ALBERT

Description: Key Points PDK1 is involved in thrombin-induced platelet activation and αIIbβ3-mediated outside-in signaling by regulating the downstream effector Gsk3β.

Description: INTRODUCTION: The molecular basis of Philadelphia chromosome negative myeloproliferative neoplasms (MPNs) is unclear. So-called "driver" mutations in JAK2, CALR, or MPL are present in the vast majority of cases, but there is no compelling evidence to explain how each mutant gene can lead to phenotypically distinct and/or overlapping disease phenotypes. In an attempt to understand the molecular events that underlay clinical characteristics of MPNs, we studied gene expression profiles and sequenced hematopoietic cells of MPN patients with a focus on myelofibrosis (MF) landscape. METHODS: Patients were consented to MI-ONCOSEQ study approved by the University of Michigan IRB. Peripheral blood or bone marrow aspirates were either enriched for CD34 expressing cells or peripheral blood or bone marrow mononuclear cells (PBMC/BMMC) were analyzed. MI-ONCOSEQ is a Next Generation Sequencing platform to identify genetic aberrations in 1,711 genes and gene expression and fusion analysis of 24,774 capture targets by transcriptome sequencing. Gene expression data was analyzed between various sub-groups of MF based upon clinical (spleen size) or molecular (mutations in Ras pathway genes) characteristics. Gene set enrichment analysis was conducted for the MF cohorts (23 CD34 enriched, 76 PBMC/BMMC) and ET/PV/PrePMF cohort (12 CD34, 35 PBMC/BMMC). RESULTS: We analyzed the genetic landscape of 163 patients with MPNs: 113 with overt MF and 50 with ET (18), PV (23), or prePMF (9). In addition to driver genes, 183 other gene variants were observed. The number of gene variants was higher in older patients (median 4 and 5 variants in those aged 70-80 and 〉80 yrs respectively) with MF as compared to ET/PV/PrePMF where the median number of variants did not exceed 3. Mutations in ASXL1, TET2, RAS and SRSF2 increased in frequency with age (Fig 1). Gene expression profiles of sub-groups of MF were further analyzed to understand aberrantly regulated molecular pathways. Hierarchical clustering of all MF patients showed that CD34 enriched samples to be distinct from the PBMC/BMMC cohort and therefore these cohorts were analyzed separately. Moreover, hierarchical clustering suggested differences in patients with large spleens. In the comparison of MF to ET/PV/PrePMF within the CD34 population gene enrichment highlights hemopoiesis, leukemia related pathways as well as endoplasmic reticulum and Golgi transport pathways. Previously, we saw that RAS pathway mutations predicted proliferative disease with high WBC counts. Therefore, we focused on RAS pathway mutated cohorts versus RAS wild type cohorts and identified dysregulated pathways by gene set enrichment analysis (Fig 2). The RAS mutated MF cohort (PBMC/BMMC fraction) showed up-regulation of cytokines IL6 (p-value 9.39E-06), IL8 (p-value 1.16E-04), and IL1beta (p-value 5.24E-04) and down-regulation of the TNF superfamily (p-value 9.98E-04). Most notably, there was up regulation of NFkB transport to the nucleus (p-value 8.69E-05) and transcription factor activity. In general, several metabolic pathways were affected and inflammatory pathways were up-regulated. Since spleen size is an indicator of disease severity, progression and response to therapy, gene set enrichment between cohorts of patients with different spleen size (〉6cm by physical exam versus

Description: Plasma high molecular weight kininogen (HK) is cleaved in inflammatory diseases by kallikrein to cleaved HK (HKa). We have reported that HKa releases cytokines (TNFα , IL-1β , IL-6) and chemokines (IL-8 and MCP-1) from isolated human peripheral blood monocytes. At a concentration of 600nM, Glutathione -S- transferase (GST) fusion proteins of kininogen domain 3 (D3), E7P - an active fragment of domain 3 (aaG255-Q292), HK domain 5 (D5), and D5 recombinant peptide HG (aa K420-D474) each stimulated secretion of IL-1β from monocytes. Receptors on monocytes including Mac-1, LFA-1, uPAR and gC1qR are required for IL-1β secretion from monocytes. We now report the signaling pathways and evidence for synthesis of IL-1 β in monocytes stimulated by HKa. Inhibitors of signaling pathways initiated by NFkB, JNK and p38, but not ERK, decreased IL-1b release from monocytes. A specific inhibitor of NFkB, MG-132 (1, 10 and 100 μM) significantly reduced IL-1β release from monocytes by 69.7, 67.3, 88.5% respectively, when stimulated by GST-E7P. The release of IL-1β by LPS (10 μg/ml), was blocked 83.4% by MG-132 (100 μM). A specific inhibitor of JNK, SP 600125 (1, 10 and 100 μM) significantly reduced IL-1β release from monocytes by 55.7, 76.3, 78.9% respectively, when stimulated by GST-E7P. The release of IL-1β by LPS (10 μg/ml), was blocked 90.23% by SP 600125 (100 μM). A specific inhibitor of p38, SB202190 (1,10 and 100 μM) significantly reduced IL-1β release from monocytes by 27.8, 14.2, 91.0% respectively, when stimulated by E7P. The release of IL-1β by LPS (10 μg/ml), was blocked 76.8 % by SB202190 (100 μM). In contrast, the ERK activation inhibitor U0126 (1, 10 and 100 μM) failed to inhibit GST-E7P-induced release of IL-1β from monocytes but the release of IL-1β with LPS (10 μg/ml), was blocked by 77.4% at 100 μM. After monocytes (4 X 106/ml) were treated with HKa (600 nM) or LPS (10 ng/ml), total RNA was extracted and RT-PCR reactions for expression of IL-1β and gC1qR mRNA using specific primers were carried out. PCR products was separated in 4% ethidium bromide-stained agarose gels and photographed. No IL-1b mRNA was detected prior to exposure to HKa or LPS but both were detected at 1 hour. In contrast, gC1qR mRNA was present without stimulation by HKa. HKa domains 3 and 5 may contribute to the pathogenesis of inflammatory diseases by stimulating the synthesis and release of IL-1β from human monocytes using intracellular signaling pathways initiated by uPAR, β 2 integrins and gC1qR receptors.

Description: The plasma kallikrein–kinin system (KKS) plays an important role in inflammation, thrombosis and vascular pathology. Activation of the KKS results in bradykinin release, which increases vascular permeability and modulates inflammatory responses. Circulating endothelial progenitor cells (EPCs) home to ischemic and inflamed tissues and participate in the vasculogenesis. However, the homing mechanism remains poorly understood. We now report that activation of the KKS regulates the homing property of EPCs using in vivo and in vitro approaches. We have previously shown that the Lewis rat, which displays a mutant kininogen (Ser511Ala), is susceptible to peptidoglycan-polysaccharide (PG-PS)- induced systemic inflammation including arthritis and displays activation of the KKS and increased bradykinin levels. CD34-positive EPCs were isolated from bone marrow of disease-free Lewis rats by Ficoll gradient centrifugation. After culture in EGM2 on collagen surfaces for 7 days, a single colony with cobblestone-like morphology appeared and was subjected to large-scale expansion. Compared with differentiated rat lung microvessel endothelial cells (rLMECs), rat EPCs expressed similar levels of endothelial cell lineage marker mRNA such as VE-cadherin, CD31, vWF and uPAR. EPCs exclusively expressed hematopoietic progenitor cell markers, Sca-1 and CD34. Both of EPCs and rLMECs expressed bradykinin type 2 receptor (B2R) but not B1R. To observe the in vivo mobilization of EPCs, EPCs were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) and injected into Lewis rats in which inflammation/arthritis was induced by PG-PS. Two weeks after PG-PS injection, pannus formed in the synovial tissues, representing the entry into the chronic phase of arthritis. The fluorescently-labeled EPCs were selectively detected in the inflamed synovium of PG-PS-treated rats, but not that of disease-free rats. As detected by confocal microscopy, implanted EPCs formed new blood vessels in the inflamed synovial tissues. To further investigate the mechanism by which the KKS regulates EPC mobilization, we observed that bradykinin (a B2R agonist), but not des-Arg9-bradykinin (a B1R agonist), stimulated EPC transmigration through confluent rLMEC layer at 100 and 1000 nM (P

Description: Circulating endothelial progenitor cells (EPCs) are involved in repairing damaged vasculature and in tumor angiogenesis, thus promoting wide interest in their therapeutic potential in ischemic diseases and cancer. Cleaved high molecular weight kininogen (HKa) potently inhibits endothelial cell (EC) functions including in vivo angiogenesis. We have shown that HKa inhibits differentiation of ECs. We now test a new hypothesis that HKa can inhibit EPC differentiation by blocking matrix metalloprotease-2 (MMP-2) activation. EPCs were isolated from human blood and cultured on collagen type I-coated surface. As detected by flow cytometry and Western-blot, they expressed MMP-2 and membrane-type 1-MMP (MT1-MMP), avb3 integrin, uPAR and caveolin-1, but did not express monocyte markers such as HLA-DR and CD14. A clone-forming assay using retroviral infection indicated that an early single EPC exhibited colony forming property, but mature ECs such as human umbilical vein endothelial cells (HUVECs) did not. Upon stimulation by VEGF, EPCs, but not HUVECs, formed vacuoles and differentiated into capillary networks. Gene silencing of either MT1-MMP or MMP-2 by siRNA completely blocked the vacuole and tube formation by EPCs, suggesting that MT1-MMP and MMP-2 are both essential for differentiation of EPCs. HKa inhibited tube formation by EPCs, as well as the conversion of pro-MMP-2 to MMP-2 as a functioning concentration in the conditioned medium. CS-1 cells expressing endogenous av subunit were transfected with human b3 cDNA. We found that HKa inhibition of MMP-2 activation in these cells is dependent on avb3 heterodimer. The binding of avb3 integrin to MMP-2 was via RGD-dependent and RGD-independent mechanisms, both of which, however, were completely blocked by HKa. Further, we found that HKa inhibited the association of MMP-2 with immunoprecipitated avb3 integrin, and prevented MMP-2 localization in caveolae in EPCs. Since HKa inhibited VEGF-stimulated proliferation of EPCs but not HUVECs on collagen-coated surface, we postulate that EPCs are more sensitive than mature ECs to HKa inhibition. Thus, HKa targets EPCs and inhibits their differentiation via blocking avb3 integrin and MMP-2 interaction. These observations provide novel insight into understanding the interactions between the kallikrein-kinin system and biologic functions of EPCs.

Description: Protein disulfide isomerase (PDI) catalyzes thiol-disulfide exchange reactions that are critical for protein function. PDI has two distinct CGHC redox-active sites, but nothing is known about which site is important in physiologic reactions including thrombosis. To study the role of PDI and its active sites in thrombosis we used two genetically-modified models, Mx1-Cre/PDIfl/fl mice with blood cells and vessel wall cells lacking PDI, and transgenic PDI(ss-oo) mice with PDI lacking the second CGHC active site. Using laser-induced cremaster arteriole injury fibrin deposition was decreased in both mouse models. Both mouse models showed attenuation of platelet accumulation using laser arterial injury and FeCl3-induced mesenteric artery injury. The defects in fibrin deposition and platelet accumulation were rescued by infusion of recombinant PDI(oo-ss) containing only a functional second active site, confirming the second active site of PDI was critical for both fibrin formation and platelet accumulation. In vitro studies showed that platelet aggregation depended on the second CGHC motif of PDI, and that binding of PDI protein to platelets depended on the presence of the αIIbβ3 integrin. Studies on platelet secretion revealed that P-selectin expression and ATP release were dependent on a functional second active site of PDI. Treatment of the platelets with eptifibatide did not affect P-selectin expression. Together these findings suggest the second active of PDI catalyzes a reaction in αIIbβ3 supporting platelet aggregation and a reaction in a non-αIIbβ3 substrate supporting secretion. In vivo studies on coagulation showed that PDI infusion rescued fibrin formation in eptifibatide-treated mice in which platelet accumulation was completely blocked, suggesting that PDI directly contributes to activation of coagulation. In summary, our results indicate that the second CGHC active site of PDI has a role in platelet function and coagulation. Targeting this site may have a dual inhibitory effect on thrombosis by decreasing both platelet and fibrin accumulation. Disclosures No relevant conflicts of interest to declare.

Description: Bradykinin is a potent activator of endothelial cells and is known to induce vasodilation, vascular permeability, and nitric oxide production. Bradykinin signaling is initiated by binding to G protein-coupled bradykinin receptors 1 and 2 (B1R and B2R). In this study we investigated whether these two receptors are involved in regulation of endothelial progenitor cell (EPC) biology. First we isolated CD34+ cells from wild-type (WT) and double B1R and B2R knockout (B1RB2R-/-) mice, and compared their capacities of proliferation, migration, as well as tube formation in a 3D cell culture system. Bone marrow-derived B1RB2R-/- EPCs displayed a reduction in proliferation and migration through collagen-coated transwell, compared with EPCs from WT mice. Moreover, implanted B1RB2R-/- EPCs into immunocompromised NOD-SCID mice showed significantly less vasculogenesis in matrigel plug. Human EPCs were isolated from peripheral blood of healthy donors using CD34+ selection. In a concentration-dependent manner, bardykinin stimulated proliferation, migration and tube formation of human EPCs. In contrast to human EPCs, human umbilicial vein endothelial cells (HUVECs) did not display a significant increase in proliferation, migration, and tube formation upon bradykinin stimulation, suggesting that bradykinin selectively enhances the function of EPCs, but not mature endothelial cells. Pre-incubation with a B2R selective antagonist (Icatibant) reduced these functions of human EPCs. When human EPCs were stimulated with [des-Arg9]-BK, a selective B1R agonist, their proliferation and tube formation remained unchanged. Moreover, preincubation of human EPCs with [des-Arg10]-Icatibant, a B1R selective antagonist, did not affect their proliferation, migration and tube formation. As detected by Western blot and real time RT-PCR, B2R was constitutively expressed in EPCs, while the expression of B1R was only induced when stimulated by inflammatory stimuli, such as TNFα and IL-1β. Take together, bradykinin regulates proliferation, migration and tube formation of EPCs through constitutively expressed B2R, whereas B1R is probably involved in inflammatory settings. Disclosures No relevant conflicts of interest to declare.

Description: Apoptosis can be induced in a variety of pathological disorders, including inflammation, autoimmune diseases, and chemotherapy. When cells undergo apoptosis, they express phosphatidylserine (PS) on cell membrane surface and thus become procoagulant. Although it has been known that the procoagulant activity of apoptotic cells are tightly associated with thrombotic disorders, such as atherothrombosis and Trousseau syndrome, the mechanisms by which apoptotic cells activate the coagulation system and enhance blood clotting are largely unknown. In this study we investigated which coagulation factor(s) is involved in this process. Using western blotting and chromogenic substrate assay, we found that incubation with apoptotic cells induced by Dexamethasone (DXMS), but not with viable cells, resulted in rapid cleavage and activation of FXII. Moreover, apoptotic cells-mediated FXII activation was significantly increased in the presence of prekallikrein (PK) and high molecular weight kininogen (HK), other two components of plasma contact system. However, incubation of apoptotic cells did not cause dramatic changes of other coagulation factors, suggesting a selective association of FXII activation with apoptotic cells. Activation of FXII by apoptotic cells was markedly inhibited by a specific anti-kallikrein antibody, indicating the activation of the contact system by apoprotic cells. Flow cytometric measurement showed that FXII bound to apoptotic cells in a concentration-dependent manner, which was inhibited by annexin V and PS liposome. A surface plasmon resonance assay showed a direct binding of FXII to PS (KD=3.9E-9 M). When challenged by apoptotic cells, clotting time of plasma from FXII-knockout mice was significantly prolonged, which was reversed by replenishment with human FXII. Moreover, an inhibitory anti-FXII antibody completely prevented apoptotic cells-induced intrinsic tenase complex formation. Consistently, apoptotic cells significantly increased thrombin production in normal plasma, which were attenuated by PS blocker annexin V, an inhibitory anti-FXII antibody, and the deficiency of FXII, respectively. Addition of human FXII to XII-deficient plasma recovered thrombin generation. As evaluated by ELISA, the levels of thrombin-antithrombin complex in circulation were significantly increased when apoptotic cells were intravenously injected into wild-type mice, but not in FXII-knockout mice. In conclusion, FXII plays an important role in apoptotic cells-mediated procoagulant activity. Disclosures No relevant conflicts of interest to declare.

Description: ERp57 is a member of protein disulfide isomerase (PDI) family. Although previous studies using antibodies have indicated ERp57 mediates platelet aggregation, the specific role of platelet-derived ERp57 in hemostasis and thrombosis is unknown. In this study we addressed this issue using a megakaryocyte/platelet specific ERp57-knockout mouse model (PF4-Cre/ERp57fl/fl). Although PF4-Cre/ERp57fl/fl mice had normal platelet counts and platelet glycoproteins expression, they had significantly prolonged tail-bleeding times, and thrombus occlusion times with FeCl3-induced carotid artery-injury. Using a mesenteric artery thrombosis model we found decreased incorporation of ERp57-deficient platelets into a growing thrombus. Upon stimulation with convulxin and thrombin, platelets lacking ERp57 had defective activation of the αIIbβ3 integrin and platelet aggregation. Addition of exogenous wild type ERp57 corrected the defect in aggregation implicating surface ERp57 in platelet aggregation. Moreover, using recombinant ERp57 mutants we found that the second active site of ERp57 targets a platelet surface substrate to potentiate platelet aggregation. Binding of Alexa 488-labeled ERp57 to thrombin-activated and Mn2+-treated platelets lacking β3 was substantially decreased, suggesting a direct interaction of ERp57 with αIIbβ3. Surface ERp57 activity in human platelets increased in response to stimulation with thrombin, concomitant with protein expression occurring in a physiologically relevant time frame. In conclusion, platelet-derived ERp57 directly interacts with αIIbβ3 during activation of this receptor and is required for incorporation of platelets into a growing thrombus. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points Intravascular ERp72 supports platelet accumulation and fibrin generation through the a and a′ active sites. ERp72 functions separately from protein disulfide isomerase and ERp57 in supporting platelet aggregation.