Holroyd, Eric

Background: The tissue factor pathway regulates vascular structure in the adult through regulation of thrombin generation and signaling. Deletion of tissue factor pathway inhibitor (TFPI) in the mouse results in early intrauterine lethality, precluding study of vascular development. In addition, the carboxyl terminus of TFPI may have a TF-independent effect on vascular structure. We sought to determine the role of TFPI and its carboxyl terminus in vascular development of zebrafish. Methods and results: TFPI mRNA localization studies demonstrated expression in the developing kidney, gut, and vasculature of the zebrafish. We further assessed the role of TFPI in zebrafish angiogenesis, by injecting translational and splice-inhibiting morpholino (MO) oligonucleotides directed against zebrafish TFPI mRNA into embryos from the double transgenic zebrafish Tg(Fli1GFP); Tg(GATA1dsRed) expressing GFP in vascular endothelium and dsRed in erythroid cells. Knockdown of TFPI protein expression was subsequently confirmed by immunoblotting. In a dose-dependent manner, multiple non-overlapping MOs targeting the translation initiation site of TFPI independently and synergistically led to the formation of abnormally targeted intersegmental arteries which crossed somite boundaries by 48 hours post fertilization (hpf). Additionally, a plethoric caudal vein plexus with increased honeycombing was present by 24 hpf. This latter aspect of the phenotype was most striking after a MO targeted against a splice site involving the Kunitz 3 and carboxyl terminus domains of zebrafish TFPI was injected alone. Conclusions: TFPI is expressed during zebrafish development. Morpholino knockdown of TFPI expression alters both arterial and venous patterning in the developing zebrafish, indicating a fundamental role for TFPI in targeting of newly-formed blood vessels. Moreover, different domains of the TFPI protein may preferentially act on distinct vascular networks.

Background: Hematopoietic stem cells and the vasculature share a unique relationship during development, in the adult bone marrow and in the setting of extramedullary hematopoiesis within diseased calcified vessels. We hypothesized that the adult vasculature may contain progenitor cells with the capacity to reconstitute multi-lineage hematopoietic cells. Methods: Aortas from normal and ApoE −/− adult mice were disaggregated with collagenase and elastase. Disaggregates were cultured in methylcellulose for quantification of short and long term hematopoietic colony forming units (CFU). Flow cytometry assessed various populations of hematopoietic progenitor cells. Aortic disaggregates (or bone marrow or blood) from ROSA26 and GFP mice were transplanted into sublethally irradiated C57BL/6 recipients (n=6) via tail vein injections. Bone marrow cells from the primary recipients were transplanted at 4 months to secondary C57BL/6 recipients (n=6). Mice were sacrificed 3 months later. Blood and bone marrow were analyzed. Results: In short term culture in methylcellulose, aortic disaggregates generated erythroid, macrophage, granulocyte and megakaryocyte CFU, with predominance of macrophage colonies, primarily from adventitia (p<0.05) and a greater frequency was found in Apo E −/− mice (p<0.05). Long term culture defined that the combined frequency of the hematopoietic precursors approximates the frequency found in bone marrow (1:47,277). Flow cytometry of the aortic disaggregates indicates that among lin-/c-kit+/sca-1+ cells a small population of hematopoietic stem cells (CD150+endoglin+) and a population of multipotent progenitor cells (CD150− endoglin−) exist. Infusion of aortic disaggregates generated bone marrow, blood and spleen chimerism in primary and secondary transplanted mice at 3– 4 months. Flow cytometry of peripheral cells demonstrated multilineage hematopoietic cells. Conclusions: For the first time, we have identified a variety of progenitor cell populations in the aorta capable of hematopoiesis. Infusion of these cells into sublethally irradiated mice results in stable chimeras. These data support the presence of novel aortic hematopoietic progenitor cells.

Background: Tissue factor pathway inhibitor (TFPI) is a key regulator of the tissue factor/factor VIIa complex. Systemic genetic disruption of TFPI exon 4 results in intrauterine lethality in mice. As such, the ability to study the biologic and tissue specific effects of TFPI deficiency has been limited. To define the tissue specific roles of TFPI we utilized a Cre-lox strategy and generated mice with flanking loxP sites surrounding exon 4 (encodes for the K1 domain). Homozygous floxed mice (TFPI Flox ) were crossed with the Tie2 Cre + or LysM Cre + mice and subsequently inbred to create homozygously floxed mice expressing Cre (TFPI Tie2 or TFPI LysM ). Methods and Results: The presence and efficiency of the deleted mRNA product was determined by RT-PCR. TFPI Tie2 and TFPI LysM mice were phenotypically similar to TFPI Flox littermates including weight, blood counts, and prothrombin and activated partial thromboplastin times. TFPI Tie2 mice have complete deletion in endothelial cells, blood cells and bone marrow cells. TFPI LysM mice show partial deletion in blood cells and complete deletion in macrophages. TFPI Tie2 mice had a 60% reduction (P < 0.0001) in circulating TFPI activity compared to TFPI Flox littermate controls. TFPI LysM mice did not have a significant reduction in circulating TFPI activity levels compared to their TFPI Flox counterparts. Neither TFPI Tie2 nor TFPI LysM mice had significant changes in hemostasis compared to TFPI Flox mice as determined by a tail bleeding assay. In an ADP-induced model of thromboembolism there were no significant changes in mortality between the TFPI Tie2 or TFPI LysM and their TFPI Flox littermates. In a ferric chloride model of arterial thrombosis, TFPI Tie2 mice had a significant reduction (P < 0.05) in time to occlusion while TFPI LysM mice did not differ compared to TFPI Flox mice. Conclusions: Deletion of TFPI from cells expressing Tie2 or LysM is consistent with normal development and hemostasis. TFPI Tie2 mice have decreased circulating levels of TFPI and an enhanced thrombotic response to ferric chloride. These data support an important regulatory role for EC TFPI in arterial thrombosis.

Abstract Abstract 4319 C-type natriuretic peptide (CNP) is known to exert pleiotropic effects on a variety of cell types through binding to its receptor, natriuretic peptide receptor type B (NPR-B) and activation of the cytoplasmic intracellular guanylyl cyclase domain. Although nitric oxide-mediated activation of cyclic guanosine monophosphate (cGMP) is known to induce megakaryocyte apoptosis and regulate platelet activation, the effects of CNP on these cells is yet to be defined. In this study for the first time we have addressed the potential role of CNP and NPR-B in megakaryocyte and platelet biology. We identified NPR-B on murine megakaryocytes by immunostaining demonstrating co-localization of CD41 and NPR-B. CNP induced cGMP production in megakaryocytes compared to a mutant form of CNP containing a single amino acid substitution (Lbab) (67.24 ± 10.65 fmol/well vs. 17.18 ± 2.089 fmol/well; P<0.05). Subsequently, CNP was shown to reduce megakaryocyte colony formation compared to Lbab and this effect was dose-dependent: Lbab 500nM 26.75 ± 5.618 CFU-M vs. 15.25 ± 2.750 CFU-M for CNP 500nM and Lbab 1000 nM 27.50 ± 2.102 CFU-M vs. 12.25 ± 1.493 CFU-M for CNP 1000nM; P<0.05). Furthermore, CNP also resulted in smaller-sized colonies, with altered cell morphology. To verify these findings, wild-type C57/BL6 mice were treated with daily injections of either saline, 1uM CNP or 1uM Lbab for one week, following which megakaryocytes were isolated and cultured. Colony formation was diminished in mice injected with CNP compared to mice receiving saline or Lbab peptide (125.3 ± 18.54 CFU-M for saline; 107.0 CFU-M ± 20.04 for Lbab and 20.00 ± 3.674 CFU-M for CNP; P<0.05). In addition platelet numbers were measured in the aforementioned mice and the CNP treated mice had significantly lower platelet counts compared to the mice treated with Lbab (832.0 ± 13.19 ×103/ul for CNP vs. 778.0 ± 12.00 ×103/ul for Lbab; P<0.05). Taken together, these data indicate that CNP may play a role in the development, maturation and/or survival of megakaryocytes. Using flow cytometry we identified that 47% of CD41+ murine platelets co-express NPR-B. In addition, this expression of NPR-B was confirmed by immunocytostaining and western blotting in both human and murine platelets. Interestingly, despite the expression of NPR-B, and unlike megakaryocytes, CNP (similar to the mutant peptide) did not increase cGMP production in platelets (1.652 ± 0.016 fmol/well for CNP and 1.606 ± 0.01550 fmol/well for Lbab). Further, although preliminary results did not indicate that CNP alters platelet secretion or aggregation, exposure to the peptide profoundly inhibited adhesion of platelets to collagen (53 ± 5 adhered platelets with Lbab, 32 ± 3 with CNP, relative reduction 42%; P<0.05). In summary, these results provide for the first time evidence that the NPR-B receptor is expressed on both megakaryocytes and platelets. Our studies suggest that CNP might have both cGMP dependent and independent functions in megakaryocytes and platelets and further studies will assist in delineating the mechanisms by which CNP exerts its actions on these cells. Disclosures: No relevant conflicts of interest to declare.

Objective— Tissue factor pathway inhibitor (TFPI) is the primary regulator of the tissue factor (TF) coagulation pathway. As such, TFPI may regulate the proangiogenic effects of TF. TFPI may also affect angiogenesis independently of TF, through sequences within its polybasic carboxyl terminus (TFPI C terminus [TFPIct]). We aimed to determine the effects of TFPI on angiogenesis and the role of TFPIct. Methods and Results— Transgenic overexpression of TFPI attenuated angiogenesis in the murine hindlimb ischemia model and an aortic sprout assay. In vitro, TFPI inhibited endothelial cell migration. Peptides within the human TFPIct inhibited endothelial cell cord formation and migration in response to vascular endothelial growth factor (VEGF) 165 but not VEGF121. Furthermore, exposure to human TFPIct inhibited the phosphorylation of VEGF receptor 2 at residue Lys951, a residue known to be critical for endothelial cell migration. Finally, systemic delivery of a murine TFPIct peptide inhibited angiogenesis in the hindlimb model. Conclusion— These data demonstrate an inhibitory role for TFPI in angiogenesis that is, in part, mediated through peptides within its carboxyl terminus. In addition to its known role as a TF antagonist, TFPI, via its carboxyl terminus, may regulate angiogenesis by directly blocking VEGF receptor 2 activation and attenuating the migratory capacity of endothelial cells.

Background— Hematopoiesis originates from the dorsal aorta during embryogenesis. Although adult blood vessels harbor progenitor populations for endothelial and smooth muscle cells, it is not known if they contain hematopoietic progenitor or stem cells. Here, we hypothesized that the arterial wall is a source of hematopoietic progenitor and stem cells in postnatal life. Methods and Results— Single-cell aortic disaggregates were prepared from adult chow-fed C57BL/6 and apolipoprotein E–null (ApoE −/− ) mice. In short- and long-term methylcellulose-based culture, aortic cells generated a broad spectrum of multipotent and lineage-specific hematopoietic colony-forming units, with a preponderance of macrophage colony-forming units. This clonogenicity was higher in lesion-free ApoE −/− mice and localized primarily to stem cell antigen-1–positive cells in the adventitia. Expression of stem cell antigen-1 in the aorta colocalized with canonical hematopoietic stem cell markers, as well as CD45 and mature leukocyte antigens. Adoptive transfer of labeled aortic cells from green fluorescent protein transgenic donors to irradiated C57BL/6 recipients confirmed the content of rare hematopoietic stem cells (1 per 4 000 000 cells) capable of self-renewal and durable, low-level reconstitution of leukocytes. Moreover, the predominance of long-term macrophage precursors was evident by late recovery of green fluorescent protein–positive colonies from recipient bone marrow and spleen that were exclusively macrophage colony-forming units. Although trafficking from bone marrow was shown to replenish some of the hematopoietic potential of the aorta after irradiation, the majority of macrophage precursors appeared to arise locally, suggesting long-term residence in the vessel wall. Conclusions— The postnatal murine aorta contains rare multipotent hematopoietic progenitor/stem cells and is selectively enriched with stem cell antigen-1–positive monocyte/macrophage precursors. These populations may represent novel, local vascular sources of inflammatory cells.

AbstractThe antithrombotic surface of endothelium is regulated in a coordinated manner. Tissue factor pathway inhibitor (TFPI) localized at the endothelial cell surface regulates the production of FXa by inhibiting the TF/VIIa complex. Systemic homozygotic deletion of the first Kunitz (K1) domain of TFPI results in intrauterine lethality in mice. Here we define the cellular sources of TFPI and their role in development, hemostasis, and thrombosis using TFPI conditional knockout mice. We used a Cre-lox strategy and generated mice with a floxed exon 4 (TFPIFlox) which encodes for the TFPI-K1 domain. Mice bred into Tie2-Cre and LysM-Cre lines to delete TFPI-K1 in endothelial (TFPITie2) and myelomonocytic (TFPILysM) cells resulted in viable and fertile offspring. Plasma TFPI activity was reduced in the TFPITie2 (71% ± 0.9%, P < .001) and TFPILysM (19% ± 0.6%, P < .001) compared with TFPIFlox littermate controls. Tail and cuticle bleeding were unaffected. However, TFPITie2 mice but not TFPILysM mice had increased ferric chloride–induced arterial thrombosis. Taken together, the data reveal distinct roles for endothelial- and myelomonocytic-derived TFPI.

Abstract Pulmonary hypertension (PH) is a commonly recognized complication of chronic respiratory disease. Enhanced vasoconstriction, pulmonary vascular remodeling, and in situ thrombosis contribute to the increased pulmonary vascular resistance observed in PH associated with hypoxic lung disease. The tissue factor pathway regulates fibrin deposition in response to acute and chronic vascular injury. We hypothesized that inhibition of the tissue factor pathway would result in attenuation of pathophysiologic parameters typically associated with hypoxia-induced PH. We tested this hypothesis using a chronic hypoxia–induced murine model of PH using mice that overexpress tissue factor pathway inhibitor (TFPI) via the smooth muscle–specific promoter SM22 (TFPISM22). TFPISM22 mice have increased pulmonary TFPI expression compared with wild-type (WT) mice. In WT mice, exposure to chronic hypoxia (28 d at 10% O2) resulted in increased systolic right ventricular and mean pulmonary arterial pressures, changes that were significantly reduced in TFPISM22 mice. Chronic hypoxia also resulted in significant pulmonary vascular muscularization in WT mice, which was significantly reduced in TFPISM22 mice. Given the pleiotropic effects of TFPI, autocrine and paracrine mechanisms for these hemodynamic effects were considered. TFPISM22 mice had less pulmonary fibrin deposition than WT mice at 3 days after exposure to hypoxia, which is consistent with the antithrombotic effects of TFPI. Additionally, TFPISM22 mice had a significant reduction in the number of proliferating (proliferating cell nuclear antigen positive) pulmonary vascular smooth muscle cells compared with WT mice, which is consistent with in vitro findings. These findings demonstrate that overexpression of TFPI results in improved hemodynamic performance and reduced pulmonary vascular remodeling in a murine model of hypoxia-induced PH. This improvement is in part due to the autocrine and paracrine effects of TFPI overexpression.