Thrombin activatable fibrinolysis inhibitor (TAFI) is a human plasma-derived zymogen that is activated through proteolytic cleavage by thrombin, thrombin in complex with thrombomodulin, or plasmin. Active TAFI attenuates fibrinolysis by removing carboxyl-terminal lysine residues from partially degraded fibrin, thereby inhibiting a potent positive feedback loop in the fibrinolytic cascade. In addition to the plasma pool of TAFI arising from expression in the liver, a distinct pool of TAFI has been reported to be present in platelets. While the antifibrinolytic effect of plasma-derived TAFI has been well-documented by in vitro and in vivo clot lysis assays, characterization of the platelet-derived form has been limited. Here, we not only confirm the presence of TAFI in the medium of washed, thrombin-stimulated platelets, but also that platelet-derived TAFI is capable of attenuating platelet-rich thrombus lysis in vitro independently of plasma TAFI using a novel thrombus lysis assay. Fluorescent thrombi were generated by suspending washed human platelets in plasma immunodepleted of TAFI containing fluorescently-labeled human fibrinogen such that the only TAFI present in the system was of platelet origin. Following platelet activation and clot retraction induced by thrombin, t-PA-dependent platelet-rich thrombus lysis was observed by removal of timed aliquots from the medium of retracted thrombi followed by measurement of fluorescence. When supplementary thrombomodulin was added to the thrombus medium, a 2.3-fold reduction in lysis rate was observed, indicating platelet-derived TAFI could attenuate the fibrinolytic cascade in vitro. Furthermore, when supplementary recombinant TAFI (rTAFI) was included in the medium, platelet-derived TAFI and rTAFI were observed to combine for greater inhibition of fibrinolysis. Taken together, these observations indicate that the secretion of platelet-derived TAFI can augment concentrations of TAFI already present in plasma to enhance attenuation of the fibrinolytic cascade. This could be significant at sites of vascular damage or regions of pathological thrombosis, where activated platelets are known to accumulate and secrete the contents of their granules. Finally, we have purified platelet-derived TAFI from platelet releasates for future characterization studies and mass spectrometry. / Thesis (Master, Biochemistry) -- Queen's University, 2009-09-24 14:22:42.5

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Background The apelin system is a novel peptidic pathway widely expressed in the heart and vasculature. In preclinical studies, apelin receptor agonism mediates nitric oxide-dependent vasodilatation, reduces ventricular preload and afterload and potently increases myocardial contractility. In preclinical models of heart failure, expression of the apelin pathway is down regulated but the haemodynamic effects of apelin receptor agonism are preserved. These changes in expression appear to be paralleled in patients with chronic heart failure but the cardiovascular actions of apelin in vivo in man are, to date, unknown. Detailed clinical investigation is therefore required to establish the role of apelin in human cardiovascular physiology and pathophysiology and to explore the therapeutic potential of apelin receptor agonism in patients with heart failure. Objectives Through a series of in vivo clinical studies: 1) to establish the direct vascular actions of apelin in the peripheral venous, peripheral arterial and coronary arterial circulations; 2) to determine the contribution of the endothelium-derived vasodilators, nitric oxide and prostacyclin, to the vascular actions of apelin; 3) to establish the effects of apelin on cardiac contractility and systemic haemodynamics; 4) to compare the direct vascular and systemic haemodynamic effects of the fulllength mature apelin peptide, apelin-36, with a shorter, biologically active carboxyl (C)-terminal fragment, (Pyr1)apelin-13); and 5) to establish whether the local vascular and systemic haemodynamic effects of apelin are altered in patients with chronic heart failure. Methods The cardiovascular effects of apelin were assessed in 32 healthy volunteers, 6 patients undergoing elective diagnostic coronary angiography, 18 patients with stable New York Heart Association (NYHA) class II-III chronic heart failure and 18 age- and sex-matched healthy controls. Dorsal hand vein tone was assessed by the Aellig hand vein technique during local intravenous infusions (0.1-3 nmol/min) of apelin-36, (Pyr1)apelin-13, and sodium nitroprusside (SNP; 0.6 nmol/min). Forearm blood flow was measured by venous occlusion plethysmography during intrabrachial infusions of apelin-36 and (Pyr1)apelin-13 (0.01-30 nmol/min) and subsequently in the presence or absence of a ‘nitric oxide clamp’ (nitric oxide synthase inhibitor, L-NG-monomethylarginine (L-NMMA; 8 μmol/min), co-infused with SNP (90-900 ng/min)), or a single oral dose of aspirin (600 mg) or matched placebo. Coronary blood flow was evaluated by quantitative coronary angiography (QCA) and Doppler flow wire, and left ventricular pressures measured by pressure wire before and after intracoronary injection of apelin-36 (20 and 200 nM), 0.9% saline and glyceryl trinitrate (GTN) (100 μg). Blood pressure, heart rate, cardiac output and peripheral vascular resistance were assessed by sphygmomanometry and thoracic electrical bioimpedance (TEB) during systemic intravenous infusion of apelin-36 and (Pyr1)apelin-13 (30-300 nmol/min). Forearm blood flow and systemic haemodynamic responses to (Pyr1)apelin-13 in patients with chronic heart failure were then compared with age- and sex-matched healthy controls. Results Although SNP caused venodilatation (P<0.0001), apelin-36 and (Pyr1)apelin-13 had no effect on dorsal hand vein diameter (P=0.2). Both apelin isoforms caused vasodilatation in forearm resistance vessels (P<0.0001) but the offset was slower with apelin-36. (Pyr1)apelin-13-mediated vasodilatation was attenuated by the nitric oxide clamp (P=0.004) but unaffected by aspirin (P=0.7). Intracoronary bolus of apelin-36 increased coronary blood flow and the maximum rate of rise in left ventricular pressure, and reduced peak and end-diastolic left ventricular pressures (all P<0.05). Both (Pyr1)apelin-13 and apelin-36 increased heart rate and cardiac output whilst reducing peripheral vascular resistance (P<0.01 for all) with no overall effect on blood pressure. Intrabrachial infusions of (Pyr1)apelin-13, acetylcholine and SNP caused forearm vasodilatation in patients and controls (P<0.0001 for all). Vasodilatation to acetylcholine (P=0.01) but not apelin (P=0.3) or SNP (p=0.9) was attenuated in patients with heart failure. Systemic infusions of (Pyr1)apelin-13 increased cardiac index and lowered mean arterial pressure and peripheral vascular resistance index in patients and matched controls (all P<0.01) but increased heart rate only in controls (P<0.01). Conclusions Although having no apparent effect on venous tone, acute apelin receptor agonism causes peripheral and coronary vasodilatation and increases cardiac contractility and output. Local vascular and systemic haemodynamic responses to apelin are preserved in patients with stable symptomatic chronic heart failure. The apelin system merits further clinical investigation to determine its role in cardiovascular homeostasis and represents a novel potential therapeutic target for patients with heart failure.

Endothelial dysfunction, present in most cardiovascular disease, results in up-regulation of inflammatory adhesion molecules/cytokines, increases in vascular permeability, and decreased vasoprotective factors leading to vascular dysfunction. A novel means of communication between almost all cells are small vesicles containing biologically active proteins, nucleic acids, and lipids known as extracellular vesicles. Despite the advances in cardiovascular biology, the role of extracellular vesicles between endothelial cells and cells of vascular wall are underexplored. Therefore, we hypothesized that endothelial activation results in the release of pro-inflammatory vesicles that initiate inflammatory remodeling of vascular smooth muscle cells of the aorta. Extracellular vesicles were released from both endothelial cells and vascular smooth muscle cells with characteristic size, shape, and content. However, serum-free collection in endothelial cells resulted in endothelial activation of cell in culture and resulted in altered function in vascular smooth muscle cells, characterized by increased monocyte adhesion, altered protein synthesis/signal transduction, and signs of pro-senescent features. These effects were not recapitulated in any combination of endothelial-vascular smooth muscle cell extracellular vesicle communication. Unbiased mass spectroscopy of vascular smooth muscle cell treated with serum-free endothelial vesicles identified several proteins significantly up- regulated, including high mobility group box 1 and 2. Pharmacologic and genetic inhibition of these molecules significantly attenuated NF-kB activation, VCAM-1 expression, and monocyte adhesion. In summation, we suggest a new axis through which endothelial activation releases vesicles that skew the function of vascular smooth muscle cells to phenotype characterized by inflammatory properties through up-regulation of high mobility group box proteins 1 and 2. This highlights the importance of extracellular vesicles as a novel communication method between cells of the vasculature and how alterations in the host cell function may change the function of these vesicles. / Biomedical Sciences

<p>The endothelial receptor tyrosine kinase (RTK) Tie1 was discovered over 20 years ago, yet its precise function and mode of action remain enigmatic. To shed light on Tie1’s role in endothelial cell biology, we investigated a potential threonine phosphorylation site within the juxtamembrane domain of Tie1. Expression of a non-phosphorylatable mutant of this site (T794A) in zebrafish (Danio rerio) significantly disrupted vascular development, resulting in fish with stunted and poorly branched intersomitic vessels. Similarly, T794A-expressing human umbilical vein endothelial cells formed significantly shorter tubes with fewer branches in three-dimensional Matrigel cultures. However, mutation of T794 did not alter Tie1 or Tie2 tyrosine phosphorylation or downstream signaling in any detectable way, suggesting that T794 phosphorylation may regulate a Tie1 function independent of its activity as a kinase. Although T794 is within a consensus Akt phosphorylation site, we were unable to identify a physiological activator of Akt that could induce T794 phosphorylation, suggesting that Akt is not the physiological Tie1-T794 kinase. However, the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), which is required for angiogenesis and capillary morphogenesis, was found to associate with phospho-T794 but not the non-phosphorylatable T794A mutant. Pharmacological activation of Rac1 induced downstream activation of p21-activated kinase (PAK1) and T794 phosphorylation in vitro, and inhibition of PAK1 abrogated T794 phosphorylation. Our results provide the first demonstration of a signaling pathway mediated by Tie1 in endothelial cells, and they suggest that a novel feedback loop involving Rac1/PAK1-mediated phosphorylation of Tie1 on T794 is required for proper angiogenesis.</p> / Dissertation

REGENERATIVE MEDICINE: VASCULARIZED SKELETAL MUSCLE Tissue engineering is a compelling strategy to create replacement tissues and in this study, skeletal muscle. One major hurdle in the field is how to vascularize large tissue-engineered constructs exceeding the nutrient delivery capability of diffusion. Endothelial colony forming cells and mesenchymal progenitor cells form blood vessels de novo and were co-injected with satellite cells in Matrigel, an extracellular matrix, or PuraMatrix, a synthetic hydrogel. Our approach focused on the ability of bioengineered vascular networks to induce murine and human satellite cells to differentiate and form organized skeletal muscle when injected. We found that perfused human blood vessels were formed in both Matrigel and PuraMatrix and that murine satellite cells differentiated and formed organized myotubes with striations, indicative of adult skeletal muscle. Mesenchymal progenitor cells also induced differentiation of satellite cells in vitro. Human Satellite cells, however, did not show signs of differentiation in either Matrigel or Puramatrix. These data have provided a proof of concept of engineering vascularized skeletal muscle using murine satellite cells. INDUCTION OF CARDIOMYOGENESIS The heart's regenerative capabilities are not robust enough to repair the amount of damaged tissue from myocardial infarction. A novel approach to relieve the ischemia is to deliver cells with vasculogenic ability, endothelial colony forming cells and mesenchymal progenitor cells, to assemble de novo blood vessels and support recovery of cardiomyocytes. In our study, we used an in vitro transwell system that prevent cell contact, but allow diffusion of soluble factors to investigate if endothelial colony forming cells or mesenchymal progenitor cells secrete factors that induce cardiomyogenesis. We found that neonatal rat cardiomyocyte proliferation is enhanced in the presence of endothelial colony forming cells and mesenchymal progenitor cells; however, presence of these cells without fetal bovine serum is not sufficient to initiate cardiomyogenesis. PERSONALIZED THERAPY FOR RENAL CELL CARCINOMA TESTING IN AN ENDOTHEIAL CELL MODEL Sunitinib and Pazopanib are both tyrosine kinase inhibitors with high specificity for vascular endothelial growth factor receptor 2 and are used in the treatment of Renal Cell Carcinoma to inhibit angiogenesis. Recent clinical findings suggest that a subset of the population with a single nucleotide polymorphism in vascular endothelial growth factor receptor 2 respond better to Pazopanib treatment. We used a standard in vitro angiogenesis assay, endothelial cell proliferation, to test the effects of the single nucleotide polymorphism on responsiveness to Sunitinib and Pazopanib. We found that cells containing the polymorphism are more sensitive to Pazopanib than Sunitinib, confirming the clinical finding. We also analyzed the inhibition of phosphorylated vascular endothelial growth factor receptor 2 and confirmed drug activity on the phosphorylated protein. These findings could have personalized clinical implications for the 3% of the population with the polymorphism.

The central role of vascular endothelial growth factor (VEGF) in physiological and pathological angiogenesis makes it attractive both as a therapeutic target for anti-angiogenic drugs in cancer treatment and as a pro-angiogenic cytokine to treat ischemic disease. Currently, it is well established that the VEGF but also other growth factors exert their angiogenic effects partly through the activation of endothelial nitric oxide synthase (eNOS). A better understanding of the VEGF/NO signaling pathway could therefore lead to the identification of new therapeutic targets to impact on angiogenesis.This thesis is based on four different articles, the principal findings of which are summarized here below. First, we demonstrated that, chronologically, endothelial cell exposure to VEGF first led to eNOS dissociation from caveolin (a hallmark of the Ca2+/CaM-mediated activation of eNOS), and then to the interaction of eNOS with the heat shock protein hsp90. We also reported that eNOS-bound hsp90 could recruit VEGF-activated (phosphorylated) Akt to the complex, which in turn could phosphorylate eNOS on the serine 1177. Finally, we found that although the VEGF-induced phosphorylation of eNOS led to a sustained production of NO independently of a maintained increase in intracellular [Ca2+], this late stage of eNOS activation was strictly conditional on the initial VEGF-induced Ca2+-dependent stimulation of the enzyme. These data established the critical temporal sequence of events leading to the sustained activation of eNOS by VEGF and suggested new ways of regulating the production of NO in response to this cytokine through the structural protein caveolin and the ubiquitous chaperone protein, hsp90. A second study identified caveolin and Hsp90 as key players in the proangiogenic action of statins and therefore as potential pharmacological targets to modulate NO-dependent angiogenesis. We found that atorvastatin stabilized endothelial tube formation from both outgrowing and isolated macrovascular ECs cultured in Matrigel through a decrease in caveolin abundance and in its inhibitory interaction with eNOS. In a similar angiogenic assay, microvascular endothelial cells appeared also responsive to statins, not through a decrease in the caveolin pool (which is (too) large in these cells) but via the increased recruitment of hsp90 in the eNOS complex and the associated eNOS phosphorylation on the serine 1177. These data provided new mechanistic insights into the NO-mediated effects of statins and underscored the potential of these drugs and other modulators of hsp90 and caveolin abundance to promote neovascularization in disease states associated or not with atherosclerosis. In a third study, we have reported a net decrease in the ability of cultured ECs expressing recombinant caveolin to migrate and to form capillary like networks (e.g. the crucial steps occurring during the angiogenic process) in presence of VEGF. We then exploited the propensity of cationic lipids to target EC lining tumor blood vessels to transfect tumor-bearing mice in vivo. A dramatic tumor growth delay associated to a decrease in tumor microvessel density in the central core of the tumor was observed in mice transfected with caveolin versus sham-transfected animals. Interestingly, we also found that in the early time after lipofection (e.g. when macroscopic effects on tumor growth were not yet detectable), caveolin expression also impaired NO-dependent tumor blood flow. These findings indicated that besides (before) acting as an anti-angiogenic agent, recombinant caveolin can modulate the endothelium phenotype and impact on the tumor blood flow, both effects leading to a decrease in tumor growth. Finally, we showed that the activation of the VEGF/NO signaling pathway led to the down-regulation of adhesion molecules and to the anergy of endothelial cells. We found, indeed, that the adhesion of human CD8+ lymphocytes on microvascular endothelial cells exposed to TNF-a was dramatically reduced in the presence of VEGF. Interestingly, we also documented that the co-administration of the NOS inhibitor L-NAME or the Hsp90 inhibitor geldanamycin could restore this adhesion to the level originally obtained with TNF-a alone. Finally, we confirmed the key role of NO in the VEGF-mediated effects on the CD8+ adhesion by tipping the balance towards more or less angiogenesis through the transfection of caveolin siRNA or caveolin plasmid, respectively. In these experiments, lymphocyte adhesion appeared directly correlated to the extent of caveolin expression, confirming that the so-called anti-angiogenic strategy can directly impact on the phenotype of (tumor) endothelial cells and instead of (before) killing them, be exploited to potentiate cancer immunotherapy. In conclusion, by dissecting the post-translational regulation of eNOS, we have identified major therapeutic targets, namely caveolin and hsp90, that may be exploited either to block or promote angiogenesis. More particulary, cDNA encoding for these proteins or their mutant form, when combined with adequate mode of delivery, appeared to exert profound effects on the vascular compartment of tumors but also of ischemic tissues.

Lintérêt actuel suscité par la contribution des prostanoïdes en général et par le TXA2 en particulier à lhoméostasie cardio-vasculaire, nous a conduit à mener une série de procédures expérimentales chez le porc. Nous avons ainsi étudié son effet sur la vasomotricité systémique qui se résume en une élévation des résistances vasculaires systémiques (R2) et une diminution de la compliance vasculaire (C). Nous avons également démontré son action pro coagulante et pro inflammatoire dans un processus dischémie myocardique induit par application topique de FeCL3. Dans ce processus, le TXA2 nexerce aucun effet sur lévolution des paramètres hémodynamiques systémiques. Enfin, il joue un rôle mineur dans lischémie-reperfusion myocardique mais il est important de rappeler que dans notre procédure expérimentale, linduction de lischémie se faisait de façon abrupte par application dun clamp coronaire ce qui rend les mécanismes thrombotiques et dont la libération de TXA2 moins prépondérants.Lutilisation d'un modulateur du thromboxane A2, le BM-573, savère efficace dans la prévention des effets de lagoniste du TXA2, lU-46619, sur les résistances vasculaires périphériques. Le BM-573 exerce de plus un effet cardioprotecteur en cas dischémie myocardique, mais aucun effet bénéfique nest observé en cas dischémie-reperfusion myocardique.Le TXA2 joue donc un rôle important dans lhoméostasie cardiovasculaire et intervient avec dautres médiateurs dans la physiopathologie de lhypertension artérielle et de lischémie myocardique. Nous navons cependant pas démontré sa contribution exacte au processus dischémie-reperfusion myocardique / Prostanoids in general and thromboxane A2 in particular appear to play a crucial role in cardiovascular homeostasis. To address this issue, we have carried out a number of experimental procedures in the pigs. We were able to show that the effects of TXA2 on sytemic vascular hemodynamic, consist in an increase of vascular resistance and a decrease of vascular compliance. Additionnaly, TXA2 had a procoagulant and proinflammatory effects in a model of myocardial ischaemia induced by topical application of FECL3 on the left anterior descending coronary artery. No effects were noticed concerning the evolution of systemic hemdynamics parameters. Finally, TXA2 appeared to play a minor role in a myocardial ischaemia-reperfusion model. The use of a TXA2 modulator, BM-573, prevented the systemic vascular effects of TXA2. It also had a cardioprotective effect in the model of myocardial ischaemia, but failed to prevent reperfusion injury in acutely ischemic pigs. We conclude that TXA2 plays an important role in cardiovascular homeostasis and that it intervenes along with others mediators in the physiopathology of hypertension and myocardial ischaemia.

Vascular endothelial cell loss initiates directional migration of medial smooth muscle cells into the arterial intima contributing to in-stent restenosis, atherosclerosis and coronary arterial by-pass graft failure. N-cadherin is a cell-cell adhesion molecule that mediates the interaction between vascular endothelial cells and the innermost smooth muscle cells to stabilize the arterial wall. Upon injury, I reasoned that relocalization of N-cadherin on the inner most smooth muscle cells to the posterior-lateral borders stimulates cell polarization to enable directional migration. Using an in vitro scratch-wound model to stimulate cell polarity and locally remove cell-cell contacts at one pole of smooth muscle cells, I found that N-cadherin localization provides signaling cues via a Cdc42/GSK pathway that promote polarized reorganization of the cytoskeleton and directional cell migration. I also found that N-cadherin was important to functions of lamellipodia at the anterior of migrating cells. In lamellipodia, actin polymerization drives protrusion of the leading edge and coincident, but more posterior, actin depolymerization results in retrograde flow of actin and associated plasma membrane structures. Using live cell imaging, I found that clusters of N-cadherin-GFP repeatedly accumulated at the leading edge specifically at the neck of large pinocytotic vesicles called macropinosomes that were internalized and transported away from the leading edge. This localization is consistent with a role for N-cadherin in closure and scission of vesicles during macropinocytosis. These are the first studies to examine polarity in migrating vascular smooth muscle cells, and advance our understanding concerning cell-cell adhesions in controlling directional cell migration. My results suggest that N-cadherin may serve as a viable target for the treatment of arterial stenosis that would limit smooth muscle cell migration and stabilize the arterial wall. Furthermore, I report on a novel localization and function of N-cadherin in the biogenesis of macropinosomes in the lamellipodia that contribute to cell protrusion.

Vascular endothelial cell loss initiates directional migration of medial smooth muscle cells into the arterial intima contributing to in-stent restenosis, atherosclerosis and coronary arterial by-pass graft failure. N-cadherin is a cell-cell adhesion molecule that mediates the interaction between vascular endothelial cells and the innermost smooth muscle cells to stabilize the arterial wall. Upon injury, I reasoned that relocalization of N-cadherin on the inner most smooth muscle cells to the posterior-lateral borders stimulates cell polarization to enable directional migration. Using an in vitro scratch-wound model to stimulate cell polarity and locally remove cell-cell contacts at one pole of smooth muscle cells, I found that N-cadherin localization provides signaling cues via a Cdc42/GSK pathway that promote polarized reorganization of the cytoskeleton and directional cell migration. I also found that N-cadherin was important to functions of lamellipodia at the anterior of migrating cells. In lamellipodia, actin polymerization drives protrusion of the leading edge and coincident, but more posterior, actin depolymerization results in retrograde flow of actin and associated plasma membrane structures. Using live cell imaging, I found that clusters of N-cadherin-GFP repeatedly accumulated at the leading edge specifically at the neck of large pinocytotic vesicles called macropinosomes that were internalized and transported away from the leading edge. This localization is consistent with a role for N-cadherin in closure and scission of vesicles during macropinocytosis. These are the first studies to examine polarity in migrating vascular smooth muscle cells, and advance our understanding concerning cell-cell adhesions in controlling directional cell migration. My results suggest that N-cadherin may serve as a viable target for the treatment of arterial stenosis that would limit smooth muscle cell migration and stabilize the arterial wall. Furthermore, I report on a novel localization and function of N-cadherin in the biogenesis of macropinosomes in the lamellipodia that contribute to cell protrusion.