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Unexpected role of anticoagulant protein C in

controlling epithelial barrier integrity and
intestinal inflammation
Stefania Vetranoa, Victoria A. Ploplisb, Emanuela Salaa, Mayra Sandoval-Cooperb, Deborah L. Donahueb,
Carmen Correalea, Vincenzo Arenac, Antonino Spinellid, Alessandro Repicia, Alberto Malescia, Francis J. Castellinob,1,2,
and Silvio Danesea,1,2
a
IBD Unit, Division of Gastroenterology, Istituto Clinico Humanitas IRCCS, Rozzano 20089 Milan, Italy; bW. M. Keck Center for Transgene Research, University
of Notre Dame, Notre Dame, IN 46556; cDepartment of Pathology, Catholic University of Rome, 00168 Rome, Italy; and dDivision of General Surgery III, Istituto
Clinico Humanitas, 20089 Rozzano Milan, Italy

Edited* by Charles A. Dinarello, University of Colorado Denver, Aurora, CO, and approved October 25, 2011 (received for review May 12, 2011)

The protein C (PC) pathway is a well-characterized coagulation epithelial cells (ECs), vascular endothelium, platelets] and co-
system. Endothelial PC receptors and thrombomodulin mediate agulation are deeply involved in the pathogenesis of IBD (6–10).
the conversion of PC to its activated form, a potent anticoagulant In particular, we recently reported that the PC pathway controls
and anti-inflammatory molecule. Here we show that the PC path- microvascular inflammation by down-regulating endothelial
way is expressed on intestinal epithelial cells. The epithelial ex- EPCR and TM expression, thereby impairing activation of PC by
pression of PC and endothelial PC receptor is down-regulated In the inflamed mucosal microvasculature (5, 10). Whether PC also
patients with inflammatory bowel disease. PC−/−/PC(Tg) mice, could be involved in intestinal barrier function or in cytopro-
expressing only 3% of WT PC, developed spontaneous intestinal tection is unclear, however (5, 10).
inflammation and were prone to severe experimental colitis. In this paper we report the surprising observation that, along
These mice also demonstrated spontaneous elevated production with its known expression in the microcirculation of the gut
of inflammatory cytokines and increased intestinal permeability. mucosa, the epithelial layer of the intestine expresses proteins of
Structural analysis of epithelial tight junction molecules revealed
the PC pathway, which play a unique role in regulating intestinal
permeability and controlling the integrity of tight junctions. Ex-
that lack of PC leads to decreased JAM-A and claudin-3 expression
pression of epithelial PC is altered in patients with CD or UC,
and an altered pattern of ZO-1 expression. In vitro, treatment of
and its deletion in mice leads to spontaneous colitis. Restoring
epithelial cells with activated PC led to protection of tight junction
epithelial PC is therapeutically effective in mice and could rep-
disruption induced by TNF-α, and in vivo, topical treatment with
resent a novel treatment approach in humans with IBD.
activated PC led to mucosal healing and amelioration of colitis. Taken
together, these findings demonstrate that the PC pathway is a Results
unique system involved in controlling intestinal homeostasis and in- Expression of EPCR, PAR-1, PC, and TM by ECs from Healthy Individuals
flammation by regulating epithelial barrier function.
and Patients with IBD. To investigate whether intestinal epithelium
expresses components of the PC system, we performed confocal
intestinal barrier integrity | DSS-induced colitis | intestinal permeability and microscopy using specific antibodies to PC, EPCR, PAR-1, and
tight junction proteins TM, and an antibody for pan-cytokeratin, a specific epithelial
marker, on sections of colon obtained from 16 healthy individ-

I nflammation and coagulation are closely linked, interdepen-
dent processes (1, 2). Under physiological conditions, the
molecules within the microcirculation of tissues act in an anti-
uals (Fig. 1A) (11). PC, EPCR, and PAR-1, but not TM, were
expressed by ECs in the intestine, as indicated by colocalization
with pan-cytokeratin (Fig. 1A). Staining for the same proteins in
coagulant and anti-inflammatory manner. However, when in- specimens from patients with active CD (n = 12) and active UC
flammation occurs, coagulation is also triggered and participates (n = 13) revealed a significant decrease in the epithelial ex-
in the spreading of inflammation. Unexpected roles of hemo- pression of EPCR and PC compared with healthy individuals
stasis in the humoral and cellular mechanisms of innate immu- (Fig. 1A). No significant difference was found in PAR-1 ex-
nity have been reported recently (2). One of the major systems pression on ECs between NL subjects and patients with CD,
that forms a bridge between inflammation and coagulation is whereas PAR-1 was significantly down-regulated in patients with
the protein C (PC) pathway (3). This pathway is composed of UC compared with NL subjects and patients with CD. We
thrombomodulin (TM), endothelial cell PC receptor (EPCR), quantified protein expression in the tissue by a semiquantitative
PC, and protease-activated receptor-1 (PAR-1). TM and EPCR score (10, 12). We found a significant decrease in the expression
are expressed mainly by vascular endothelial cells and form a of epithelial EPCR in tissue samples from patients with UC
complex on the surface of these cells that converts circulating PC (0.8 ± 0.2) and those with CD (0.8 ± 0.2) compared with mucosa
into its active form (3). from normal individuals (2.7 ± 0.1) (P < 0.001). Similar results
Although the function of the PC pathway is classically con- were obtained for PC expression, which was significantly
sidered anticoagulative, mounting evidence indicates that this
pathway also plays a dominant role in inflammation, with each
component of the pathway displaying remarkably potent anti- Author contributions: F.J.C. and S.D. designed research; S.V., E.S., M.S.-C., D.L.D., C.C.,
inflammatory activity (2–4). Indeed, PC is now emerging as a V.A., A.S., A.M., and A.R. performed research; F.J.C. contributed new reagents/analytic
tools; S.V., V.A.P., and F.J.C. analyzed data; and S.V. and S.D. wrote the paper.
participant in the pathogenesis of acute and chronic inflam-
matory diseases, such as sepsis, asthma, inflammatory bowel The authors declare no conflict of interest.
disease (IBD), atherosclerosis, and lung and heart inflammation, *This Direct Submission article had a prearranged editor.
and might represent a previously unexpected therapeutic target 1
F.J.C. and S.D. contributed equally to this work.
for intervention (3, 5). 2
To whom correspondence may be addressed: E-mail: fcastell@nd.edu or sdanese@
Crohn’s disease (CD) and ulcerative colitis (UC) are the two hotmail.com.
major forms of IBD. Although these are immune-mediated This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
diseases, we and others have shown that nonimmune cells [e.g., 1073/pnas.1107140108/-/DCSupplemental.

19830–19835 | PNAS | December 6, 2011 | vol. 108 | no. 49 www.pnas.org/cgi/doi/10.1073/pnas.1107140108

the low-PC mice exhibited spontane- ous intestinal inflammation characterized by the presence of B A B Fig. ECs in healthy subjects expressed PC. PC and EPCR expres- sion was decreased by 47%.A intestine. PAR-1. (A) Immunofluorescent staining shows the expression and colocalization of EPCR. we used PC−/−/PC(Tg) (“low-PC”) mice. PNAS | December 6. The degree of colitis increased after administration of dextran sodium sulfate (DSS). and PAR-1. *P < 0. PAR-1. In patients with IBD. and PAR-1 expression was decreased by 30% (Fig. Decreased expression of components of the PC pathway in intestinal epithelial cells of patients with IBD. Histological images were taken using a 20× objective. S1). To study the consequences mice. After mice were killed.6 ± 0. The expression of mRNA for the colon of WT and low-PC mice before (day 0) and after 7 d (day 7) of 2% DSS administration demonstrates the spontaneous mucosal hyperemia and TM was undetectable (Fig. When mice (6–8 wk old) underwent colonoscopy.05. No decrease in PC and E F EPCR staining was found in specimens from patients in an in- active disease state. (E) Red arrows indicate the presence of small lymphoid follicles at day 0 and increased leukocyte infiltrate at day 7 in the mucosa of low PC Spontaneous Colitis in PC-Deficient Mice. Bleeding and the presence of mucosal erosions that were absent in WT mice characterized this damage. 2 C and D). patients with CD (n = 6).2) compared with mucosa from control individuals (2.4 NA Plan-Apochromat. and that Trends toward greater body weight loss (C) and disease activity index (DAI) this expression is significantly reduced in the inflamed intestines (D) in low-PC mice compared with their littermates over the course of DSS of patients with active IBD. but not of TM (in green) with the EC marker pan-cytokeratin (in red) in normal subjects (NL). These results suggest that inflammation down- regulates the expression of EPCR and PC on ECs within the intestine.001). decreased in the epithelium of patients with UC (0. low-PC mice had an endoscopic colitis score indicating the presence of spontaneous colitis characterized by mucosal hyper- emia and friability (P < 0. 2. and TM by RT-PCR. 14).4 ± 0. We next analyzed the expression of the PC pathway in primary ECs by FACS analysis. Spontaneous inflammation and increased susceptibility to DSS-in- confirmed down-regulation of mRNA for EPCR. PC. PC. treatment. In addition. (A) Endoscopic images of mucosal damage in in patients with active CD or UC. PC. 2 A and B). indicated by the white arrows. Furthermore. We also investigated the expression of EPCR. Indeed. 1B). The images were acquired with an oil immersion objective (60×. Vetrano et al. Results from primary ECs isolated from in- testinal tissue of healthy subjects and patients with active IBD Fig. and PAR-1 were detected in the colon of C D patients with active Crohn’s disease (CD) and patients with active ulcerative colitis (UC) with DAPI nuclear stain (blue).1) and those with CD (0. and TM relative to GAPDH in NL subjects (n = 10). TM was not expressed by the intestinal epithelium. PC. which express only 3% of the amount of PC expressed by WT mice (13. EPCR. 108 | no. 49 | 19831 . and PAR-1. 1.5 ± 0.05). (B) Rela- tive changes in mRNA expression of EPCR. and patients with UC (n = 6). these results demonstrate that the intestinal SYSTEMS BIOLOGY doscopic score in WT and low-PC mice before and after treatment. Olympus). No decrease in PC or EPCR friability in low-PC mice and the presence of mucosal erosions at day 7. PC. (C and D) epithelium of healthy subjects expresses PC and EPCR. admin- istration of 3% DSS led to 100% mortality in low-PC mice after 3–4 d of administration. Given that the genetic deletion of PC leads to death soon after birth. Marked reductions in EPCR. with more significant colon damage seen in low-PC mice (P < 0. (B) Quantification of mucosal injury by en- Taken together. minimal doses of DSS were sufficient to induce significant clinical colitis in low-PC mice. we evaluated the susceptibility to experimental colitis in PC-deficient mice.05) (Fig. 2011 | vol. characterized by a sig- nificant loss in body weight and a progressive increase in the overall disease activity index (Fig. 1. as mRNA was found in ECs from patients with quiescent IBD. and PAR-1 duced colitis in low-PC mice. (F) Histological damage scoring at day 0 and day 7 in low-PC and WT of the decreased expression of epithelial PC in the inflamed mice.1) (P < 0.

There was no between-group difference in 0. Interestingly. and ZO-1 tight junc- tion proteins (green) in the colon of low- PC and WT mice before DSS administra- tion (day 0). Changes in TEER were measured over 48 h. 4A). tally abrogated the effects of TNF-α on TEER (TNF + aPC.74 Ω/cm2) compared with low-PC aPC disappeared in the presence of anti-EPCR antibodies. These data are representative of three indepen- dent experiments. JAM-A.05). hanced TEER values similarly to APC (1 μg/mL). Caco- with WT mice (all mediators. After 3 d of DSS signaling.20 Ω*cm2. and EC Increased Mucosal Cytokine Production and Intestinal Permeability in Proliferation. down-regulating epithelial tight junctions. and 12 h of claudin-3. ZO-1. After 7 d of DSS ad. and actively contributes to the disruption of barrier function by macrophage inflammatory protein 2. t = 48: 304. To investigate the role logical findings indicating the presence of spontaneous in. but not TM (Fig.05). As expected. In addition. (A and B) Secretion of IL-6. 3. These data claudin-3 and JAM-A compared with WT mice (P < 0.31 Ω/cm2. To assess this hypothesis. characterized by total crypt loss and diffuse leukocyte in.05). The tween-group difference in the overall expression of tight junc- finding that deletion of PC leads to spontaneous colitis unveils tions after 7 d of DSS-induced colitis. phosphate (S1P). . However. Changes in mucosal cytokine production and the integrity of intestinal barrier function in low-PC mice.) Values represent mean ± SEM. 6.8 ± 26. we investigated paracellular TNF-α (t = 0: 304 ± 23.0001) (Fig. (C and D) Immunofluorescent staining and bar graph showing the expression of JAM-A. Thus. low-PC mice. 3B). (Scale bar: 30 μm. n = 13 for WT mice and n = 20 for low-PC mice. we evaluated the investigated the expression levels of the tight junction proteins changes in TEER values in Caco-2 cells after 0.05) (Fig. and claudin-10 in low-PC and WT stimulation with S1P (1 μM). Consistent with the observed intestinal permeability. Increased leakiness of the intestinal barrier is con- PC-Deficient Mice. 3. These differences were maintained over 7 d of DSS We then cultured Caco-2 cells with and without stimulation by administration (Fig. 2 E and F). *P < 0. KC. a signaling sphingolipid that promotes in- Epithelial permeability is governed by tight junctions. We next measured the mucosal production of sidered a crucial event in the pathogenesis of IBD. the mucosa of low-PC mice displayed a significantly altered and irregular in low-PC mice compared with significantly higher degree of histological inflammation (P < WT mice (Fig. together. Because the aPC–EPCR complex cross-activates sphingosine 1 PC mice. TEER values were markedly diminished in both thelial permeability in an EPCR-dependent manner. nuclei were stained with DAPI (blue). 3B). mice (29. 19832 | www.pnas. when the cells were test. t = sistance (TEER) using a Ussing chamber and the Evans blue dye 48: 381. the addition of exogenous aPC to- DSS administration by measuring transepithelial electrical re. P < 0. even before DSS ad.66 Ω/cm2) (P = 0. with no significant differences (Fig. we hypothesized that PC could play a decrease in TEER was observed after 48 h of stimulation with a key role in this process. we investigated whether alterations in the expression of the proteins forming these aPC induces the potent epithelial barrier protective effects via junctions might lead to a leaky intestinal barrier (15). we S1P activation. intestinal permeability. **P < 0.57 epithelial permeability in low-PC and WT mice before and after Ω*cm2) (Fig. CL-10. support the evidence suggesting the involvement of S1P in the A B Fig. PC Controls Epithelial Tight Junction Integrity. of PC in epithelial barrier function. a role for PC in intestinal inflammation.05. ministration.75 ± 6.85 ± 3.small lymphoid follicles in the mucosa and submucosa mainly in 3C). these data indicate a leaky intestinal barrier in low. Moreover. and TNF-α various inflammatory mediators. TNF-α in the absence and presence of exogenous activated PC Given that PC is expressed by ECs. No effects groups of mice. suggesting that aPC exerts a direct effect on paracellular epi- ministration. claudin-10 expression (Fig. an intact intestinal barrier.9 ± 3. S2). + TNF. t = 48: 78. PAR-1 and EPCR. we first analyzed the ex- flammation.1073/pnas. the expression pattern of ZO-1 was after DSS challenge. 4A). Low-PC mice demonstrated a spontaneous increase in pared with nontreated cells (+ aPC. t = 48: 552 ± 34. with significantly lower values of TEER control. a finding that could explain the leaky intestinal barrier in the colon but not in the small bowel (Fig. P < 0. Notably. 3C).5 Ω*cm2. S3A). The protective effects of higher in WT mice (45. low.org/cgi/doi/10. CL-3. Wound Healing. epithelial permeability was still significantly and absence of anti-EPCR antibodies. which function to maintain (aPC). S1P not only en- mice. Consistent with our histo. there was no be- filtration that was not observed in WT mice (Fig. we performed the same experiments in the presence administration. and creased endothelial barrier protection. 3A). including IL-6.66 ± 2 Ω/cm2) compared with WT mice (88 ± 2 Ω/cm2) the protective effect observed with aPC was dependent on EPCR before DSS challenge (P < 0. but also ab- PC mice showed a significant decrease in the expression of rogated TNF-dependent TEER reduction (Fig.01.0005). IL-8 (KC). To test whether (28.31 Ω*cm2) (Fig. In addition.1107140108 Vetrano et al. 3C). low-PC mice displayed a significant increase in the pression of the PC pathway in a monolayer of the colonic EC line spontaneous production of all inflammatory mediators compared (Caco-2) by immunofluorescence. TEER was monitored as an indicator of epithelial barrier treated only with aPC an increase of TEER was observed com- integrity. 2 E and F). and 7 of DSS administration. 2 cells expressed PC. Thus.8 ± 23. Similar to primary ECs. and macrophage inflammatory protein 2 by the mucosa of the colon (A) and TEER measured from C D the colon (B) of low-PC and WT mice on days 0. Taken on TEER values were seen in the presence of anti-EPCR alone.

TNF. EPCR. PAR-1. Taken together. differences were observed between control and FTY720 alone. sion. Mucosal healing is believed to be an important target for the treatment of IBD (16). Because creased number of ulcers (Fig. immuno. pretreatment with α-EPCR alone did not induce any effects on ECs. individually Vetrano et al. (Fig. Because intestinal fluorescence analysis of JAM-A and ZO-1 (in green) was performed on Caco-2 inflammation leads to down-regulation of epithelial PC expres- cells grown on Transwell filters under different conditions: control. treated mice (P < 0. a [3H]-thymidine incorporation assay revealed that a low con- centration of aPC (1 μg/mL) induced a high rate of Caco-2 cell proliferation. 5E). PNAS | December 6. we tested whether aPC can in- duce intestinal wound closure and cell proliferation. a signif- antibody inhibiting EPCR activity (5 μg/mL). S4C). φP < 0. S3D). S3 B and C). which is particularly true in IBD (8). In the last several years. ζP < 0. 4 B and C). reinforcing the hypothesis that aPC signals through EPCR (Fig. and mice expressed high but physiological levels of PC. 6. DAPI nuclear stain of aPC could improve colitis by favoring epithelial healing. 5 A and B). SphK1. 5A). The cells pretreated with α-EPCR and stimulated with aPC dis- C D played significantly reduced cellular proliferation. Indeed. 49 | 19833 . of the major tight junction proteins in EC lines in the presence The specific hemostasis-related molecules involved in inflam- SYSTEMS BIOLOGY and absence of aPC and before and after stimulation with TNF-α. S4 A and B). and aPC. 4 B and C). The PC pathway Consistent with the in vivo findings in low-PC mice.05 (control vs.01. We began by investigating the expression of epi- by EPCR. S4 A and B). S1P regulates the en- significantly less severe histological score.01(aPC vs. and 12 h. 50 ng/mL) (Fig. coagulation cascade in inflammatory processes. Endoscopically. TM. It has been reported that once demonstrated significantly less colon shortening compared with formed after the phosphorylation of sphingosine by enzyme saline-treated mice (P < 0. Topical aPC Treatment Leads to Improvement of Colitis. with each component of the antibodies totally inhibited such modification of tight junctions. 4. supporting the suggestion of a functional role for EPCR in aPC-mediated epithelial migration (Fig.05) (Fig. whereas the stim- ulation with α-EPCR alone did not alter the expression of JAM- A or ZO-1. (B–D) After 48 h of stimulation. we investigated the expression levels terrelated processes. To in- vestigate wound closure. Inflammation is the result of an imbalance between proinflam- FTY720 treatment displayed no inhibitory effects on aPC ac.A regulation of tight junctions (Fig. and it has been To further explore the mechanism by which aPC reinforces firmly established that coagulation and inflammation are in- epithelial barrier function. tivity. we found that is a major pathway bridging inflammation and coagulation (3). *P < 0. Furthermore. the use of anti-EPCR dominant role in inflammation. and the mice that received intrarectal aPC epithelial barrier protection. To explore the therapeutic potential of our in vitro findings. TNF + aPC + EPCR. control).) Values are mean ± SEM. TNF and TNF aPC by a significant increase in body weight compared with saline- EPCR). pathways involved in hemostasis (1. as indicated tative of three independent experiments. θP < 0. 2011 | vol. it has become clear that inflammation involves unique pathways rogated (P < 0. (Scale bar: 60 μm. matory and anti-inflammatory pathways. at a level comparable to that induced by epithelial growth factor (EGF. Evidence These results suggest that aPC-enhanced epithelial barrier pro. and to heal the disrupted barrier. In contrast. After 5 h of pretreatment with aPC in intestinal inflammation. in with (TNF aPC EPCR) or without (aPC EPCR) a 1-h pretreatment with the mice developing colitis induced by DSS administration. No that are not classically acknowledged as inflammatory systems. This finding suggests that the Caco-2 cell line expresses S1P1. 17. for example. supporting this hypothesis has pointed to the involvement of the tection is S1P1-independent. we seeded Caco-2 cells onto plates in designated areas in the absence or presence of exogenous aPC (1 μg/mL) and monitored cell migration for 48 h after creation of B a scratch in the monolayers. Mice (blue). the ECs of healthy WT TNF-α (25 ng/mL) in the presence (TNF aPC) or absence of aPC (1 μg/mL).05 (aPC vs. the data are represen- receiving aPC recovered more rapidly from colitis. characterized by a de- dothelial barrier function mediating the S1P1 receptor. Importantly. Finally. aPC inhibits the altered expression of JAM-A and ZO-1 induced Mounting evidence also indicates that the PC pathway plays a by TNF-α (Fig. Finally. (A) Values of TEER were mea. Along with their crucial role in formation of tight junctions to maintain the epithelial barrier. S5). mation are only now beginning to be elucidated. Similar to human samples. ECs are able to ac- tively respond to injury by migrating when wounds occur. Confluent Caco-2 cells were treated without (control) and with thelial PC.01) TNF-dependent TEER values (Fig. aPC. and aPC + EPCR. icant decrease in PC expression was detected by day 3 and per- sured over 48 h of stimulation. 18). Protective effects of aPC on epithelial barrier function are mediated mental colitis. we investigated whether topical intrarectal administration TNF + aPC. pathway displaying remarkably potent anti-inflammatory activity suggesting a functional role for EPCR in aPC-mediated (3. 108 | no. FTY720 (1 μM). which is known to inhibit the S1P1 receptor. TNF and TNF aPC EPCR). aPC in the presence of FTY720 significantly ab. Indeed. we verified whether the effects of aPC on these data support the potential therapeutic efficacy of topical TEER were S1P1-dependent. Thus.05) (Fig. we investigated whether the intrarectal administration of aPC would exert a therapeutic effect in mice with experi- Fig. comparable to that induced by EGF (50 ng/mL). the Caco-2 cells were stimulated with and without TNF-α (25 ng/mL) Discussion in the presence or absence of aPC (1 μg/mL) for 0. and SphK2 mRNA aPC has a beneficial and therapeutic effect. 2). 5 C and D). ad- ministration of aPC significantly ameliorated mucosal damage (Fig. sisted to the end of the experiment (Fig. Significant wound closure was seen in the presence of a PC compared with the control. Pretreatment with α-EPCR antibody (5 μg/mL) 1 h before the stimulation decreased epithelial wound closure. quantification of mucosal inflammation revealed that aPC-treated mice had a sphingosine kinase (SphK1 and SphK 2).

The aPC/EPCR/PAR-1 complex cross-activates scribed the interactions between immune mucosal cells and ECs S1P. suggesting the potential for development of the release of proinflammatory cytokines and limiting leukocyte novel therapeutic approaches for a wide range of inflammatory adhesion (10). When PC activation is impaired. ** P < 0. indicating the likely presence of cross-talk be. even if they synthesize PC. Why the ECs express the inactive form of PC but are not able That study demonstrated that ECs express S1P and that the to covert it remains unclear. It has been reported that S1P is that contribute to intestinal homeostasis and intestinal epithelial involved in regulating the proliferation. let alone the role of PC in the pathogenesis mice induce a proinflammatory state. Low-PC patients with CD and UC. Recently. results. must sequent cleavage and activation of protease-activated receptor-1 obtain aPC from other types of cells. (B) After 8 d of DSS administra- tion. consistent active IBD. In addition. However.pnas. We found that low-PC mice had In this paper we report the surprising finding that beyond its altered or reduced expression of ZO-1. 23). 8). we found that that the aPC’s beneficial protective effects in controlling in- intestinal ECs are positive for all PC pathway components except testinal barrier function are EPCR-dependent. a recent study indicated that S1P regu- form. In the present study. and migration of many types of cells. (PAR-1) (3). permeability (19). but notably. differentiation.and aPC-treated mice on day 16.1073/pnas. Therefore. which is an adherens junction (24). 17. display altered the intestinal immune response (7. we also found a down-regulation of epithelial PC in mice This indicates that aPC controls the intestinal barrier function during DSS-induced colitis. which reported that most of the cytoprotective effects of aPC are me- has been widely demonstrated to exert anti-inflammatory and diated by EPCR. Tight junction proteins strictly control pathway has been implicated in the control of microvascular intestinal permeability. the PC intestinal barrier function. 21). 5. Exogenous administration of aPC ameliorates recovery of intestinal mucosa in DSS-induced colitis.05. Furthermore. derived from the endothelial membrane. this effect was abrogated by recombinant aPC. aPC plays an signaling pathway of S1P is involved in maintaining the integrity 19834 | www. function. It has been TM impairs the conversion of PC into its active form. In high levels of proinflammatory cytokines at baseline. a protein crucial for aPC conversion. (A) Immunohistochemi- cal staining of PC on sections of colon from WT mice before and after adminis- tration of 3% DSS. alterations of the coagulative system mice. PAR-1. and the other group received recombinant murine aPC (100 μg/kg) intrarectally in a volume of 80 μL using a straight gavage needle every other day after colitis was established. besides being associated with high mortality and pro- and of the cells involved in coagulation play important roles in thrombotic and proinflammatory phenotypes. *P < 0. have emerged as crucial controllers of inflammation important role in governing intestinal homeostasis by inhibiting in multiple organs. its presence was able to inhibit the altered ex- suggest that inflammation leads to dysregulation of PC system pression of ZO-1 and JAM-A induced by TNF-α. These findings tions in vitro. neous intestinal inflammation. as in low-PC disorders (3. However. We demon. aPC. aPC had a strong effect on tight junc- ECs from patients in an inactive disease state. Values are mean ± SEM. and together. and that the expression of PC system is lost in patients with intestinal barrier permeability (11. These data are repre- sentative of two independent experiments. 10). . Previous reports have de. WT mice were divided into two groups: One group received saline.1107140108 Vetrano et al. we have demonstrated cytoprotective properties (3. It is plausible to hypothesize that ECs are mediated through its interaction with EPCR and the sub- expressing EPCR and PAR-1. a signaling sphingolipid. and their alteration can lead to sponta- inflammation of the intestine (5.01. suggesting that TM. these data EPCR expressed by the epithelium is functionally active and is suggest that although the epithelium expresses EPCR. Loss of body weight was monitored daily during treatment. 18). have not been investigated. In particular. but whether this form is functional is unknown (20). However. expression of the proteins of the PC pronounced experimental colitis phenotype after challenge with pathway and their roles in controlling intestinal barrier integrity DSS. Interestingly. consistent with these human ity. components in intestinal ECs. similar to what is seen in intestinal TNF-α stimulation induced an increase in epithelial permeabil- endothelial cells (10). Fig. PC. including endothelial cells tween ECs and other cells expressing TM. and claudin-3. it does not provide an appropriate microenvironment for The protective effects of aPC on endothelial barrier integrity aPC production. the PC pathway was suggested to mice. classical role in vascular biology. 10). and the transgenic be involved in endothelial barrier integrity and in cytoprotection mice develop spontaneous intestinal inflammation and a more (18). Once activated. As expected. survival. a TM-soluble (22. molecule that we recently identified as a crucial mediator of way. the expression of E-cadherin. a strate that the intestinal epithelial layer expresses the PC path. n = 6 mice for all groups. reinforcing the evidence that the low PC levels in these and cytoprotection. The altered expression of EPCR and enhancing the expression of tight junction proteins. Arrows indicate the strong expression of PC on ECs in healthy colon and its dramatic reduction after DSS treatment. JAM-A. is released in lates the function of the intestinal epithelial barrier by altering plasma. PC also acts as an unexpected three tight junction proteins required for intestinal barrier mediator of intestinal epithelial barrier function. as also indicated by the of the two major forms of IBD. Most notable was the down-regulation of JAM-A. (E) Evaluation of the number of ulcers on day 16 after topical aPC or saline treatment.org/cgi/doi/10. Images were taken using a 20× objective. up to now. the intestinal homeostasis is altered. (C and D) Endoscopic images and evaluation of mucosal damage in the colons of healthy and saline. whereas no changes in the PC pathway were found in with the in vivo findings. and essential for the regulation of cellular permeability.

Exp Cell Res 299:119– ternal reproductive capabilities. In particular. J Thromb Haemost 1:1343–1348. Lay AJ. (2007) Crucial role of the protein C pathway in governing micro. Healthy areas of intestine taken from patients investigated whether aPC promoted this function in intestinal admitted for bowel resection because of polyps or diverticulosis were used as ECs as well. flammation occurs. we tested whether the aPC-dependent in. Groschwitz KR. topical administra. Gonzalez-Cabrera PJ (2007) Tipping the gatekeeper: disease pathogenesis. 20. PNAS | December 6. were approved by the Ethical Committee of Istituto Clinico Humanitas. IBD Research Founda- four patients found that treatment of chronic leg ulcers with tion (mini Grant 2010). abrogating the TNF-dependent decrease in TEER. 49 | 19835 . vascular thrombosis and inflammation. sepsis. Vetrano S. especially when in. Am J Gastroenterol 102:174–186. (2009) VEGF-A links angiogenesis and inflammation in in- 26. et al. 10. We thank Sarah A. Thus. Specimens were formalin-fixed and paraffin-embedded or frozen in erated wound closure and induced proliferation of ECs similarly Cryoblock Compound (DiaPath) on dry ice and stored at −80 °C. (2009) Persistent signaling induced by FTY720-phosphate is 135:173–184. the epithelium must be coagulant may help guide the development of new treatments for able to proliferate and migrate after injury. European Crohn’s and Colitis Organization (ECCO) topical aPC stimulated wound healing by activating skin ree. mediated by internalized S1P1 receptors. taining mucosal homeostasis in inflammatory bowel disease. pithelialization (27). Ann N Y Acad Sci 1165:308–313. but found no difference in TEER values PC pathway controls the process of mucosal homeostasis that is in the presence or absence of FTY720. herens junction protein E-cadherin and enhances intestinal epithelial cell barrier 11. Lust M. Human studies to EGF. Xue M. the local aPC treatment was and National Institutes of Health Grant HL019982 (to F. Danese S. et al. and coagulation. It is plausible to suppose mediated by ECs through the maintenance of barrier function that aPC in ECs activates S1P signaling. Sanna MG. tissue inflammation and injury: Pathogenic role and therapeutic implications. ceptor. et al. Vetrano S. thelial proliferation. 5. (Grant 2009). migration and wound closure.V. lium by acting on a molecule classically recognized as an anti- To maintain intestinal homeostasis.. (2006) Angiogenesis as a novel component of inflammatory bowel 23. 9. et al. ScientificWorldJournal 6:946–966. 2011 | vol. Majerus PW (1985) Thrombomodulin is present in human plasma and urine. 4. inhibits apoptosis and upregu- SYSTEMS BIOLOGY C display prothrombotic and proinflammatory phenotypes and compromised ma. Rutgeerts P. Ishii H. et al. Greenspon J. our study provides evidence of an unexpected ternalization of S1P1 in endothelial cells after 60 min of treat- ment (25). Because aPC promotes wound closure and intestinal epi. suggesting Importantly. 1. 7. Intestinal tissues were obtained from surgical specimens taken from keratinocyte proliferation in a dose-dependent manner. aPC accel. Blood 109:1984–1991. (2007) Inflammation and coagulation in inflammatory bowel disease: 22. the intrarectal administration of aPC did not in- that the cytoprotective effects of aPC that we observed in ECs crease hemorrhage. Danese S (2008) Nonimmune cells in IBD pathogenesis: From victim to villain. further studies are needed to provide evidence of the involve. Rosen ED. Cahalan SM. compared with mice receiving saline. Mosnier LO. Vetrano et al. Fondazione Cariplo (Grant 2010-0733). De La Rue of Readable Science for partially dependent on the reduced expression of PC and her assistance with the manuscript. both S1P2 and the gut leads to spontaneous intestinal inflammation. 24. Castellino FJ (2010) The protein C pathway in disease: Impossible ideal or therapeutic target? Gut 56:453–455. 6. the S1P1 using FTY720 did not abolish the effect of aPC. 14. aPC was found to increase Patients. et al. The cell-signaling properties of aPC are known to Consistent with these findings. A clinical study of zione Italiana Ricerca Cancro (IG-2010 10205) to S. Turner JR (2009) Intestinal mucosal barrier function in health and disease. and Ministero della Salute (GR -2009 convenzione 76) to S. in an EPCR-dependent manner. the inhibition of tions associated with the administration of i. We found that at low concentrations. Crit Care Med 29:S48–S51. lates MMP-2 activity in cultured human keratinocytes. Scaldaferri F. S1P1 and cytoprotection. Zhang L. Van Brocklyn JR (2006) Signal transduction of sphingosine-1-phosphate G The clot thickens. Furthermore. Blood 109:3161–3172. et al. Trends Immunol 25:536–542. (2003) Platelets trigger a CD40-dependent inflammatory response in J Clin Invest 76:2178–2181. et al. Altered PC expression in the epithelium of receptor is not a unique receptor for S1P ligand. Esmon CT (2003) Inflammation and thrombosis. revealing the ties that bind. Van Assche G (2007) Mucosal healing in inflammatory bowel 3. Donahue D. Innovative Medicines Initiative (IMI) (115142). Vetrano S. after colitis was established displayed faster recovery of body testinal barrier protective effects were mediated by S1P1. 16. 13.v. viously unrecognized component of the pathogenesis of IBD. Proc Natl 237–244. but not via S1P1. our in vivo studies demonstrated enhance vascular endothelial barrier function via the S1P1 re. aPC. Restoring the function of the PC pathway in intestinal epithe- ment of S1P receptors in aPC effects. S1P regulation of endothelial barrier function. et al. Scaldaferri F. Griffin JH (2006) The cytoprotective protein C pathway. Trends Mol Med 14: gration and barrier function by a chymase/Mcpt4-dependent mechanism. J Clin Invest 117:1951–1960. 108 | no. Detailed information is provided in SI Materials and Methods. however. migration. Esmon CT (2004) Interactions between the innate immune and blood coagulation Immunol 9:799–809. Trends Immunol 28:102–107. the capacity of aPC treatment to favor mucosal healing. Nat Chem Biol 5:428–434. Thus. Evidence points to the capacity of aPC to stimulate proliferation.D. Thus. et al.. Both weight loss and endoscopic and histological changes. IBD and mucosal inflammation. Dig Dis Sci 56:1342–1353. et al. a pre- S1P3 are expressed by ECs at higher levels than S1P1 (22). Here we pretreated the cells with FTY720 for 5 h function of the anticoagulative PC pathway in the intestine. the microvasculature of inflammatory bowel disease patients. protein-coupled receptors. Danese S (2008) The protein C pathway in inflammatory bowel 19. Blood 17. Arch Dermatol 144:1479–1483. Young N. The before aPC stimulation. Jackson C (2004) Activated protein C stimulates flammatory bowel disease pathogenesis. Given the increase in bleeding complica- can be mediated by S1P signaling. FTY720 is intrarectal route could provide a safer route of administration. Danese S. (2008) Unique role of junctional adhesion molecule-a in main- function. 127. Thompson P. with no adverse effects or complications (27). Gastroenterology 130:2060–2073. Castellino FJ (2005) Mice with a severe deficiency in protein proliferation. Immunol 29:555–564. Danese S (2009) The role of JAM-A in inflammatory bowel disease: Un- 1249–1264. (1999) Inflammation. systems. Whitmont K. (2009) Mast cells regulate homeostatic intestinal epithelial mi- disease: The missing link between inflammation and coagulation. 8. Poplis VA. protein C. and wound closure in human Materials and Methods keratinocytes (26). Danese S. an immunomodulator drug that has been proven to induce in- In conclusion. Liang Z. Rosen H. these effects. Vulcano M.J.of the epithelial barrier. Gastroenterology 124: 21. Esmon CT (2001) Protein C anticoagulant pathway and its role in controlling micro- 115:1121–1130. Although the level of S1P1 mRNA expression was very mice treated with intrarectal injection of aPC every other day low in Caco-2 cells. Castellino FJ (2007) Acute inflammation is exacerbated in 27. This study was supported by grants from EPCR by the intestinal epithelium. Gastroenterology 136:585–595. Haematologica 84: 18. Trends Acad Sci USA 106:22381–22386. Mullershausen F. Nat Rev 2.C. Esmon CT. completely duced number of ulcers. Indeed. (2008) Treatment of chronic leg ulcers with topical activated mice genetically predisposed to a severe protein C deficiency. with a re- S1P and aPC stimulation exerted similar effects.). Kelso I. J Clin Invest 115:1552–1561. Vermeire S. 254–259. we patients with active CD or UC. Zlokovic BV. 15. it is plausible that the prolonged healing process seen in patients with UC and CD could be at least ACKNOWLEDGMENTS. Danese S. Tsai MJ. but it did not address the mechanism of well tolerated. Lay AJ. 12. and Associa- tion of aPC could promote mucosal healing. Gastroenterology 25. controls. Ministero della Salute (GR- 2007 convenzione 23). (2011) Sphingosine-1-phosphate regulates the expression of ad- vascular inflammation in inflammatory bowel disease.