Search

Tissue-type plasminogen activator (t-PA) is a pivotal enzyme in the mammalian fibrinolytic system. It exerts its action by converting the zymogen plasminogen into the proteolytically active serine protease plasmin, which, in turn, degrades fibrin clots. Because of its affinity for fibrin, t-PA is

Fibrosis is an abnormal fibroblast-activation-associated pathological manifestation in injured organs where excessive non-physiological synthesis and accumulation of extracellular matrix (ECM) proteins by activated/differentiated fibroblasts disrupts tissue homeostasis. Like other eukaryotic genes, expression of ECM protein genes not only depends on its gene sequences in the regulatory region but also influenced by non-genetic factors called epigenetic regulators including acetyltransferases, deacetylases, methyltransferases and microRNAs. The acetyltransferase p300 (ATp300), a transcriptional coactivator, is a major player in the epigenetic regulation of genes whose products are involved in cellular growth, proliferation, apoptosis and essential for embryonic development. ATp300 acetylates specific lysine residues in histones and transcription factors (KAT) and as a transcriptional coactivator it forms a bridge between upstream regulatory element binding protein complex and basal transcriptional machinery. Abnormal coactivator activity-associated diseases are known as coactivator diseases. Abnormalities in ATp300 activities in adults are associated with numerous diseases. Here, we review the significant roles of ATp300 in epigenetic regulation of collagen synthesis and deposition in extracellular spaces, matrix remodeling and tissue fibrogenesis. The present day understanding on the distinct role of acetyltransferases, deacetylases, and deacetylase inhibitors on epigenetic regulation of matrix remodeling and fibrosis has also been discussed.

Fibroblasts are responsible for producing the majority of collagen and other extracellular matrix (ECM) proteins in tissues. In the injured tissue, transforming growth factor-β (TGF-β)-activated fibroblasts or differentiated myofibroblasts synthesize excessive ECM proteins and play a pivotal role in the pathogenesis of fibrosis in heart, kidney and other organs. Recent studies suggest that fibroblast-like cells, derived from endothelial cells by endothelial-to-mesenchymal transition (EndMT), contribute to the pathogenesis of cardiac fibrosis. The molecular basis of EndMT, however, is poorly understood. Here, we investigated the molecular basis of EndMT in mouse cardiac endothelial cells (MCECs) in response to TGF-β2. MCECs exposed to TGF-β2 underwent EndMT as evidenced by morphologic changes, lack of acetylated-low density lipoprotein (Ac-LDL) uptake, and the presence of alpha-smooth muscle actin (α-SMA) staining. Treatment with SB431542, a small molecule inhibitor of TGF-β-receptor I (TβRI) kinase, but not PD98059, a MEK inhibitor, completely blocked TGF-β2-induced EndMT. The transcript and protein levels of α-SMA, Snail and β-catenin as well as acetyltransferase p300 (ATp300) were elevated in EndMT derived fibroblast-like cells. Importantly, microRNA (miRNA) array data revealed that the expression levels of specific miRNAs, known to be dysregulated in different cardiovascular diseases, were altered during EndMT. The protein level of cellular p53, a bonafide target of miR-125b, was downregulated in EndMT-derived fibroblast-like cells. Here, we report for the first time, the differential expression of miRNAs during cardiac EndMT. These results collectively suggest that TβRI serine-threonine kinase-induced TGF-β signaling and microRNAs, the epigenetic regulator of gene expression at the posttranscriptional level, are involved in EndMT and promote profibrotic signaling in EndMT-derived fibroblast-like cells. Pharmacologic agents that restrict the progression of cardiac EndMT, a phenomenon that is found in adults only in the pathological conditions, in targeting specific miRNA may be helpful in preventing and treating cardiac fibrosis.

The plasminogen activator (i.e., fibrinolytic) system is one of the key endogenous defense mechanisms against intravascular thrombosis. Thrombolytic agents represent the only direct way of augmenting fibrinolytic activity in humans, and have proven to be of value in the treatment of acute myocardial infarction and stroke. Although these agents are efficacious in the acute setting, they are not a viable option for long-term use. Net fibrinolytic activity is plasma is largely determined by the balance between tissue-type plasminogen activator (t-PA) and its natural, fast-acting inhibitor, plasminogen activator inhibitor-1 (PAI-1). The recent development of specific PAI-1 antagonists promises to expand the limits of understanding of the role of the fibrinolytic system in human disease, and to break through the current confines of therapeutic options that can effectively restore and augment the activity of the fibrinolytic system.

Women generally have a reduced risk of cardiovascular disease (CVD). However, this protection of gender diminishes rapidly after menopause and with advancing age, particularly in obese women. Alterations in vascular function are thought to a key early step in the development of atherosclerosis. In this review, we will describe the features of endothelial dysfunction in the post-menopausal obese female and discuss the interplay of aging, estrogen withdrawal, and obesity. The objectives include (1) a review of endothelial biology and endothelial dysfunction, and (2) a discussion how the endothelial function is altered in the context of aging, hormonal changes and insulin resistance. The clinical consequences of endothelial dysfunction and CVD will also be reviewed.

Livedoid vasculopathy (LV) is a chronic, recurrent, painful cutaneous disease with distinctive clinical features and an uncertain etiology. The skin lesions are recognizable by focal purpura, depigmentation and shallow ulcers. Thrombophilic conditions occur frequently in patients with LV. While no definitive treatment exists for LV, smoking cessation, antiplatelet therapy, immunosuppressive treatment, and anabolic steroids are often included in the therapeutic ladder. Recently, a possible link between LV and impaired fibrinolysis was established as cutaneous LV lesions responded to tissue plasminogen activator (t-PA) infusion suggesting that inhibition of the fibrinolysis through plasminogen activator inhibitor-1 (PAI-1) activity may determine the disease course in patients with LV. In this study, we investigated PAI-1 antigen (Ag) and activity levels in 20 patients with biopsy proven LV (mean age 26 ± 11, M/F = 7/13, median disease duration 3.5 years). All patients received antiplatelet treatment with aspirin and/or dipyrimadole and 14 patients received anabolic steroids or immunosuppressive treatment. Fasting PAI-1 Ag and activity levels were measured at 9 AM in all patients. Both Ag (34 (26) ng/ml) (median (interquartile range)) and specific activity (17 (23) IU/fmole) levels of PAI-1 were moderately elevated in LV patients compared to the controls, however, PAI-1 kinetic studies demonstrated markedly enhanced stability of PAI-1 activity in plasma from patients with LV. Specific activity at 16 h was significantly higher than expected specific activity levels (7 (11) vs. 0.07 (0.09) IU/fmole, P < 0.01). While the exact mechanism of increased stability of PAI-1 activity is not known, it may be due to post-translational modifications or increased binding affinity for a stabilizing cofactor. In conclusion, enhanced stability of PAI-1 may contribute to the pathophysiology of LV, and systemic or local treatment with PAI-1 inhibitors may offer a potential treatment alternative in patients with LV.

Elevated levels of endogenous tissue-type plasminogen activator (t-PA) appear to be a marker for preclinical atherosclerosis. At present, however, data describing potential relations between plasma t-PA level and established lipid risk factors for coronary atherosclerosis are sparse. To explore these potential relations, we measured plasma levels of t-PA antigen (t-PA:ag) in 633 apparently healthy men in the Physicians' Health Study as well as total cholesterol, high-density lipoprotein (HDL) cholesterol, HDL2 cholesterol, HDL3 cholesterol, and apolipoproteins A-I, A-II, and B-100. Overall, plasma t-PA:ag levels were inversely correlated with HDL cholesterol (r = -.1616, P < .0005), HDL2 cholesterol (r = -.1632, P < .0005), and HDL3 cholesterol (r = -.0927, P = .019). In stratified analyses, the inverse association between t-PA:ag and HDL cholesterol was present among frequent (P trend = .002) and infrequent (P trend = .004) consumers of alcohol as well as among the subgroups of frequent exercisers (P trend < .001), men in the lower half of body mass index (P trend = .001), and nonsmokers (P trend < .001). In contrast, there was no association between t-PA:ag and total cholesterol (r = .0219, P = .58), whereas relations of t-PA:ag with apolipoproteins A-I, A-II, and B-100 were minimal and of borderline significance. These data indicate that plasma levels of endogenous t-PA:ag are inversely related to HDL cholesterol as well as HDL2 and HDL3 cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)

“I'm goin' home And when I wanna go home I'm goin' mobile Well, I'm gonna find a home And we'll see how it feels Goin' mobile Keep me movin'” —Pete Townshend, The Who, 1971 [(1)][1] Although mortality following myocardial infarction (MI) has decreased significantly over the past

Left ventricular dyssynchrony is an adverse consequence of ST-elevation myocardial infarction (STEMI) and bears an unfavorable prognosis. Mechanical dyssynchrony as measured by phase analysis from gated single photon emission computed tomography (GSPECT) correlates well with other imaging methods of assessing dyssynchrony but has not been studied in STEMI. We hypothesized that systolic dyssynchrony as measured by GSPECT would correlate with adverse remodeling after STEMI. In 28 subjects suffering STEMI, GSPECT with technetium-99m sestamibi was performed immediately after presentation (day 5) and remotely (6 months). Parameters of left ventricular dyssynchrony (QRS width, histogram bandwidth (HBW) and phase standard deviation (PSD)) were measured from GSPECT using the Emory Cardiac Toolbox. Left ventricular volumes, ejection fraction (LVEF) and infarct size were also assessed. After successful primary percutaneous coronary intervention to the infarct-related artery, subjects had an LVEF of 46.4% ± 11% and a resting perfusion defect of 27.4% ± 16% at baseline. Baseline QRS width was normal (91.5 ± 17.5 ms). Subjects with STEMI had dyssynchrony compared with a cohort of 22 normal subjects (age 57.2 ± 10.6 years

Background— Elevated levels of plasminogen activator inhibitor-1 (PAI-1), a potent inhibitor of urokinase plasminogen activator and tissue plasminogen activator, are implicated in the pathogenesis of tissue fibrosis. Paradoxically, lack of PAI-1 in the heart is associated with the development of cardiac fibrosis in aged mice. However, the molecular basis of cardiac fibrosis in aged PAI-1–deficient mice is unknown. Here, we investigated the molecular and cellular bases of myocardial fibrosis. Methods and Results— Histological evaluation of myocardial tissues derived from aged PAI-1–deficient mice revealed myocardial fibrosis resulting from excessive accumulation of collagen. Immunohistochemical characterization revealed that the levels of matrix metalloproteinase-2, matrix metalloproteinase-9, and transforming growth factor-β1/2 and the number of Mac3-positive and fibroblast specific protein-1–positive cells were significantly elevated in aged PAI-1–deficient myocardial tissues compared with controls. Zymographic analysis revealed that matrix metalloproteinase-2 enzymatic activity was elevated in PAI-1–deficient mouse cardiac endothelial cells. Real-time quantitative polymerase chain reaction analyses of RNA from myocardial tissues revealed the upregulation of profibrotic markers in aged PAI-1–deficient mice. The numbers of phosphorylated Smad2–, phosphorylated Smad3–, and phosphorylated ERK1/2 MAPK–, but not pAkt/PKB–, positive cells were significantly increased in PAI-1–deficient myocardial tissues. Western blot and immunocytochemical analysis revealed that PAI-1–deficient mouse cardiac endothelial cells were more susceptible to endothelial-to-mesenchymal transition in response to transforming growth factor-β2. Conclusions— These results indicate that spontaneous activation of both Smad and non-Smad transforming growth factor-β signaling may contribute to profibrotic responses in aged PAI-1–deficient mice hearts and establish a possible link between endothelial-to-mesenchymal transition and cardiac fibrosis in PAI-1–deficient mice.

Background The objective of the study was to determine whether the effects of infarct-related artery (IRA) infusion of autologous bone marrow–derived CD34 + cells after ST elevation myocardial infarction (STEMI) are dependent on the dose (quantity and mobility) of the cells infused. Beneficial effects of IRA infusion of mononuclear cells after STEMI have been inconsistent, possibly because of differences in timing, cell type, quantity, and mobility of infused cells. Methods Patients were randomized to bone marrow harvest (n = 16) or control (n = 15). At a median of 8.3 days after coronary stenting for STEMI, CD34 + cells were infused via the IRA at 3 dose levels (5, 10, and 15 × 10 6 ) in cohorts of 5 patients each. Baseline and follow-up imaging and ex vivo CD34 + cell mobility were performed. Results Cell harvest and infusion were safe. Quantitative rest hypoperfusion score measured by single-photon emission computed tomography improved at 6 months in the ≥10 million cohorts compared with controls (−256 vs +14, P = .02). There was a trend toward improved ejection fraction at 6 months (+4.5%) in the ≥10 million cohorts compared with no change in the controls and 5 million cohort (+0.7%). Improved perfusion and infarct size reduction correlated with the quantity and mobility of the infused CD34 + cells. Conclusions The effects of CD34 + cell IRA infusion during the repair phase after STEMI are dose dependent and, at a threshold dose of 10 million CD34 + cells, associated with a significant improvement in perfusion that may limit deterioration in cardiac function (IRA infusion of CD34 + cells in patients with acute myocardial infarction [AMR-01] NCT00313339).

Plasminogen activator inhibitor-1 (PAI-1), the primary physiological inhibitor of tissue-type plasminogen activator (t-PA) in plasma, is a serine proteinase inhibitor (serpin) that forms a 1:1 stoichiometric complex with its target proteinase leading to the formation of a stable inactive complex. The active, inhibitory form of PAI-1 spontaneously converts to a latent form that can be reactivated by protein denaturants. In the present study we have isolated another molecular form of intact PAI-1 that, in contrast with active PAI-1, does not form stable complexes with t-PA but is cleaved at the P1-P1' bond (Arg346-Met347). Other serine proteinases, e.g. urokinase-type plasminogen activator and thrombin, also cleaved this "substrate" form of PAI-1. Fluorescence spectroscopy revealed conformational differences between the latent, active, and substrate forms of PAI-1. This observation confirms our hypothesis that the three functionally different forms of PAI-1 are the consequence of conformational transitions. Thus PAI-1 may occur in three interconvertible conformations: latent, inhibitor, and substrate PAI-1. The identification of two distinct conformations of PAI-1 which interact with their target protease either as an inhibitor or as a substrate is a previously unrecognized phenomenon among the serpins. Conversion of substrate PAI-1 to its inactive degradation product may constitute a pathway for the physiological regulation of PAI-1 activity.

SummaryThe effects of human recombinant plasminogen activator inhibitor (rPAI-1) on thrombolysis with recombinant tissue-type plasminogen activator (rt-PA) were studied in a rabbit model of jugular vein thrombosis. Two functionally distinct rPAI-1 preparations were used in these experiments, including latent rPAI-1 (~2 units of t-PA neutralizing activity per µg protein) and reactivated rPAI-1 (~150 units/µg).Simultaneous intravenous infusion over 4 h of 1.7 mg/kg of reactivated rPAI-1 (inhibitory capacity ~0.5 mg/kg rt-PA) with 0.5 mg/kg of rt-PA completely prevented lysis of a jugular venous thrombus, whereas an equivalent amount of latent PAI-1 did not significantly influence clot lysis. These findings demonstrate that reactivated human rPAI-1 efficiently neutralizes thrombolysis with rt-PA in vivo. Since previous studies have suggested that elevated endogenous levels of PAI-1 do not attenuate the thrombolytic potency of rt-PA in the endotoxin-treated model, we compared the stability of complexes formed by 125I-rt-PA with reactivated human rPAI-1 and with rabbit PAI-1 in vitro. Our findings indicate that both forms of PAI-1 form SDS-stable complexes following incubation with 125I-rt-PA. Thus, it seems likely that elevated levels of active PAI-1 can negate the thrombolytic effects of rt-PA in vivo and argues against the possibility that t-PA can dissociate from PAI-1 and have its activity restored in the presence of a thrombus. We propose that the present model may be a valuable tool in monitoring and evaluating the in vivo thrombolytic efficacy of various t-PA mutants designed to be less sensitive to the inhibitory effects of PAI-1.

Obesity is an increasingly important public health issue reaching epidemic proportions. Visceral obesity has been defined as an important element of the metabolic syndrome and expansion of the visceral fat mass has been shown to contribute to the development of insulin resistance and cardiovascular disease. To identify novel contributors to cardiovascular and metabolic abnormalities in obesity, we analyzed the adipose proteome and identified soluble epoxide hydrolase (sEH) in the epididymal fat pad from C57BL/6J mice that received either a regular diet or a "western diet." sEH was synthesized in adipocytes and expression levels increased upon differentiation of 3T3-L1 preadipocytes. Although normalized sEH mRNA and protein levels did not differ in the fat pads from mice receiving a regular or a "western diet," total adipose sEH activity was higher in the obese mice, even after normalization for body weight. Furthermore, peroxisome proliferator-activated receptor gamma (PPARgamma) agonists increased the expression of sEH in mature 3T3-L1 adipocytes in vitro and in adipose tissue in vivo. Considering the established role for sEH in inflammation, cardiovascular diseases, and lipid metabolism, and the suggested involvement of sEH in the development of type 2 diabetes, our study has identified adipose sEH as a potential novel therapeutic target that might affect the development of metabolic and cardiovascular abnormalities in obesity.

Several previous studies have demonstrated that administration of autologous bone marrow–derived mononuclear cells (BMMNCs) improves cardiac function in patients after acute myocardial infarction (AMI). However, optimum timing of administration has not been investigated in a clinical trial. The Cardiovascular Cell Therapy Research Network was developed and funded by the National Heart, Lung, and Blood Institute to address important questions such as timing of cell delivery and to accelerate research in the use of cell-based therapies. The TIME trial is a randomized, phase II, double-blind, placebo-controlled clinical trial. The 5 member clinical sites of the Cardiovascular Cell Therapy Research Network will enroll 120 eligible patients with moderate-to-large anterior AMIs who have undergone successful percutaneous coronary intervention of the left anterior descending coronary artery and have a left ventricular (LV) ejection fraction ≤45% by echocardiography. Participants will have bone marrow aspirations and intracoronary infusions of 150 × 10 6 BMMNCs or placebo on day 3 or day 7 post-AMI. Objectives of this study are (1) to evaluate effects of BMMNCs on regional and global LV function compared to placebo therapy in patients with acute AMI as assessed by cardiac magnetic resonance imaging at 6 months and (2) to assess whether effects of BMMNC infusion on global and regional LV function and safety are influenced by the time of administration. This study will provide further insight into the clinical feasibility and appropriate timing of autologous BMMNC therapy in high-risk patients after AMI and percutaneous coronary intervention.

The currently available thrombolytic agents are widely perceived to be suboptimal in terms of both efficacy and safety. This perception has in turn stimulated efforts to design and construct novel plasminogen activators endowed with improved biochemical and pharmacologic properties. There is as yet no consensus as to the properties of an “ideal” thrombolytic agent, and the failure of comparative clinical trials to identify a superior agent has contributed to the controversy. Although an improved plasminogen activator has not yet been constructed, it is clear that efforts to do so have advanced our knowledge of the complex structure-function relationships within plasminogen activators.

The rate and strength of beating of the heart is under the reciprocal control of the adrenergic (sympathetic) and cholinergic (parasympathetic) systems. Increased strength (inotropy) in cardiac beating in response to blood-borne epinephrine or to neurally delivered norepinephrine is mediated by β-adrenergic receptors. These G protein-coupled receptors have served as a key model for defining the molecular events linking receptor occupancy to effector regulation via G proteins.

To examine the temporal effects of plasmin generated in vivo on platelet function, we infused tissue-type plasminogen activator (t-PA) in rabbits over 3 hours and measured ex vivo platelet aggregation. We noted an initial increase in the aggregation response to ADP occurring 30 minutes after the start of infusion. This enhanced response was short-lived and by 180 minutes was reduced, compared with pretreatment levels. Baseline aggregation response was restored by 240 minutes. This pattern of aggregation response to t-PA infusion was also seen with thrombin as the agonist. Coinfusion of either prostaglandin I2 or prostaglandin E1 abolished the initial hyperaggregable phase induced by t-PA; the hypoaggregable phase occurred earlier (after 60 minutes) and persisted throughout the 1-hour recovery period. Similarly, streptokinase infused for 1 hour also increased platelet aggregation at early times and then reduced aggregation responses after the first hour. Plasma plasmin activity increased as expected with t-PA infusion alone, peaking at 30 minutes and returning to baseline by the first hour. Interestingly, prostaglandin E1 blunted the rise in plasma plasmin activity. This same dose of prostaglandin E1 or prostaglandin I2 used alone did not appreciably alter platelet function at any time during the experiment. Our data show that therapeutic doses of t-PA or streptokinase produce a biphasic effect on platelet aggregation response in the rabbit. Coinfusion of either of the antiplatelet agents, prostaglandin E1 or prostaglandin I2, abolishes the hyperaggregable phase and prolongs the inhibitory effects on platelet aggregation produced by t-PA. These data suggest that the effects of thrombolytic agents on platelet function are complex and can be modulated by antiplatelet prostaglandins.

Summary Specific receptors for urokinase have previously been identified on a number of cell types, including peripheral blood monocytes and endothelial cells. In the present study, the existence of specific receptors for single chain urokinase-type plasminogen activator (scu-PA) on gel-filtered human platelets was investigated. Platelets were found to bind 125 I-labelled recombinant scu-PA ( 125 I-rscu-PA) rapidly, specifically, saturably and reversibly, with a binding capacity of 92000±10000 molecules (mean±SEM) per platelet, and an apparent K d of 130±20nM. Unlabelled rscu-PA and high molecular weight urokinase efficiently completed with binding, whereas low molecular weight urokinase (lacking the epidermal growth factor and kringle domains) did not. Other molecules known to bind to platelets, including fibrinogen, tissue-type plasminogen activator and plasminogen, had little effect on the binding of 125 I-rscu-PA. Binding was not significantly altered by the addition of 1.5 mM Ca ++ or by 5mM EDTA. Thus, platelets have a high density, specific mechanism that binds scu-PA and potentially localises fibrinolytic activity to their surface.

Background Gonadotropin releasing hormone (GnRH) agonists are the cornerstone of metastatic prostate cancer treatment. Cardiovascular effects of GnRH agonists are unclear. In this study, we investigated the short term effects of GnRH agonists on plasma fibrinolytic parameters in patients with metastatic prostate cancer. Methods Eleven patients (mean age 69.3 ± 6.5) with metastatic prostate cancer and a clinical indication for GnRH agonist therapy were selected. Plasma plasminogen activator inhibitor (PAI-1) antigen (Ag), tissue plasminogen activator (t-PA) Ag and thrombin-activatable fibrinolysis inhibitor (TAFI) activity levels were measured at baseline and at 4 weeks after the first dose of GnRH agonist, Goserelin Acetate (Zoladex®, subcutaneous administration, 10.8 mg). Results Serum prostate specific antigen (PSA) levels significantly decreased from 36.6 ± 19.3 to 1.1 ± 0.3 ng/ml after Goserelin acetate treatment (P = 0.005). Significant changes occurred in the fibrinolytic parameters. GnRH agonists decreased plasma t-PA Ag levels (16.3 ± 4.9 vs. 12.2 ± 2.8 ng/ml, P = 0.047) and increased PAI-1/t-PA molar ratio (4.8 ± 3.6 vs. 6.6 ± 3.4, P = 0.16), on the other hand, plasma PAI-1 Ag (59.0 ± 48.5 vs. 56.4 ± 30.5 ng/ml, P = 0.8), and TAFI levels (130.6 ± 9.5 vs. 124.2 ± 26.5% activity, P = 0.3) did not change significantly. Conclusion This study provides evidence that GnRH agonists may inhibit fibrinolytic system by decreasing t-PA levels.

Intracoronary delivery of endothelial progenitor cells (EPCs) is an emerging concept for the treatment of cardiovascular disease. Enhancement of EPC adhesion to vascular endothelium could improve cell retention within targeted organs. Because extracellular adenosine is elevated at sites of ischemia and stimulates neovascularization, we examined the potential role of adenosine in augmenting EPC retention to cardiac microvascular endothelium. Stimulation of adenosine receptors in murine embryonic EPCs (eEPCs) and cardiac endothelial cells (cECs) rapidly, within minutes, increased eEPC adhesion to cECs under static and flow conditions. Similarly, adhesion of human adult culture-expanded EPCs to human cECs was increased by stimulation of adenosine receptors. Furthermore, adenosine increased eEPC retention in isolated mouse hearts perfused with eEPCs. We determined that eEPCs and cECs preferentially express functional A 1 and A 2B adenosine receptor subtypes, respectively, and that both subtypes are involved in the regulation of eEPC adhesion to cECs. We documented that the interaction between P-selectin and its ligand (P-selectin glycoprotein ligand-1) plays a role in adenosine-dependent eEPC adhesion to cECs and that stimulation of adenosine receptors in cECs induces rapid cell surface expression of P-selectin. Our results suggest a role for adenosine in vasculogenesis and its potential use to stimulate engraftment in cell-based therapies.

Susceptibility to arterial thrombosis has a significant genetic component that is partly due to the expression of two proteins, tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1), that directly influence thrombus formation and degradation. We have initiated a large-scale population-based study to characterize the genetic architecture of plasma t-PA and PAI-1 in Blacks from Sunyani, Ghana.The design of the study is based on the recruitment of 2000 unrelated subjects who are ascertained without regard to chronic disease status. The analyses of the results will be done by dividing the data into two parts, a modeling set and a validation data set. This study design will facilitate the identification of genetic, environmental, and demographic factors that contribute to inter-individual variation in plasma levels of t-PA and PAI-1 in the population at-large.We report the specifics of the study design, as well as phenotype information on the first 1000 subjects. Our results show that females and males differ significantly in several key measures, including PAI-1, BMI, total cholesterol, and systolic blood pressure.The data collected from this population-based study demonstrate significant sex differences in PAI-1 and critical factors that may influence risk of thrombosis. These samples will serve to inform the genetic analyses of t-PA and PAI- levels.

At present, thrombolytic agents represent the only direct way of augmenting fibrinolytic activity in humans. While these agents are proven to be efficacious in the treatment of acute thrombotic events, they are not a viable option for long-term administration. There are numerous drugs available that indirectly to increase fibrinolytic activity by reducing plasma levels of plasminogen activator inhibitor-1 (PAI-1), including ACE inhibitors, insulin-sensitizing agents, and hormone replacement therapy in women. At present, efforts are underway to develop and test synthetic, selective PAI-1 antagonists. The potential applications of PAI-1 antagonists include thrombotic disorders (arterial and venous), amyloidosis, obesity, polycystic ovarian syndrome, and perhaps even type 2 diabetes mellitus. The availability of specific PAI-1 antagonists promises to expand the limits of understanding the role the fibrinolytic system plays in human disease and break through the current confines of therapeutic options that can effectively restore and augment the activity of the fibrinolytic system.

The plasminogen activation system (PAS) and its principal inhibitor, plasminogen activator inhibitor-1 (PAI-1), are recognized modulators of matrix. In addition, the PAS has previously been implicated in the regulation of bone homeostasis. Our objective was to study the influence of active PAI-1 on geometric, biomechanical, and mineral characteristics of bone using transgenic mice that over-express a variant of human PAI-1 that exhibits enhanced functional stability. Femora were isolated from male and female, wildtype (WT) and transgenic (PAI-1.stab) mice at 16 and 32 weeks of age ( n = 10). Femora were imaged via DEXA for BMD and μCT for cortical mid-slice geometry. Torsional testing was employed for biomechanical properties. Mineral composition was analyzed via instrumental neutron activation analysis. Female femora were further analyzed for trabecular bone histomorphometry ( n = 11). Whole animal DEXA scans were performed on PAI-1.stab females and additional transgenic lines in which the functional domains of the PAI-1 protein were specifically disrupted. Thirty-two week female PAI-1.stab femora exhibited decreased mid-slice diameters and reduced polar moment of area compared to WT, while maintaining similar cortical bone width. Greater biomechanical strength and stiffness were demonstrated by 32 week PAI-1.stab female femora in addition to a 52% increase in BMD. PAI-1.stab trabecular bone architecture was comparable to WT. Osteoid area was decreased in PAI-1.stab mice while mineral apposition rate increased by 78% over WT. Transgenic mice expressing a reactive-site mutant form of PAI-1 showed an increase in BMD similar to PAI-1.stab, whereas transgenic mice expressing a PAI-1 with reduced affinity for vitronectin were comparable to WT. Over-expression of PAI-1 resulted in increased mineralization and biomechanical properties of mouse femora in an age-dependent and gender-specific manner. Changes in mineral preceded increases in strength/stiffness and deterred normal cross-sectional expansion of cortical bone in females. Trabecular bone was not altered in PAI-1.stab mice whereas MAR increased significantly, further supporting mineral changes as the underlying factor in strength differences. The primary influence of PAI-1 occurred during a period of basal bone remodeling, attributing a role for this system in remodeling as opposed to development. Comparison of transgenic lines indicates that PAI-1's influence on bone is dependent on its ability to bind vitronectin, and not on its proteolytic activity. The impact of PAI-1 on mouse femora supports a regulatory role of the plasminogen activation system in bone homeostasis, potentially elucidating novel targets for the treatment of bone disease.

Objective Endothelial progenitor cells (EPCs) mobilize from the bone marrow secondary to a stimulus and home to sites of injury, where they differentiate into endothelial cells and contribute to the repair of damaged vasculature. We hypothesized that growth hormone (GH) administration would increase the number of circulating EPCs in adults and thereby represent a mechanism to enhance vascular health. Design A prospective trial of low-dose GH (0.03 mg/kg/week for 4 weeks followed by 0.06 mg/kg/week for a maximum of four additional weeks) in 10 healthy adults (6 males and 4 females; mean age 37 years, range 26–65). Primary outcomes measured included the number of circulating EPCs as assessed by colony-forming unit (CFU) assay and flow cytometry. Secondary outcomes included plasma measurements of known mediators of EPC mobilization and indices of nitric oxide (NO). Outcomes were measured at baseline and at study completion. Results GH administration increased serum IGF-1 (143 ng/mL [IQR 121–164] to 222 [IQR 194–244]; P = 0.005). The increase in early-outgrowth EPCs (13 CFU per high-power field [IQR 6–24] to 19 [IQR 13–40]; P = 0.005) correlated with the peak IGF-1 after adjustment for the baseline number of early-outgrowth EPCs ( r = 0.719 [95% CI 0.06, 0.93]; P = 0.027). The number of late-outgrowth EPCs as well as CD34+, VEGFR2(KDR)+, and AC133+ cells did not significantly change. Other mediators of EPC mobilization were stable while plasma nitrite trended upwards (1.3 μmol/L [IQR 0–2.5] to 3.7 [IQR 2.2–8.9]; P = 0.052). Conclusions GH administration selectively augments the early-outgrowth EPC population in healthy individuals. These findings both support GH replacement in the setting of GH deficiency to maintain vascular integrity and have implications for the use of GH in future regenerative cell-based therapies. Furthermore, the decrease in EPCs observed with aging may in part be explained by the declining somatotropic axis, and thereby contribute to cardiovascular senescence.

Context: Adults with GH deficiency (GHD) have multiple cardiovascular risk factors, including an unfavorable lipid profile and body composition as well as impairments in endothelial function and cardiac performance. We hypothesized that GHD is associated with elevated levels of plasminogen activator inhibitor-1 (PAI-1), the major inhibitor of plasminogen activation in the circulation. Objective: The objective of the study was to determine the fibrinolytic profile of adults with GHD in comparison with controls. Study Design and Participants: This was a prospective, observational study including 12 adults with GHD. Twelve gender-, age-, and body mass index-matched adults served as controls. Main Outcome Measures: The primary outcome measures were circadian plasma PAI-1 antigen with corresponding tissue-plasminogen activator (tPA) activity values. Endothelial function was assessed by flow-mediated vasodilation and fibrinolytic potential by venous occlusion test. Results: Adults with GHD exhibited an unfavorable 24-h fibrinolytic profile characterized by a mean 62% elevation in PAI-1 antigen (2.77 ng/ml after adjustment for baseline PAI-1; P = 0.049) in the setting of a mean 24% reduction in tPA activity (−0.17 IU/ml after adjustment for baseline tPA; P = 0.003). Fibrinolytic response was defective in GHD, as demonstrated by a sustained elevation in PAI-1 activity greater than 4 IU/ml after venous occlusion [7.2 IU/ml (interquartile range 0.8–17.4); P = 0.018]. Endothelial function was impaired in GHD, as quantified by percent flow-mediated vasodilation over 120 sec [area under the curve 3.8 (interquartile range −2.4 to 7.9) vs. 12.8 (interquartile range 2.1–19.4); P = 0.043]. Conclusions: Adults with GHD demonstrate alterations in plasma fibrinolytic balance, including elevated levels of PAI-1 antigen with decreased tPA activity. These changes may contribute to the increased cardiovascular morbidity within this population.

Obesity is commonly associated with development of insulin resistance and systemic evidence of inflammation. Macrophages contribute to inflammatory amplification in obesity and may contribute directly to insulin resistance and the development of nonalcoholic fatty liver disease through the production of inflammatory cytokines, including tumor necrosis factor (TNF)-α. To test this hypothesis, we transplanted male wild-type (WT) and TNF-α deficient (KO) mice with either TNF-α-sufficient (TNF-α+/+) or TNF-α-deficient (TNF-α−/−) bone marrow. After consuming a high-fat diet for 26 wk, metabolic and morphometric characteristics of the animals were analyzed. While there were no differences in terms of relative weight gain, body composition analysis yielded a lower relative adipose and higher relative lean mass in mice lacking TNF-α, which was partially explained by reduced epididymal fat pad and liver weight. TNF-α−/−→KO mice exhibited enhanced insulin sensitivity compared with that observed in TNF-α+/+→KO mice; remarkably, no protection against insulin resistance was provided by transplanting TNF-α−/−bone marrow in WT mice compared with TNF-α+/+→WT. The preserved insulin sensitivity seen in TNF-α−/−→KO mice provided protection against the development of hepatic steatosis. Taken together, these data indicate that macrophage-derived TNF-α contributes to the pattern and extent of fat accumulation and insulin resistance in diet-induced obesity; however, this contribution is negligible in the presence of host-derived TNF-α.

We examined the effect of −58 C/T and BE1 +9/−9 polymorphisms in the bradykinin B2 receptor gene on forearm vascular resistance (FVR) before and during intrabrachial artery infusion of the B2 receptor-, endothelium-dependent agonist bradykinin and the endothelium-independent agonist sodium nitroprusside in 228 normotensive subjects. In 166 white Americans, systolic blood pressure (SBP) and pulse pressure were highest in the BE1 +9/+9 group (118±2 and 51±2 mm Hg, respectively; P

We investigated for evidence of early metabolic syndrome irrespective of body mass index (BMI) in subjects with premature pubarche (PP). Ten children with PP were compared with controls matched for age, sex, ethnicity, and BMI. Congenital adrenal hyperplasia and other known causes of PP were excluded by standard methods. Anthropometry, blood pressure (BP), dual-energy x-ray absorptiometry body scan, fasting blood lipid profile, and cytokines were obtained. The children were divided into 2 groups: (1) the total group of children with PP, and their age-, sex-, ethnicity-, and BMI-matched controls and (2) those with PP and normal BMI (

The acute physiologic release of tissue-type plasminogen activator (t-PA) from the endothelium is critical for vascular homeostasis. This process is prostacyclin- and nitric oxide (NO)-independent in humans. It has been suggested that calcium signaling and endothelial-derived hyperpolarizing factors (EDHF) may play a role in t-PA release. G-protein-coupled receptor-dependent calcium signaling is typically Galphaq-dependent. EDHFs have been functionally defined and in various tissues are believed to be various regioisomers of the epoxyeicosatrienoic acids (EETs). We tested the hypothesis in vitro that thrombin-stimulated t-PA release from human microvascular endothelial cells (HMECs) is both Galphaq- and EDHF-dependent. Conditioned media was harvested following thrombin stimulation, and t-PA antigen was measured by ELISA. Thrombin-induced t-PA release was limited by a membrane-permeable Galphaq inhibitory peptide, the PLC-beta antagonist U73122, and the IP3 receptor antagonist 2-aminoethoxyphenylborane, while the Galphaq agonist Pasteurella toxin modestly induced t-PA release. The cytochrome P450 (CYP450) inhibitor, miconazole, and the arachidonic acid epoxygenase inhibitor MS-PPOH inhibited thrombin-stimulated t-PA release, while 5,6-EET-methyl ester stimulated t-PA release. The 5,6- and 14,15-EET antagonist, 14,15-epoxyeicosa-5(Z)-enoic acid, inhibited t-PA release at the 100 microM concentration. However, thrombin-stimulated t-PA release was unaffected by the prostacyclin and NO inhibitors ASA and L-NAME, as well as the potassium channel inhibitors TEA, apamin and charybdotoxin. These studies suggest that thrombin-stimulated t-PA release is Galphaq-, PLC-beta-, IP3-, and 5,6-EET-dependent while being prostacyclin-, NO- and K+ channel-independent in HMECs.

Nω-nitro-L-arginine methyl ester (L-NAME) treatment induces arteriosclerosis and vascular senescence. Here, we report that the systemic inhibition of nitric oxide (NO) production by L-NAME causes pulmonary emphysema. L-NAME-treated lungs exhibited both the structural (alveolar tissue destruction) and functional (increased compliance and reduced elastance) characteristics of emphysema development. Furthermore, we found that L-NAME-induced emphysema could be attenuated through both genetic deficiency and pharmacological inhibition of plasminogen activator inhibitor-1 (PAI-1). Because PAI-1 is an important contributor to the development of senescence both in vitro and in vivo, we investigated whether L-NAME-induced senescence led to the observed emphysematous changes. We found that L-NAME treatment was associated with molecular and cellular evidence of premature senescence in mice, and that PAI-1 inhibition attenuated these increases. These findings indicate that NO serves to protect and defend lung tissue from physiological aging.

This study tested the hypothesis that angiotensin promotes oxidative stress and inflammation in humans via aldosterone and the mineralocorticoid receptor. We measured the effect of intravenous aldosterone (0.7 μg/kg per hour for 10 hours followed by 0.9 μg/kg per hour for 4 hours) and vehicle in a randomized, double-blind crossover study in 11 sodium-restricted normotensive subjects. Aldosterone increased interleukin (IL)-6 (from 4.7±4.9 to 9.4±7.1 pg/mL; F=4.94; P =0.04) but did not affect blood pressure, serum potassium, or high-sensitivity C-reactive protein. We next conducted a randomized, double-blind, placebo-controlled, crossover study to measure the effect of 3-hour infusion of angiotensin II (2 ng/kg per minute) and norepinephrine (30 ng/kg per minute) on separate days after 2 weeks of placebo or spironolactone (50 mg per day) in 14 salt-replete normotensive subjects. Angiotensin II increased blood pressure (increase in systolic pressure: 13.7±7.5 and 15.2±9.4 mm Hg during placebo and spironolactone, respectively; P 2 ; P P =0.002). Angiotensin II transiently increased free plasma F 2 -isoprostanes similarly during placebo and spironolactone. Angiotensin II increased serum IL-6 concentrations during placebo (from 1.8±1.1 to 2.4±1.4 pg/mL; F=4.5; P =0.04) but spironolactone prevented this effect (F=6.4; P =0.03 for spironolactone effect). Norepinephrine increased blood pressure and F 2 -isoprostanes but not aldosterone or IL-6. Aldosterone increases IL-6 in humans. These data suggest that angiotensin II induces IL-6 through a mineralocorticoid receptor–dependent mechanism in humans. In contrast, angiotensin II–induced oxidative stress, as measured by F 2 -isoprostanes, is mineralocorticoid receptor independent and may be pressor dependent.

Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) directly influence thrombus formation and degradation and thus risk for arterial thrombosis. We report here results from a genetic analysis of plasma t-PA and PAI-1 levels in a large population-based sample from the PREVEND study in Groningen, the Netherlands (n = 2,527). We measured polymorphisms from genes of the fibrinolytic system, the renin-angiotensin system (RAS), and the bradykinin system. We found that males had higher levels of natural-log transformed t-PA, and PAI-1 (P0.01) compared to females. When stratifying females by menopausal status, PAI-1 levels were only significantly different between pre-menopausal females and males (p0.001). Furthermore, we found that age, body mass index, and waist-to-hip ratio were significant predictors of t-PA and PAI-1 in both females and males, and that the regression relationships between these factors and plasma t-PA and PAI-1 were dependent on gender. In addition, we found that the PAI-1 4G/5G polymorphism was a significant predictor of PAI-1 levels in both females and males, that the angiotensin II type I receptor A1166C was a significant predictor of t-PA and PAI-1 levels in females, and that the bradykinin receptor B2 58CT polymorphism was a significant predictor of t-PA levels in females. In conclusion, this large population-based study showed that t-PA and PAI-1 levels are determined by several demographic and genetic factors involved in the fibrinolytic, RAS and bradykinin system. In addition, the results support the idea that the biology of t-PA and PAI-1 is different between females and males.

Plasma levels of plasminogen activator inhibitor-1 (PAI-1) are elevated in obesity and correlate with body mass index. The increase in PAI-1 associated with obesity likely contributes to increased cardiovascular risk and may predict the development of type 2 diabetes mellitus. Although adipocytes are capable of synthesizing PAI-1, the bulk of evidence indicates that cells residing in the stromal fraction of visceral fat are the primary source of PAI-1. We hypothesized that bone marrow-derived PAI-1, e.g. derived from macrophages located in visceral fat, contributes to the development of diet-induced obesity. To test this hypothesis, male C57BL/6 wild-type mice and C57BL/6 PAI-1 deficient mice were transplanted with either PAI-1(-/-), PAI-1(+/-), or PAI-1(+/+) bone marrow. The transplanted animals were subsequently fed a high fat diet for 24 weeks. Our findings show that only the complete absence of PAI-1 protects from the development of diet-induced obesity, whereas the absence of bone marrow-derived PAI-1 protects against expansion of the visceral fat mass. Remarkably, there is a link between the PAI-1 levels, the degree of inflammation in adipose tissue, and the development of obesity. Based on these findings we suggest that bone marrow-derived PAI-1 has an effect on the development of obesity through its effect on inflammation.

Bradykinin 1-5 is a major stable metabolite of bradykinin, formed by the proteolytic action of angiotensin-converting enzyme. In vitro and animal studies suggest that bradykinin 1-5 possesses biological activity. This study tests the hypothesis that bradykinin 1-5 affects vasodilation, fibrinolysis, or platelet aggregation in humans. Graded doses of bradykinin (47-377 pmol/min) and bradykinin 1-5 (47-18,850 pmol/min) were infused in the brachial artery in random order in 36 healthy subjects. Forearm blood flow (FBF) was measured, and simultaneously obtained venous and arterial plasma samples were analyzed for tissue plasminogen activator (t-PA) antigen. In seven subjects each, alpha- and gamma-thrombin-induced platelet aggregation was measured in platelet-rich plasma obtained from antecubital venous blood at baseline and during peptide infusions. Bradykinin caused dose-dependent increases in FBF and net t-PA release (P0.001 for both). Bradykinin 1-5 did not affect FBF (P = 0.13) or net t-PA release (P = 0.46) at concentrations1500 times physiologic. In contrast, both bradykinin and bradykinin 1-5 inhibited alpha-and gamma-thrombin-induced platelet aggregation (P0.01 versus baseline). Bradykinin 1-5 inhibited gamma-thrombin-induced platelet aggregation 50% at a calculated dose of 183 +/- 3 pmol/min. Neither bradykinin nor bradykinin 1-5 affected thrombin receptor-activating peptide-induced platelet aggregation, consistent with the hypothesis that bradykinin and bradykinin 1-5 inhibit thrombin-induced platelet aggregation by preventing cleavage of the thrombin receptor and liberation of thrombin receptor-activating peptide. This study is the first to demonstrate biological activity of bradykinin 1-5 following in vivo administration to humans. By inhibiting thrombin-induced platelet aggregation without causing vasodilation, bradykinin 1-5 may provide a model for small molecule substrate-selective thrombin inhibitors.

Hepatic veno-occlusive disease (VOD) is a common complication of high-dose chemotherapy associated with bone marrow transplantation. While the pathogenesis of VOD is uncertain, plasminogen activator inhibitor-1 (PAI-1) has emerged as a diagnostic marker and predictor of VOD in humans. In this study, we investigated the role of PAI-1 in a murine model of VOD produced by long-term nitric oxide synthase inhibition using L-NAME. After 6 weeks, wild-type (WT) mice developed extensive fibrinoid hepatic venous thrombi and biochemical evidence of hepatic injury and dysfunction. In contrast, PAI-1–deficient mice were largely protected from the development of hepatic vein thrombosis. Furthermore, WT mice that received tiplaxtinin, an antagonist of PAI-1, were effectively protected from L-NAME–induced thrombosis. Taken together, these data indicate that NO and PAI-1 play pivotal and antagonistic roles in hepatic vein thrombosis and that PAI-1 is a potential target in the prevention and treatment of VOD in humans.

This study tests the hypothesis that spironolactone influences plasminogen activator inhibitor-1 (PAI-1) concentrations through mineralocorticoid receptor antagonism rather than through changes in potassium. Effects of spironolactone (50 mg per day) and triamterene (50 mg per day) on fibrinolytic balance were compared in 18 normotensive and 20 hypertensive subjects pretreated with hydrochlorothiazide (HCTZ; 12.5 mg per day). Blood pressure and serum potassium were similar in spironolactone and triamterene treatment groups. The effect of the 2 drugs on the renin-angiotensin-aldosterone system was also similar. In contrast, spironolactone and triamterene exerted opposing effects on PAI-1 antigen ( P =0.006 for drug effect). In normotensive subjects, triamterene (from 10.1±7.8 to 16.9±9.9 ng/mL at 9 am , P =0.019; from 7.6±5.4 to 11.5±7.3 ng/mL at 11 am , P =0.027; from 9.3±7.7 to 13.7±8.5 ng/mL for average of all time points, P =0.054) but not spironolactone significantly increased PAI-1 antigen. In hypertensive subjects, spironolactone significantly decreased PAI-1 antigen (from 22.0±23.4 to 16.7±19.0 ng/mL at 10 am , P =0.041; from 17.5±21.7 to 12.7±16.8 ng/mL at 11 am , P =0.043; from 20.3±22.6 to 16.6±19.7 ng/mL for average of all time points, P =0.014), whereas there was no effect of triamterene. Only spironolactone significantly decreased the molar ratio of PAI-1 to tissue-type plasminogen activator (t-PA) in hypertensive subjects. By regression analysis, predictors of mean PAI-1 response were spironolactone versus triamterene ( P =0.014), hypertension ( P =0.002), and PAI-1 response to HCTZ ( P =0.019), with a trend for aldosterone ( P =0.061). Mineralocorticoid receptor antagonism prevents the effect of activation of the renin-angiotensin-aldosterone system on PAI-1 antigen in normotensive subjects and improves fibrinolytic balance in hypertensive subjects through a potassium-independent mechanism.

The vascular endothelium maintains blood fluidity by inhibiting blood coagulation, inhibiting platelet aggregation, and promoting fibrinolysis. Endothelial cells lose these nonthrombogenic properties on exposure to proinflammatory stimuli. We recently identified the Kruppel-like factor KLF2 as a novel regulator of endothelial proinflammatory activation. Here it is found that KLF2 differentially regulates key factors involved in maintaining an antithrombotic endothelial surface. Overexpression of KLF2 strongly induced thrombomodulin (TM) and endothelial nitric oxide synthase (eNOS) expression and reduced plasminogen activator inhibitor-1 (PAI-1) expression. Furthermore, overexpression of KLF2 inhibited the cytokine-mediated induction of tissue factor (TF). In contrast, siRNA mediated knockdown of KLF2 reduced antithrombotic gene expression while inducing the expression of pro-coagulant factors. The functional importance of KLF2 was verified by in vitro clotting assays. By comparison to control infected cells, KLF2 overexpression increased blood clotting time as well as flow rates under basal and inflammatory conditions. In contrast, siRNA-mediated knockdown of KLF2 reduced blood clotting time and flow rates. These observations identify KLF2 as a novel transcriptional regulator of endothelial thrombotic function. The full text of this article is available online at http://circres.ahajournals.org.

Thrombospondin-1 (THBS1) is a large extracellular matrix glycoprotein that affects vasculature systems such as platelet activation, angiogenesis, and wound healing. Increases in THBS1 expression have been liked to disease states including tumor progression, atherosclerosis, and arthritis. The present study focuses on the effects of thrombin activation of the G-protein-coupled, protease-activated receptor-1 (PAR-1) on THBS1 gene expression in the microvascular endothelium. Thrombin-induced changes in gene expression were characterized by microarray analysis of approximately 11,000 different human genes in human microvascular endothelial cells (HMEC-1). Thrombin induced the expression of a set of at least 65 genes including THBS1. Changes in THBS1 mRNA correlated with an increase in the extracellular THBS1 protein concentration. The PAR-1-specific agonist peptide (TFLLRNK-PDK) mimicked thrombin stimulation of THBS1 expression, suggesting that thrombin signaling is through PAR-1. Further studies showed THBS1 expression was sensitive to pertussis toxin and protein kinase C inhibition indicating G(i/o)- and G(q)-mediated pathways. THBS1 up-regulation was also confirmed in human umbilical vein endothelial cells stimulated with thrombin. Analysis of the promoter region of THBS1 and other genes of similar expression profile identified from the microarray predicted an EBOX/EGRF transcription model. Expression of members of each family, MYC and EGR1, respectively, correlated with THBS1 expression. These results suggest thrombin formed at sites of vascular injury increases THBS1 expression into the extracellular matrix via activation of a PAR-1, G(i/o), G(q), EBOX/EGRF-signaling cascade, elucidating regulatory points that may play a role in increased THBS1 expression in disease states.

Objective— To test the hypothesis that pharmacological plasminogen activator inhibitor (PAI)-1 inhibition protects against renin-angiotensin-aldosterone system-induced cardiovascular injury, the effect of a novel orally active small-molecule PAI-1 inhibitor, PAI-039, was examined in a mouse model of angiotensin (Ang) II-induced vascular remodeling and cardiac fibrosis. Methods and Results— Uninephrectomized male C57BL/6J mice were randomized to vehicle subcutaneus, Ang II (1 μg/h) subcutaneous, vehicle+PAI-039 (1 mg/g chow), or Ang II+PAI-039 during high-salt intake for 8 weeks. Ang II caused significant medial, adventitial, and aortic wall thickening compared with vehicle. PAI-039 attenuated Ang II-induced aortic remodeling without altering the pressor response to Ang II. Ang II increased heart/body weight ratio and cardiac fibrosis. PAI-039 did not attenuate the effect of Ang II on cardiac hypertrophy and increased fibrosis. The effect of PAI-039 on Ang II/salt-induced aortic remodeling and cardiac fibrosis was comparable to the effect of genetic PAI-1 deficiency. Ang II increased aortic mRNA expression of PAI-1, collagen I, collagen III, fibronectin, osteopontin, monocyte chemoattractant protein-1, and F4/80; PAI-039 significantly decreased the Ang II-induced increase in aortic osteopontin expression at 8 weeks. Conclusions— This study demonstrates that pharmacological inhibition of PAI-1 protects against Ang II-induced aortic remodeling. Future studies are needed to determine whether the interactive effect of Ang II/salt and reduced PAI-1 activity on cardiac fibrosis is species-specific.

To test the hypothesis that NO influences aldosterone production in humans, we examined the effect of N G -nitro- l -arginine methyl ester ( l -NAME) on aldosterone concentrations in the presence and absence of the NO precursor l -arginine (3 g TID) and the angiotensin-converting enzyme inhibitor ramipril (10 mg QD). Ten normal subjects were given l -NAME (66 μg/kg per min for 30 minutes) or vehicle in random order on separate days during placebo and after randomized, double-blind treatment with l -arginine, ramipril, or l -arginine plus ramipril. Infusion of l -NAME significantly increased systolic blood pressure (all P P ≤0.02) during all 4 treatment arms. After placebo pretreatment, serum aldosterone was significantly higher during l -NAME infusion than during vehicle (6.6±1.7 versus 3.3±0.5 ng/dL; P =0.045). Combined treatment with l -arginine plus ramipril abolished this effect. There was no effect of l -NAME on plasma renin activity (PRA; P =0.297) or angiotensin II concentrations ( P =0.537). However, there was a significant interactive effect of l -NAME and time on serum potassium ( P =0.039). There was a significant linear relationship between PRA and aldosterone concentration after vehicle infusion ([aldosterone]=3.9·PRA+1.9; r 2 =0.476; P =0.027) and l -NAME infusion ([aldosterone]=7.2·PRA+3.1; r 2 =0.457; P =0.032), and the intercepts of these lines were different ( P =0.029). There was a significant linear relationship between serum potassium and aldosterone during l -NAME ([aldosterone]=8.2 · [potassium]−28.9; r 2 =0.609; P =0.008) but not during vehicle ( P =0.313). These data suggest that endogenous NO modulates aldosterone synthesis in humans.

In the current Adult Treatment Panel guidelines for cardiovascular risk detection,1 the plasma-based markers recommended for use in global risk assessment or in the definition of the metabolic syndrome are low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol, and triglycerides. It is widely recognized, however, that more than half of all future vascular events occur in individuals without overt hyperlipidemia. For example, in a recent large-scale analysis of >27 000 healthy American women, 77% of all future events occurred in those with LDL-C levels

To investigate the molecular mechanisms involved in the estrogen-dependent control of plasminogen activator inhibitor-1 (PAI-1) gene expression in vascular cells, we compared the transactivation properties of estrogen receptors (ERalpha and ERbeta) in regulating the activity of a human PAI-1 promoter reporter construct in transfected bovine aortic endothelial cells (BAECs). ERalpha increased PAI-1 promoter activity in BAECs by an estrogen-dependent mechanism, whereas ERbeta suppressed PAI-1 promoter activity by an estrogen-independent mechanism. The suppressive activity of ERbeta was dominant over the inductive activity of ERalpha. Mutation of a putative estrogen response element (ERE) located at position -427 in the proximal promoter abolished the ERalpha action without influencing the suppressive effects of ERbeta. Mutation of either AP1-like site did not eliminate the ERalpha or ERbeta actions at the PAI-1 promoter, suggesting that other promoter elements are involved in these responses. These mutations significantly reduced the -3.4kbp PAI-1 promoter response to serum. We concluded that ERalpha and ERbeta exert differential effects on the PAI-1 promoter activity in transfected BAECs. ERalpha activated the PAI-1 promoter through a proximal ERE (-427) and possibly additional EREs located within the PAI-1 promoter, whereas ERbeta suppressed the promoter construct via an unidentified mechanism. This is the first demonstration of the differential regulation of a vascular gene promoter by ERalpha and ERbeta.

Increased plasminogen activator inhibitor 1 (PAI-1) has been linked to not only thrombosis and fibrosis but also to obesity and insulin resistance. Increased PAI-1 levels have been presumed to be consequent to obesity. We investigated the interrelationships of PAI-1, obesity, and insulin resistance in a high-fat/high-carbohydrate (HF) diet-induced obesity model in wild-type (WT) and PAI-1-deficient mice (PAI-1(-/-)). Obesity and insulin resistance developing in WT mice on an HF diet were completely prevented in mice lacking PAI-1. PAI-1(-/-) mice on an HF diet had increased resting metabolic rates and total energy expenditure compared with WT mice, along with a marked increase in uncoupling protein 3 mRNA expression in skeletal muscle, likely mechanisms contributing to the prevention of obesity. In addition, insulin sensitivity was enhanced significantly in PAI-1(-/-) mice on an HF diet, as shown by euglycemic-hyperinsulinemic clamp studies. Peroxisome proliferator-activated receptor (PPAR)-gamma and adiponectin mRNA, key control molecules in lipid metabolism and insulin sensitivity, were maintained in response to an HF diet in white adipose tissue in PAI-1(-/-) mice, contrasting with downregulation in WT mice. This maintenance of PPAR-gamma and adiponectin may also contribute to the observed maintenance of body weight and insulin sensitivity in PAI-1(-/-) mice. Treatment in WT mice on an HF diet with the angiotensin type 1 receptor antagonist to downregulate PAI-1 indeed inhibited PAI-1 increases and ameliorated diet-induced obesity, hyperglycemia, and hyperinsulinemia. PAI-1 deficiency also enhanced basal and insulin-stimulated glucose uptake in adipose cells in vitro. Our data suggest that PAI-1 may not merely increase in response to obesity and insulin resistance, but may have a direct causal role in obesity and insulin resistance. Inhibition of PAI-1 might provide a novel anti-obesity and anti-insulin resistance treatment.

Background— Angiotensin-converting enzyme (ACE) inhibition potentiates the tissue-type plasminogen activator (t-PA) response to exogenous bradykinin. This study tested the hypothesis that ACE inhibition increases endothelial t-PA release through endogenous bradykinin. Methods and Results— We measured the effect of intra-arterial enalaprilat (5 μg/min) on forearm blood flow (FBF) and net t-PA release before and during intra-arterial infusion of bradykinin (25 to 400 ng/min) and methacholine (3.2 to 12.8 μg/min) in 24 smokers pretreated with bradykinin receptor antagonist HOE 140 (100 μg/kg intravenously) or vehicle. There was no specific effect of HOE 140 on FBF or forearm vascular resistance (FVR, 29.9±3.6 versus 29.7±3.6 mm Hg · mL −1 · min −1 · 100 mL −1 after vehicle and HOE 140, respectively, P =0.956 between groups). Resting FVR decreased during enalaprilat compared with vehicle or HOE 140, but not compared with baseline, and the effect was similar in the 2 groups (22.0±2.7 and 24.1±2.9 mm Hg · mL −1 · min −1 · 100 mL −1 , respectively, P =0.610). In contrast, enalaprilat significantly increased resting net t-PA release (from 0.6±0.4 to 1.7±0.6 ng · min −1 · 100 mL −1 , P =0.002); this effect was abolished by HOE 140 (0.1±0.3 ng · min −1 · 100 mL −1 , P =0.036 versus enalaprilat alone). Enalaprilat increased the effect of exogenous bradykinin on FBF 60% (from 17.5±2.5 to 28.1±4.0 mL · min −1 · 100 mL −1 during 100 ng/min bradykinin, P =0.001) and on t-PA release 14-fold (from 21.2±7.9 to 317.4±118.9 ng · min −1 · 100 mL −1 , P =0.024). Enalaprilat increased the t-PA response to bradykinin to a greater extent than the FBF response, shifting the relationship between net t-PA release and FBF ( P =0.005). HOE 140 blocked these effects. There was no effect of enalaprilat or HOE 140 on the FBF or t-PA response to methacholine. Conclusion— ACE inhibition increases constitutive endothelial t-PA release through endogenous bradykinin.