Simari, Robert D.

Advances in molecular and cellular biology have supported the development of new therapeutic approaches to limiting ischemia. Most often these approaches target patients with ischemia post coronary artery bypass grafting (CABG). Currently, there are few data available on the incidence and outcomes of such patients. To understand this important patient cohort and to estimate the potential need for new therapies, we performed a population-based study of post CABG patients who underwent coronary and graft angiography for angina from 2001 – 2005. Patients who underwent coronary artery and bypass graft angiography at Mayo Clinic from 2001 – 2005 were identified. Medical records were reviewed to determine indication for angiography, and angiographic analysis was performed in all patients. The study cohort was then followed prospectively until March 1 2008. Among 133,000 residents of Olmsted County, Minnesota, 347 post bypass patients with angina underwent coronary angiography from 2001 – 2005. Of those, 51% (177 patients) received further revascularization (endovascular and surgical) and the remaining 170 patients were managed medically. There was no statistical difference in terms of all cause mortality and cardiac mortality between the group that received further revascularization and the group that did not. Adjusting for age and sex for differences between the Olmsted County population and the US white 2000 population yields a yearly incidence rate of 17.9 (95% CI 14.2–21.6) to 33.2 (95% CI 28.2 – 38.3) patients with non-revascularizable angina following CABG per 100,000 population. There was no difference in all cause or cardiac mortality in post-CABG patients presenting with angina whether they received further endovascular/surgical revascularization or not. We estimate that non-revascularizable coronary artery disease post CABG arises in approximately 50,000 (95% CI 40,000 – 61,000) to 93,000 (95% CI 79,000 – 110,000) patients per year extrapolating to the entire US population as a whole.

Abstract Objective. —Vascular gene transfer provides unique opportunities for novel therapies for cardiovascular disease. To define the limitations and expectations of translating preclinical strategies to clinical therapy, the kinetics and extent of vascular transgene expression must be determined. Data Sources. —Published clinical and preclinical studies of vascular gene transfer. Data Extraction. —Kinetics and extent of vascular transgene expression. Data Synthesis. —In spite of ongoing clinical studies in vascular gene transfer, data regarding the kinetics and extent of transgene expression are very limited. Conclusions. —For the efficient translation of preclinical to clinical vascular gene transfer studies, the kinetics and extent of transgene expression should be determined. Novel imaging technologies and unique clinical scenarios for obtaining tissue should be considered.

Abstract TFPI is an endothelium associated anticoagulant protein that directly inhibits tissue factor (TF) pro-coagulant activity and prevents disease produced by intravascular thrombosis. TFPI is produced in three alternatively spliced isoforms in mice, α, β and γ, that differ in domain structure and mechanism for association with endothelium. TFPIα has three Kunitz-type serine protease inhibitory domains and a basic C-terminal region. It produces anticoagulant activity by direct inhibition of factor Xa (fXa) via the second Kunitz domain (K2) and, in a fXa dependent manner, inhibition of TF-fVIIa via the first Kunitz domain (K1). The third Kunitz domain (K3) and C-terminal region do not directly inhibit proteolysis. However, alignment of sequences from various vertebrate species demonstrates that K3 has maintained a high degree of sequence conservation for over 430 million years suggesting that K3 maintains a physiological function. TFPIα indirectly associates with endothelium by binding to a glycosylphosphatidyl inositol (GPI) anchored protein and binds non-specifically to glycosaminoglycans. TFPIβ has K1 and K2 but lacks K3 and has a distinct C-terminal region that encodes a GPI-anchor attachment sequence. TFPIγ has K1 and K2 but lacks K3 and has a distinct C-terminal region that results in production of a secreted protein. It is not known if these structurally diverse forms of TFPI with different mechanisms for cell surface association have variable efficacy in their ability to inhibit TF activity in vivo. We characterized TFPI isoform expression in mouse tissues in order to define specific physiological functions for alternative splicing of TFPI and how it may hinder diseases mediated by intravascular TF activity. Message for TFPIα is ~16-fold more abundant than message for TFPIβ or TFPIγ in mouse tissues. In situ hybridization studies of mouse heart and lung found that the isoforms are expressed in the same pattern, primarily in endothelial cells. No cell or vascular bed exclusively producing a single isoform was identified. Western blot analyses revealed the somewhat surprising finding that TFPIβ is the predominant isoform produced by adult mouse tissues. Thus, protein synthesis of TFPI isoforms in mice is regulated at least in part at the level of protein translation. Mouse plasma TFPI levels increase only 10–20% following heparin infusion. These data contrast with the 2- to 4-fold increase in plasma TFPI observed following heparin infusion in humans. The presence of only small amounts of heparin releasable TFPI in mouse plasma is consistent with TFPIβ as the major isoform because TFPIβ is directly GPI-anchored to endothelium and lacks the highly basic C-terminal region that allows TFPIα to associate non-specifically with endothelium glycosaminoglycans. To our knowledge, these are the first data demonstrating TFPIβ production in vivo. In contrast to the adult tissues, western blot analysis of mouse placenta revealed that it produces primarily TFPIα. This finding led us to examine mouse fetal tissue to determine if there is developmental regulation of the production of different TFPI isoforms. These studies demonstrated that TFPIα and TFPIβ are produced in approximately equal amounts in E14.5 embryos. The relative proportion of TFPIα in mouse tissues then decreases during development and very little is produced at 12 weeks of age. The evolutionary conservation of TFPIα and its production in mouse placenta and embryonic tissues suggests that unique features of TFPIα, such as K3, the basic C-terminal region and/or its binding to the endothelial surface through a GPI-anchored co-receptor, perform specific functions in the regulation of intravascular TF activity during vasculogenesis/angiogenesis that are not performed by TFPIβ or TFPIγ.

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.

Abstract Introduction: Tissue factor pathway inhibitor (TFPI) is an anticoagulant protein that inhibits tissue factor and prothrombinase during early stages of the procoagulant response. It is produced in alternatively spliced isoforms. In humans, TFPIα is within platelets and released upon activation and also is present in plasma, while TFPIβ is bound to the endothelial surface. Mice differ from humans in that they do not have TFPIα in plasma, making them an optimal model system for study of the function of platelet TFPIα. Studies of murine hematopoietic cell TFPI using fetal liver transplantation models have found that platelet TFPI dampens platelet accumulation in a growing thrombus following vascular injury and alters bleeding severity in mice with hemophilia. However, results of these studies are potentially confounded by altered TFPI expression on other hematopoietic cells, such as monocytes. To better understand the function of platelet TFPI, murine models of total TFPIα deletion (TFPIα KO), of platelet-specific TFPIα deletion (pTFPIα KO) and platelet-specific TFPIα over expression (pTFPIα high) were produced and characterized. Methods: A TFPIα deleted mouse was generated by inserting a stop codon within exon 10 of the TFPI gene effectively blocking translation of the highly conserved basic C-terminal region of TFPIα, but not altering TFPIβ. The PF4-Cre transgene was bred into the floxed-TFPI(K1) mouse to produce mice specifically lacking platelet TFPIα. A transgenic mouse over expressing platelet TFPIα under control of the GP1b promoter was produced. Mice were characterized for platelet and plasma TFPI antigen by ELISA, for activity by TF-FVIIa inhibition and whole blood platelet aggregation, and for bleeding by tail clip. Results: All 3 strains were viable and reproduced. Platelet TFPI antigen was decreased approximately 25-fold in the TFPIα KO and pTFPIα KO mice and elevated approximately 5-fold in the pTFPIα high mice with corresponding changes in TF-FVIIa inhibitory activity. Plasma TFPI antigen was no different between pTFPIα KO (20.6±4.4 nM) and wild type (21.5±6.2 nM) littermates. Plasma thrombin-antithrombin complex levels were not elevated in any strain. In tail clip bleeding time assays, there was no difference between pTFPIα KO (0.23±0.16 mg/ml Hb) and wild type (0.23±0.37 mg/ml Hb), but there was a trend towards increased blood loss in pTFPIα high mice (1.25±1.8 mg/ml Hb). The pTFPIα high mice also had altered whole blood platelet aggregation that was evident primarily in a prolonged time for initiation of aggregation with 6.50uM ADP (81±12 sec vs. 6.1±5.6 sec for control) or 0.8ug/ml collagen (35±17 sec vs. 16±21 sec for control). Finally, the pTFPIα high transgene was bred into TFPI heterozygous mice which were then bred to determine if over expression of platelet TFPIα would rescue the TFPI null embryonic lethal phenotype. However, no live TFPI null pups were observed following genotyping of over 50 pups from these matings. Conclusions: Three new mouse models of altered TFPI expression have been made providing valuable tools to study the role of platelet TFPI in bleeding and clotting disorders. Plasma TFPI is unchanged in mice lacking platelet TFPI demonstrating that platelet TFPI does not contribute to plasma TFPI in mice. Platelet TFPIα antigen and activity correlate in all three models. Deletion of the basic C-terminal region in TFPIα in the TFPIα KO mice produces a TFPIα hypomorphic mouse, with only trace amounts of truncated TFPIα present in platelets. This lack of TFPIα does not produce a generalized procoagulant state. The 5-fold excess TFPIα antigen in the pTFPIα high mice substantially delays the onset of platelet aggregation in whole blood assays initiated with collagen or ADP. Results from tail clip bleeding assays suggest that over expression of platelet TFPIα may prolong bleeding in adult mice, but it is not sufficient to rescue the embryonic lethality of TFPI null mice. Disclosures Mast: Novo Nordisk: Honoraria, Research Funding.

The field of gene transfer has developed in an era of expanding biomedical knowledge. The potential for gene transfer to treat cardiovascular disease is great, yet identified and unidentified barriers remain. Gene transfer and its ultimate application, gene therapy, require extensive details of not only the mechanism of disease but the biological implications of the vectors used to deliver the therapeutic genes as well. Many of these details are becoming available via the study of genomics. Genomics, the study of complete genetic sequences, holds the potential for enabling and amplifying the therapeutic hopes for gene transfer. Identification of new therapeutic genes, new regulatory sequences, and establishing the patterns of gene expression from tissues exposed to vectors and transgenes will rapidly advance the application of gene transfer. Finally, there are historical and ongoing lessons learned from the development of gene transfer that may be applicable to the challenging field of genomics and may enable its future success.

Chronic passive hepatic congestion (congestive hepatopathy) leads to hepatic fibrosis; however, the mechanisms involved in this process are not well understood. We developed a murine experimental model of congestive hepatopathy through partial ligation of the inferior vena cava (pIVCL). C57BL/6 and transgenic mice overexpressing tissue factor pathway inhibitor (SM22α‐TFPI) were subjected to pIVCL or sham. Liver and blood samples were collected and analyzed in immunohistochemical, morphometric, real‐time polymerase chain reaction, and western blot assays. Hepatic fibrosis and portal pressure were significantly increased after pIVCL concurrent with hepatic stellate cell (HSC) activation. Liver stiffness, as assessed by magnetic resonance elastography, correlated with portal pressure and preceded fibrosis in our model. Hepatic sinusoidal thrombosis as evidenced by fibrin deposition was demonstrated both in mice after pIVCL as well as in humans with congestive hepatopathy. Warfarin treatment and TFPI overexpression both had a protective effect on fibrosis development and HSC activation after pIVCL. In vitro studies show that congestion stimulates HSC fibronectin (FN) fibril assembly through direct effects of thrombi as well as by virtue of mechanical strain. Pretreatment with either Mab13 or Cytochalasin‐D, to inhibit β‐integrin or actin polymerization, respectively, significantly reduced fibrin and stretch‐induced FN fibril assembly. Conclusion : Chronic hepatic congestion leads to sinusoidal thrombosis and strain, which in turn promote hepatic fibrosis. These studies mechanistically link congestive hepatopathy to hepatic fibrosis. (H epatology 2015;61:648‐659)