Activated Protein C Strengthens Cardiac Tolerance to Ischemic Insults in Aging

Background: Activated protein C (APC) is a plasma serine protease with anticoagulant and anti-inflammatory activities. Endothelial protein C receptor (EPCR) is associated with APC's activity and mediates its downstream signaling events. APC exerts cardioprotective effects during ischemia and reperfusion (I/R). This study aims to characterize the role of the APC-EPCR axis in ischemic insults in aging. Methods: Young (3-4 months) and aged (24-26 months) wild type C57BL/6J mice, as well as EPCR point mutation (EPCR R84A/R84A ) knock-in C57BL/6J mice incapable of interaction with APC and its wild type of littermate C57BL/6J mice, were subjected to I/R. Wild type APC, signaling-selective APC-2Cys, or anticoagulant-selective APC-E170A were administrated before reperfusion. Results: The results demonstrated that cardiac I/R reduces APC activity, and the APC activity was impaired in the aged versus young hearts possibly attributable to the declined EPCR level with aging. Serum EPCR measurement showed that I/R triggered the shedding of membrane EPCR into circulation, while administration of APC attenuated the I/R-induced EPCR shedding in both young and aged hearts. Subsequent echocardiography showed that APC and APC-2Cys but not APC-E170A ameliorated cardiac dysfunction during I/R in both young and aged mice. Importantly, APC elevated the resistance of the aged heart to ischemic insults through stabilizing EPCR. However, all these cardioprotective effects of APC were blunted in the EPCR R84A/R84A mice versus its wild-type littermates. The ex vivo working heart and metabolomics results demonstrated that AMP-activated protein kinase (AMPK) mediates acute adaptive response while protein kinase B (AKT) is involved in chronic metabolic programming in the hearts with APC treatment. Conclusions: I/R stress causes shedding of the membrane EPCR in the heart, and administration of APC prevents I/R-induced cardiac EPCR shedding that is critical for limiting cardiac damage in aging.

Abstract 12715: Administration of Activated Protein C Stabilizes Membrane EPCR and Increases Resistance of Heart to Ischemic Insult in Aging

Introduction: Activated protein C (APC) is a circulating protease with anti-coagulant property. Endothelial protein C receptor (EPCR) mediates APC's downstream events. We have revealed that APC exerts cardioprotective effects during ischemia and reperfusion (I/R). Hypothesis: There is an impaired APC signaling in aging and administration of APC restores APC signaling of aged heart to improve cardiac tolerance to ischemic insult caused by I/R stress. Methods: Young C57BL/6J (3-4 months), aged C57BL/6J (24-26 months), and EPCR R84A/R84A (3-4 months, C57BL6/J background) transgenic mice without APC binding affinity were subjected to ischemia (45 min) and reperfusion (24 hrs) by ligation/release of left anterior descending coronary artery. APC, signaling selective APC-2Cys, or anticoagulant selective APC-E170A was injected through jugular vein five-minutes before reperfusion. Results: I/R stress triggers APC signaling in both young and aged hearts, but APC activity was significantly impaired in the aged versus young hearts. Serum EPCR measurement demonstrated that membrane EPCR's shedding into circulation was dramatically promoted during I/R stress. Intriguingly, administration of APC significantly reduced EPCR shedding caused by I/R stress in both young and aged hearts but not EPCR R84A/R84A heart. The echocardiography data showed that APC and APC-2Cys but not APC-E170A ameliorated cardiac dysfunction by I/R insult in both young and aged mice. Interestingly, APC and APC-2Cys restored aged heart's resistance to a comparable level as young heart to ischemic damage. The cardioprotective effects of APC and ACP-2Cys are diminished in EPCR R84A/R84A mice. Furthermore, metabolomics analysis and working perfusion heart results demonstrated that AMP-activated protein kinase (AMPK) signaling mediates acute adaptive metabolic response while protein kinase B (Akt) signaling pathway is involved in chronic metabolic programming in the heart with APCs treatments. Conclusions: I/R stress causes membrane EPCR shedding in the heart and APC's cardioprotection against I/R injury is not related with its anti-coagulant activity. Administration of APC prevents I/R-induced cardiac EPCR shedding that is critical for limiting cardiac damage in aging.

Zingerone Suppresses the Shedding of Endothelial Protein C Receptor

Zingerone (ZGR), a phenolic alkanone found in Zingiber officinale, has been reported to have various pharmacological activities including anti-inflammatory and anti-apoptotic activities. The endothelial cell protein C receptor (EPCR) plays an important role in the cytoprotective pathway and activation of protein C EPCR can be shed from the cell surface, which is mediated by tumor necrosis factor-α converting enzyme (TACE). However, little is known about the effects of ZGR on EPCR shedding. We investigated this by monitoring the effects of ZGR on phorbol-12-myristate 13-acetate (PMA)-, tumor necrosis factor (TNF)-a, and interleukin (IL)-1p-induced EPCR shedding in human umbilical vein endothelial cells (HUVECs), and cecal ligation and puncture (CLP)-mediated EPCR shedding in mice, as well as by analyzing the underlying mechanisms. Here, ZGR triggered potent inhibition of PMA-, TNF-α-, IL-1β-and CLP-induced EPCR shedding through the inhibition of phosphorylation of mitogen-activated protein kinases (MAPKs) such as p38, janus kinase (JNK), and extracellular signal-regulated kinase (ERK) 1/2. ZGR also inhibited PMA-induced TACE expression and activity in HUVECs, suggesting that p38, ERK1/2, and JNK could be molecular targets of ZGR. These results demonstrate the potential of ZGR as an agent against PMA- and CLP-mediated EPCR shedding.

Suppressive Effects of Pelargonidin on Endothelial Protein C Receptor Shedding via the Inhibition of TACE Activity and MAP Kinases

Beyond its role in the activation of protein C, the endothelial cell protein C receptor (EPCR) plays an important role in the cytoprotective pathway. EPCR can be shed from the cell surface, which is mediated by tumor necrosis factor-[Formula: see text] converting enzyme (TACE). Pelargonidin is a well-known red pigment found in plants, and has been reported to have important biological activities that are potentially beneficial to human health. However, little is known about the effects of pelargonidin on EPCR shedding. We investigated this issue by monitoring the effects of pelargonidin on phorbol-12-myristate 13-acetate (PMA)-, tumor necrosis factor (TNF)-[Formula: see text]-, interleukin (IL)-1β-, and cecal ligation and puncture (CLP)-mediated EPCR shedding and by investigating the underlying mechanism of pelargonidin action. Data demonstrate that pelargonidin induced potent inhibition of PMA-, TNF-[Formula: see text]-, IL-1β-, and CLP-induced EPCR shedding by inhibiting the phosphorylation of mitogen-activated protein kinases (MAPKs) such as p38, janus kinase (JNK), and extracellular signal-regulated kinase (ERK) 1/2. Pelargonidin also inhibited the expression and activity of PMA-induced TACE in endothelial cells. These results demonstrate the potential of pelargonidin as an anti-EPCR shedding reagent against PMA- and CLP-mediated EPCR shedding.

EPCR/PAR1 Signaling Navigates Long-Term Repopulating Hematopoietic Stem Cell Bone Marrow Homing to Thrombomodulin-Enriched Blood Vessels

Bone marrow (BM) homing and lodgment of long-term repopulating hematopoietic stem cells (LT-HSCs) is an active and essential first step in clinical stem cell transplantation. EPCR is expressed by murine BM LT-HSCs endowed with the highest repopulation potential and its ligand, activated protein C (aPC), has anticoagulant and anti-sepsis effects in EPCR+/PAR1+ endothelial cells. We recently found that signaling cascades, traditionally viewed as coagulation and inflammation related, also independently control EPCR+ LT-HSC BM retention and recruitment to the blood via distinct PAR1 mediated pathways. EPCR/PAR1 signaling retains LT-HSCs in the BM by restricting nitric oxide (NO) production and Cdc42 activity, promoting VLA4 affinity and adhesion. Conversely, thrombin/PAR1 signaling overcome EPCR+ LT-HSC BM retention by initiating NO production, leading to TACE-‎mediated EPCR shedding, CXCR4 and PAR1 upregulation and parallel CXCL12 secretion by PAR1+ BM stromal cells, enhancing stem cell migration and mobilization. Since EPCR shedding is essential for BM LT-HSC recruitment, we tested EPCR role in LT-HSC BM homing. EPCR+ LT-HSC exhibited reduced in vitro migration towards CXCL12 and enhanced CXCL12-dependent adhesion to fibronectin. Unexpectedly, transplanted EPCR+ LT-HSCs preferentially homed ‎to the host BM, while immature progenitors were equally distributed between the BM and spleen. Specificity of BM homing was further confirmed by EPCR neutralizing antibody treatment, which blocks binding to aPC, leading to attenuated EPCR+ LT-HSC homing to the BM but not to the spleen. Importantly, short term aPC pretreatment inhibited NO production and dramatically increased EPCR+ LT-HSC BM homing. Since EPCR navigates LT-HSC to the BM, we studied the role of EPCR signaling in LT-HSC BM repopulation. Mimicking EPCR signaling by in vivo NO inhibition induced preferential expansion of blood and bone-forming stem cells and gave rise to higher donor type EPCR+ LT-HSCs in competitive repopulation assays. Similarly, repeated treatment with aPC expanded BM EPCR+ stem cells and increased competitive LT-repopulation. Importantly, loss of EPCR function reduced HSC long-term repopulation ability while maintaining their short-term repopulation activity. BM HSCs obtained from Procrlow mice, expressing markedly reduced surface EPCR, failed to compete with normal stem cells in competitive long-term repopulation assays. Consistent with inferior HSC BM repopulation, Procrlow mice exhibited reduced numbers of BM LT-HSC with reduced adhesion capacity. Additionally, these mice displayed increased HSC frequencies in the blood circulation and the spleen, which were pharmacologically corrected by inhibiting NO generation with L-NAME treatment. BM retention is essential for quiescent HSC protection from chemotherapy. Mice treated with NO donor SNAP, or with blocking EPCR antibody as well as Fr2-/-mice lacking PAR1 expression, were more susceptible to hematological failure and mortality induced by 5-FU treatment compared to control mice. Together, these results indicate a functional aPC/EPCR/PAR1 signaling pathway, regulating EPCR+ LT-HSC BM homing, adhesion and long-term repopulation potential. The thrombin-thrombomodulin (TM) complex converts protein C to its activated form aPC, facilitating high affinity binding to its receptor EPCR. To further address the preferential homing of EPCR+ LT-HSCs to the BM, we found that TM is exclusively expressed by a unique BM endothelial cell (BMEC) subpopulation, but not in the spleen. Moreover, EPCR+ LT-HSCs were found adjacent to TM+/aPC+ BMECs, imposing their adhesion and retention. Interestingly, similar to BMECs, BM EPCR+ LT-HSC also express surface TM, implying the possibility of autocrine aPC generation. Herein we define EPCR as a guidance molecule, navigating slow migrating LT-HSC in the blood flow specifically to TM+ BMEC supporting niches, maintaining NOlow stem cell retention, long-term blood production and protection from myelotoxic insult. Conversely, thrombin/PAR1 signaling oppositely increase NO generation and EPCR shedding allowing increased CXCR4-dependent LT-HSC migration and mobilization. Harnessing EPCR signaling may improve clinical stem cell transplantation, increasing LT-HSC specific BM homing and repopulation by aPC pretreatment, as well as potentially to overcome malignant stem cell chemotherapy resistance. Disclosures No relevant conflicts of interest to declare.

EPCR Limits Nitric Oxide Levels, Mediating Human and Murine Stem Cell Adhesion and Retention In The Bone Marrow, By Conjugating PAR1 and CXCR4 Signaling

Long term repopulating hematopoietic stem cells (LTR-HSC) in the murine bone marrow (BM) highly express endothelial protein C receptor (EPCR), yet the function of EPCR in HSC is incompletely defined. EPCR is expressed primarily on endothelial cells and has anti coagulation and anti inflammatory roles. While physiological stress due to injury or bleeding is a strong inducer of HSC mobilization and leukocyte production, a role for the coagulation protease thrombin, and its major receptor PAR1 in regulation of HSC has not yet been identified. We hypothesized that thrombin plays a role in HSC mobilization in the context of injury and that, conversely, signaling involving EPCR and its ligand activated protein C (aPC) play a regulatory role in HSC maintenance. Herein, we report that murine BM EPCRhigh stem cells display enhanced CXCL12 mediated adhesion and reduced migration capacitie, while motile circulating HSC in the murine blood and spleen lack high EPCR expression. Mechanistically, we found that EPCR is a negative regulator of nitric oxide (NO) levels. EPCRhigh stem cells display low intracellular NO levels, low motility, and increased adhesion to BM stroma. Furthermore, EPCRlow transgenic mouse cells displayed reduced stem cell adhesion to BM stroma and increased motility, manifested by reduced EPCRlow HSC in the BM and their corresponding increased levels in the blood. In vitro stimulation with the EPCR ligand, aPC, which we found to be physiologically expressed adjacent to small murine BM blood vessels, augmented EPCRhigh HSC adhesion and further limited their intracellular NO content by increasing eNOS phosphorylation at Thr495 in BM HSC, causing reduced production of NO. Conversely, administration of the pro-coagulant protease thrombin to mice induced PAR1 mediated EPCR shedding from BM HSC, followed by CXCR4 upregulation on HSC, and PAR1-mediated CXCL12 secretion by BM stromal cells. Together, these events lead to loss of retention and rapid stem cell mobilization to the blood. Interestingly, shedding of EPCR was found to be mediated by elevation of intracellular NO content, leading to EPCR co-localization with Caveolin. Correspondingly, thrombin failed to induce EPCR shedding and mobilization in eNOS and PAR1 deficient mice. Additionally, we found that BM LTR-HSC functionally express the metalloproteinase TACE (ADAM17) on the cell membrane, and that in- vitro inhibition of TACE activity by a newly developed selective inhibitor, reduces thrombin- mediated EPCR shedding, suggesting the involvement of TACE in EPCR shedding and HSC mobilization. Moreover, EPCR shedding was also CXCR4 dependent, revealing a crosstalk between EPCR, PAR1 and CXCR4. HSPC mobilized by thrombin possessed increased long-term repopulation capability following transplantation into lethally irradiated recipient mice and re-synthesis of EPCR by donor HSC in the engrafted host BM. In addition, EPCR expression was re-induced on circulating stem cells following in vitro treatment with eNOS inhibitor. Interestingly, bypassing eNOS by directly injecting NO donor, induced EPCR shedding, CXCR4 upregulation and rapid HSPC mobilization in both wild type and eNOS KO mice. Importantly, we found that similar to mice, EPCR was selectively and highly expressed by primitive human BM CD34+CD38- HSC, but not in the blood circulation of clinical G-CSF mobilized stem cells or in motile cord blood stem cells. Human BM CD34+/CD38- HSC are functionally EPCRhigh cells, maintaining low levels of intracellular NO which mediates their increased adhesion, while EPCR shedding was important for their migration and mobilization. In the functional pre-clinical NOD/SCID mouse model, G-CSF mobilization induced EPCR shedding, up-regulation of PAR1 and CXCR4 on human stem and progenitor cells, while NO signaling inhibition blocked G-CSF induced mobilization and increased both murine and human EPCRhigh stem cell accumulation in the murine BM. Our results define functional roles for EPCR, on both human and murine HSC, and suggest that regulation of EPCR expression is linked to NO, PAR1 and CXCR4 signaling as a pivotal mechanism determining HSC localization and function. Our study reveals that activation of coagulation in the context of cell injury controls stem cells retention and motility, and suggests that targeting this system may be useful in improving clinical stem cell mobilization and transplantation protocols. Disclosures: No relevant conflicts of interest to declare.

Coagulation Factor Thrombin Regulates Hematopoietic Stem and Progenitor Cell Egress and Mobilization Via PAR-1 & CXCR4 Upregulation, SDF-1 Secretion and EPCR Shedding

Abstract 2341 Hematopoietic stem and progenitor cell (HSPC) egress from the bone marrow (BM) to the circulation is tightly regulated and is accelerated during stress conditions. The G-protein-coupled receptor protease-activated receptor-1 (PAR-1) and its activator thrombin play an important role in coagulation following injury and bleeding. We report that a single injection of thrombin induced rapid HSPC mobilization within one hour, increasing circulating leukocytes, predominantly CFU-C and primitive Lin−/Sca-1+/c-Kit+ (SKL) progenitor cells. This rapid mobilization was preceded by a dramatic decrease of SDF-1 (CXCL12) in BM stromal cells, including rare Nestin+ mesenchymal stem cells (MSC) which functionally express PAR-1 and release SDF-1. Thrombin injection also increased expression of PAR-1 and CXCR4 by BM HSPC. These results suggest involvement of the coagulation cascade of thrombin & PAR-1 in rapid SDF-1 secretion from niche supporting BM stromal cells as part of host defense and repair mechanisms. Administration of a PAR-1 specific antagonist (SCH79797) upregulated BM SDF-1 levels and significantly reduced the amounts of circulating CFU-C and primitive SKL progenitor cells. In vitro stimulation of BM mononuclear cells with thrombin for 1 hour led to increased CXCR4 expression by Lin−/c-Kit+ progenitors, accompanied by enhanced spontaneous and SDF-1 induced migration. Of note, specific PAR-1 inhibition in vitro significantly reduced SDF-1-directed migration of Lin-/c-Kit+ progenitors. Mechanistically, we found that thrombin - activated PAR-1 induced the downstream p38 MAPK and eNOS (nitric oxide synthase) signaling pathways. Long term repopulating hematopoietic stem cells (HSC) in murine BM highly express endothelial protein C receptor (EPCRhigh) (Balazs & Mulligan et al Blood 2006; Kent & Eaves et al Blood 2009). EPCR is expressed primarily on endothelial cells (EC) and has anti coagulation and anti inflammatory roles. Surface EPCR expression on EC is downregulated by many factors, including PAR-1 activation by thrombin, a process which is termed shedding and is not fully understood. Importantly, we found that over 90% of BM CD45+/EPCRhigh long-term HSC express PAR-1 and that circulating primitive HSPC in the blood and spleen lack EPCRhigh expression. In addition, in-vivo thrombin administration downregulated EPCR from BM HSC via eNOS signaling, thus allowing the release of stem cells from their BM microenvironment anchorage to the circulation. Correspondingly, in eNOS deficient mice, thrombin failed to induce PAR-1 upregulation, EPCR shedding, and HSPC mobilization. Recently, we reported that the antioxidant NAC inhibits G-CSF induced mobilization (Tesio & Lapidot et al Blood 2011). Co-administration of G-CSF with NAC prevented PAR-1 upregulation, concomitantly with reduced HSPC mobilization and increased levels of EPCRhigh HSC in the BM. Treatment of PAR-1 antagonist with G-CSF inhibited PAR-1 and CXCR4 upregulation on BM leukocytes and immature Lin−/c-Kit+ cells accompanied by increased levels of BM EPCRhigh HSC and reduced HSPC mobilization. Tissue factor (TF) is the main initiator of the coagulation system via the formation of an enzymatic “prothrombinase complex” that converts prothrombin to active thrombin. Unexpectedly, we found a unique structure of cell clusters expressing TF, located preferentially in the trabecular-rich area of the femoral metaphysis in murine bone tips, a region highly exposed to osteoclast/osteoblast bone remodeling. In vitro, immature osteoclasts exhibited increased TF expression in cell fusion areas, suggesting that in vivo osteoclast maturation activates the coagulation thrombin/PAR-1 axis of HSPC migration to the circulation. Finally, mimicking bacterial infection a single injection of Lipopolysaccharide (LPS), rapidly and systemically upregulated TF in the murine BM. LPS treatment prompted an increase in thrombin generation and subsequently HSPC mobilization, which was blocked by the PAR-1 antagonist. In conclusion, our study reveals a new role for the coagulation signaling axis, which acts on both hematopoietic and stromal BM cells to regulate steady state HSPC egress and enhanced mobilization from the BM. This thrombin/PAR-1 signaling cascade involves SDF-1/CXCR4 interactions, immature osteoclast TF activity, Nestin+/PAR-1+ MSC secretion of SDF-1 and EPCR shedding from hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.