determine which GRKs function in APC/ PAR1 protection against TNF-α mediated inflammatory responses, we first used the publicly available transcriptomics Endothelial Cell DataBase (EndoDB) RNA-seq gene expression datasets to examine GRK expression and potential modulation by TNF-α in five separate RNA-seq studies including four HUVEC datasets and an aortic endothelial cell dataset.GRK5 and GKR2 appeared to be more highly expressed in human endothelial cells compared to GRK3 and GRK6 expression (Figures 5A-D), similar to that previously reported for GRK expression in cardiac tissue (Rockman et al., 1996;Penela et al., 2003;Dzimiri et al., 2004;Matkovich et al., 2006).Interestingly, TNF-α failed to modulate expression of the highly expressed GRK5 and GRK2 as well as the moderately expressed GRK3 and GRK6 in human endothelial cells (Figure 5), suggesting that GRK gene expression is not regulated by the pro-inflammatory TNF-α cytokine.
To assess the role of GRKs in APC/PAR1 protection against TNFα-stimulated inflammatory responses, we first used siRNA to deplete endothelial cells of endogenous GRK5 and GRK2 expression.Surprisingly, APC-mediated attenuation of TNF-α-stimulated VCAM-1 expression was virtually ablated in endothelial cells deficient in GRK2 expression, compared to control non-specific siRNA transfected cells (Figure 6A).In addition, depletion of endogenous GRK5 expression failed to effect APC's capacity to attenuate TNF-α stimulated VCAM-1 expression (Figure 6A), consistent with a preferential role for GRK2.To further examine the role of GRK2 in APC/PAR1 endothelial anti-inflammatory responses, we utilized CMPD101, a membrane permeable selective inhibitor of GRK2/GRK3 activity.In DMSO vehicle pretreated endothelial cells, APC exhibited robust cytoprotective against TNFα stimulated VCAM-1 expression (Figure 6B), which was blocked by the PAR1 antagonist vorapaxar.However, in CMPD101 pretreated endothelial cells, APC failed to protect against TNF-α-induced VCAM-1 expression (Figure 6B).To further evaluate GRK2 function, the role of GRK2 in APC-induced β-arr2 recruitment to PAR1 was examined using BRET assays in HEK293 cells.In control DMSO pretreated cells, APC stimulated an increase in β-arr2 recruitment to PAR1 that appeared to peak at 20 min (Figure 6C).In contrast, APC-stimulated β-arr2 recruitment to PAR1 was significantly inhibited in CMPD101 treated cells (Figure 6C).Together these studies report an unexpected regulation of APC/PAR1-mediated protection against TNF-αinduced VCAM-1 expression that is mediated by PAR1/ S1PR1 and GRK2 and likely controlled by AP-1, p38 MAPK and NF-κB transcription factor pathways (Figure 7).

Discussion
In this study, we define a new pathway by which APC modulates TNF-α cytokine induced VCAM-1 expression in human cultured endothelial cells.We found that APC stimulation alters phosphorylation of several TNF-α signaling mediators associated with AP-1, p38 MAPK and NF-κB transcription factor pathways assessed by mass spectrometry.In addition, we verified using a repository of endothelial cell RNA-seq datasets that TNF-α induces expression of VCAM-1, ICAM-1 and E-Selectin, which are known to be transcriptionally regulated by AP-1, p38 MAPK and NF-κB pathways and mediate leukocyte recruitment and inflammatory responses (Pober, 2002).We further show that APC/ PAR1 attenuates TNF-α-induced VCAM-1 expression independent of mRNA stability.Using a combination of pharmacological inhibitors and siRNAs, we demonstrate a critical role for S1PR1 and GRK2 in APC/PAR1 protection against TNF-α induced VCAM-1 expression.This study suggests that different APC/PAR1 cytoprotective responses are mediated by discrete βarrestin-2-driven signaling pathways that are specified by distinct co-receptors and GRKs.
An important finding from our study is that different GPCR coreceptors drive distinct β-arrestin-2 mediated cytoprotective responses.Co-receptor interaction is known to alter GPCRinduced β-arrestin biased signaling (Shen et al., 2018), but is understudied in biased signaling.We found that GPCR coreceptor interaction is relevant to APC/PAR1 cytoprotection.Here we show that S1PR1 functions as a GPCR co-receptor to facilitate APC/PAR1's ability to suppress TNF-α induced VCAM-1 expression (Figure 4), an anti-inflammatory response.These findings are consistent with S1PR1 capacity to decrease leukocyte adhesion molecule expression (Galvani et al., 2015;Burg et al., 2018).Previous studies established a role for PAR3 in APC/PAR1 mediated endothelial barrier protection in vitro (Burnier and Mosnier, 2013).Other studies demonstrated a function for PAR3 in APC/PAR1mediated neuroprotection and cytoprotective signaling in podocytes in vivo (Guo et al., 2004;Guo et al., 2009;Madhusudhan et al., 2012).While S1PR1 has been shown to contribute to basal integrity of the endothelial barrier (Feistritzer and Riewald, 2005), the role of S1PR1 in APC/PAR1-induced cytoprotective responses are less clear.We recently showed that S1PR1 is required for APC/ PAR1-induced Akt-mediated anti-apoptotic activities induced by TNF-α in human cultured endothelial cells (Molinar-Inglis et al., 2021).Here, we now show that S1PR1 is also necessary for APC/ PAR1 protection against TNF-α-stimulated VCAM-1 expression, suggesting common regulatory pathways.These studies indicate that different GPCR co-receptors modulate distinct APC/ PAR1 cytoprotective responses.
The mechanisms by which GPCR co-receptors influence APC/PAR1-stimulated β-arrestin-2-mediated downstream signaling are not well defined.In previous work, we reported that APC/PAR1 signals independent of Gi protein (Soh and Trejo, 2011) but rather activates β-arrestin-2-dependent polymerization of dishevelled-2 (Dvl-2), ERK1/2 and Rac-1 FIGURE 5 GRK2,3 and GRK5,6 expression in endothelial cells is not modified by TNF-α.GRK mRNA expression was examined in five independent studies including four HUVEC RNA-seq datasets and one aortic endothelial cell RNA-seq data sets treated with or without TNF-α.Data for analysis of GRK2 (A), GRK3 (B), GRK5 (C) and GRK6 (D) expression in endothelial cells with or without TNF-α treatment were obtained from Endothelial Cell DataBase (EndoDB).The relative mRNA levels, including replicates, of each of the 5 independent studies were analyzed by box-and-whisker plot with median and range from minimum-to-maximum.N. D is not detected.
We found that APC modulates multiple TNF-α regulated signaling components that control AP-1, p38 MAPK and NF-κB transcription factor pathways based on our mass spectrometry analysis (Figure 1), which is consistent with reported studies (Joyce et al., 2001;Guitton et al., 2011).However, it is not known how APC/PAR1 signaling modulates AP-1, p38 MAPK and NF-κB regulated transcription factor pathways to control VCAM-1 expression induced by TNF-α, while not affecting TNF-αstimulated ICAM-1 expression.The VCAM-1 and ICAM-1 gene promoter regions contain several inducible transcription factor binding sites including sites for AP-1, NF-κB, Sp1 and other transcription factors (Cook-Mills et al., 2011).In addition, TNF-α signals through multiple pathways including AP-1, p38 MAPK and NF-κB to regulate VCAM-1 and ICAM-1 gene expression (Figure 1), but precisely how these pathways converge to enable APC to suppress cytokine-induced VCAM-1 and not ICAM-1 expression in HUVEC-derived EA. hy926 cells is not known (Figure 7).Our findings indicate that APC/ PAR1 likely controls VCAM-1 gene transcription since APC failed to modulate VCAM-1 mRNA stability (Figure 3D), consistent with APC/PAR1 phosphoproteome association with processes such as DNA binding and chromatin regulators identified in gene ontology analysis (Lin et al., 2020).
Classic biased agonists use different GRKs to distinctly phosphorylate activated GPCRs, which promotes different β-arrestin binding modes and functions (i.e.desensitization, internalization and signaling) (Kim et al., 2005;Ren et al., 2005;Shenoy et al., 2006;Drube et al., 2022).Of the seven GRKs, GRK2/GRK3 and GRK5/GRK6 are ubiquitously expressed and relevant to endothelial biology (Tiruppathi et al., 2000;Roy et al., 2016).Indeed, analysis of endothelial RNA-seq datasets indicate that GRK5 and GKR2 are highly expressed in human endothelial cells (Figure 5).GRK5 was shown previously to desensitize thrombin/PAR1 signaling in human cultured endothelial cells (Tiruppathi et al., 2000).However, the function of GRKs in regulation of APC/ PAR1 cytoprotective responses is not well understood.A previous study implicated GRK5 in APC/PAR1 protection against endothelial barrier permeability (Roy et al., 2016).Intriguingly, we found that GRK2 and not GRK5 mediates APC/PAR1 protection against TNF-α induced VCAM-1 expression (Figure 6).However, the regulation of APC/ PAR1 cytoprotective signaling is more complex with GRK5 displaying dual functions controlling both thrombin signaling as well as APC/PAR1 cytoprotective signaling.The mechanism responsible for divergent GRK activity at APCactivated PAR1 is not known.In addition, our studies suggest that both GRK5 and GRK2 can promote APC/PAR1-stimulated β-arrestin-2-dependent signaling.However, it is unclear if GRK5 and GRK2 have additional roles is desensitization of either APC-activated PAR1 or the S1PR1 receptor.

Conclusion
In summary, this study illustrates the complexity of APC/PAR1 β-arrestin-2 signaling that is controlled by different GPCR coreceptors and GRKs that drive distinct endothelial cytoprotective responses.Understanding APC/PAR1 signaling pathways that enable the endothelium to acquire resilience to resist injury and disruption is critical for the advancement of new targets for therapeutic development.

FIGURE 1
FIGURE 1 APC modulates TNF-α stimulated proinflammatory pathways in endothelial cells.(A) TNF-α signals through AP-1, p38 MAPK (MAPK14), and NF-κB pathways to promote inflammation and apoptosis in endothelial cells.(B) Heat map of TNF-α pathways components illustrates phospho-peptide abundance from endothelial cells treated with APC for the indicated times from three biological replicates, where increases and decreases in phosphopeptide abundance are indicated by the red and blue intensities, respectively and gray is not detected (N.D.).Specific changes in NF-κB (C), AP-1 (D) and p38 MAPK (E) pathway components phospho-peptide abundance detected in endothelial cells treated with or without APC are shown as floating bar graphs of the average and minimum-to-maximum of the individual replicates.(F) APC stimulation of p38 MAPK phosphorylation in endothelial cells was validated by immunoblot.Data (mean ± S.D.) are from three independent experiments and expressed as fold over control (0 min) analyzed by ANOVA; *, p < 0.05; ***, p < 0.001; ****, p < 0.0001.

FIGURE 2
FIGURE 2 TNF-α induces VCAM1, ICAM1 and SELE mRNA expression in endothelial cells.Relative abundance of (A) VCAM1, (B) ICAM1 and (C) SELE (E-Selectin) mRNA transcripts in endothelial cells treated with or without TNF-α were obtained from the online repository Endothelial Cell DataBase (EndoDB) endothelial cell RNA-seq transcriptomics datasets.The relative mRNA levels, including replicates, of each of the 5 independent studies were analyzed by box-and-whisker plot with median and range from minimum-to-maximum.

FIGURE 3
FIGURE 3 APC attenuates TNF-α-induced VCAM-1 expression in endothelial cells.(A) Endothelial cells were stimulated with TNF-α for the indicated times.Cell lysates were immunoblotted for expression of VCAM-1 and ICAM-1 and GAPDH as a loading control.Quantifications of VCAM-1 and ICAM-1 induction are shown as the mean ± S.D. from three independent experiments analyzed by one-way ANOVA; *, p < 0.05; ***, p < 0.001; ****, p < 0.0001.(B,C) Endothelial cells were pre-treated with or without APC and then stimulated with or without TNF-α for 24 h.Cell lysates were immunoblotted for VCAM-1, ICAM-1 and GAPDH expression.Quantification of VCAM-1 and ICAM-1 induction (mean ± S.D.) from three independent experiments was analyzed by one-way ANOVA; ***, p < 0.001; n. s, not significant.(D) Endothelial cells were pre-treated with or without APC, stimulated with TNF-α, and then treated with Actinomycin D for indicated times.Relative VCAM-1 mRNA expression was quantified by RT-qPCR and normalized to 18S ribosomal mRNA expression and is represented as the fold-change relative to TNF-α-induced VCAM-1 expression without Actinomycin D treatment (mean ± S.D.) from three independent experiments.Data were analyzed by one-way ANOVA.(E) Endothelial cells were treated with APC or TNF-α for 24 h and changes in TNF-α mRNA transcripts were quantified by RT-qPCR, normalized to 18S mRNA expression and represented as the fold change relative to untreated control.Data (mean ± S.D.) from three independent experiments were analyzed by one-way ANOVA; ****, p < 0.0001.

FIGURE 7
FIGURE 7Model of APC/PAR1-S1PR1 mediated protection against TNF-α induced VCAM1 expression.In endothelial cells, APC bound to EPCR cleaves and activates PAR1 to stimulate β-arr2 mediated cytoprotection against TNF-α induced upregulation of adhesion molecule VCAM-1 expression.Interestingly, APC/PAR1-mediated cytoprotection against TNF-α-induced VCAM-1 expression further requires the co-receptor S1PR1 and unexpectedly is dependent on GRK2.These studies highlight the complexity of APC/PAR1 cytoprotective signaling, which utilizes multiple co-receptors and different GRKs to promote distinct β-arr2-dependent cytoprotective responses in endothelial cells.The mechanism by which APC/PAR1 signaling intersects with the TNFα-stimulated AP-1, p38 MAPK and NF-κB pathways to promote gene transcription of VCAM-1 expression is not known.