Corrie A. Painter

Age-Dependent Spontaneous Coronary Arterial Thrombosis in Transgenic Mice That Express a Stable Form of Human Plasminogen Activator Inhibitor-1

Background—Plasminogen activator inhibitor-1 (PAI-1) regulates fibrinolysis and has been reported to be an independent risk factor for ischemic cardiovascular events. This study describes the age-dependent development of spontaneous coronary arterial thrombi that are associated with evidence of subendocardial myocardial infarction in mice transgenic for human PAI-1. Methods and Results—We generated two independent transgenic mice founder lines that express a stable variant of active human PAI-1 under control of the murine preproendothelin-1 (mPPET-1) promoter. Backcrossed homozygous transgenic animals from founder line I had plasma PAI-1 levels of 23±12 ng/mL. PAI-1 transgenic animals younger than 4 months do not exhibit any evidence of arterial or venous thrombosis. Ninety percent of transgenic animals (n=10) older than 6 months developed spontaneous occlusions of typically multiple, penetrating coronary arteries, with histological evidence of subendocardial infarction identified in 50% of animals. Conclusions—This study shows that chronically elevated levels of PAI-1 are associated with age-dependent coronary arterial thrombosis in mice transgenic for human PAI-1. This is the first study of a murine model of coronary thrombosis that occurs in the absence of severe hypercholesterolemia or multiple genetic manipulations. These findings provide new evidence to support the hypothesis that PAI-1 excess contributes to the development of coronary arterial thrombosis.

Regulation of the PAI-1 promoter by circadian clock components: differential activation by BMAL1 and BMAL2.

Circadian variation in plasminogen activator inhibitor-1 (PAI-1) production likely contributes to increased risk of myocardial infarction and decreased efficacy of thrombolytic therapy during the morning. In this study, we characterize the abilities of fundamental molecular components of intrinsic circadian clocks to regulate the human PAI-1 promoter in transfected endothelial cells. Both CLOCK:BMAL1 and CLOCK:BMAL2 heterodimers activate the PAI-1 promoter through requisite proximal (-565 to -560 bp) and distal (-680 to -675 bp) E-box enhancers. Although the distal E-box overlaps the 4G/5G polymorphism of the PAI-1 promoter, allelic variation at this site does not influence CLOCK:BMAL1-and CLOCK:BMAL2-mediated transactivation. Together, CLOCK:BMAL1 and CLOCK:BMAL2 make additive contributions to PAI-1 gene transcription. While the abilities of these heterodimers to activate gene expression differ by twofold, the susceptibilities of these circadian activators to inhibition by period and cryptochrome proteins are equivalent and redox independent. Given that BMAL1 and BMAL2 differ in their spatiotemporal distributions, such distinctions may allow intrinsic circadian clocks to modulate the amplitudes of their oscillators, while maintaining circadian periodicity. In this way, fundamental circadian clock components may drive circadian variation in PAI-1, which in turn influences the pathogenesis, timing, and treatment of acute atherothrombotic events.

ACE Inhibition Versus Angiotensin Type 1 Receptor Antagonism. Differential Effects on PAI-1 Over Time

Abstract—ACE inhibition reduces plasminogen activator inhibitor-1 (PAI-1), a risk factor for myocardial infarction, whereas the effect of angiotensin receptor antagonism on PAI-1 is uncertain. The present study compares the time course of effects of ACE inhibition and angiotensin type 1 (AT1) receptor antagonism on morning plasma PAI-1 antigen. Blood pressure and endocrine, metabolic, and fibrinolytic variables were measured in 20 insulin-resistant (defined by fasting glucose >8.3 mmol/L, body mass index >28 kg/m2, or fasting serum triglyceride ≥2.8 mmol/L) hypertensive subjects (mean age, 47.9±2.1 years) (1) before and after 1 week of hydrochlorothiazide 12.5 mg/d, and (2) before and 1, 3, 4, and 6 weeks after addition of ramipril (escalated to 10 mg/d) or losartan (escalated to 100 mg/d). Hydrochlorothiazide decreased systolic (P =0.011) and diastolic (P =0.019) pressure. Ramipril (from 133.6±5.1/94.5±2.4 to 127.0±3.1/91.4±3.3 mm Hg) or losartan (from 137.0±3.9/93.1±2.9 to 123.7±2.6/86.4±2.1 mm Hg) further reduced systolic (P =0.009) and diastolic (P =0.037) pressure. The pressure effects of the 2 drugs were similar. Hydrochlorothiazide increased plasma PAI-1 (P =0.013) but not tissue-type plasminogen activator (tPA) (P =0.431) antigen. Addition of either ramipril or losartan significantly decreased plasma PAI-1 antigen (P =0.046). However, the effect of losartan on PAI-1 antigen was not sustained throughout the 6-week treatment period, such that there was a significant drug×time interaction (P =0.043). tPA antigen decreased during either ramipril or losartan (P =0.032), but tPA activity decreased only during losartan (P =0.018). Short-term interruption of the renin-angiotensin-aldosterone system by either ACE inhibition or AT1 receptor antagonism decreases PAI-1 antigen, but the duration of this effect is greater for ACE inhibition than for AT1 receptor antagonism.

Ethnicity Affects Vasodilation, but Not Endothelial Tissue Plasminogen Activator Release, in Response to Bradykinin

Previous studies indicate that the vasodilator response to bradykinin (BK) and other endothelium-dependent and -independent agonists is decreased in black Americans compared with white Americans. The purpose of the present study was to determine the effect of ethnicity on fibrinolytic function in humans. Graded doses of BK (100, 200, and 400 ng/min), acetylcholine (15, 30, and 60 &mgr;g/min; N=20), or methacholine (3.2, 6.4, 12.8 &mgr;g/min; N=20), and sodium nitroprusside (0.8, 1.6, and 3.2 &mgr;g/min) were infused via brachial artery in 19 white and 21 black age-matched normotensive subjects. Forearm blood flow (FBF) was measured by plethysmography, and venous and arterial samples were collected for tissue plasminogen activator (tPA) antigen. Compared with whites (increase in FBF from 3.7±0.5 to 23.9±2.5 mL · min−1 · 100 mL−1), blacks (increase in FBF from 2.8±0.3 to 15.2±1.9 mL · 100 mL−1 · min−1) exhibited a blunted FBF response to BK (P =0.035). Responses to sodium nitroprusside and methacholine or acetylcholine were similarly decreased. In contrast, there was no effect of ethnicity on net tPA antigen release in response to BK (increase from −0.2±0.4 to 67.3±15.2 ng · min−1 · 100 mL−1 in blacks; from 0.04±0.9 to 65.9±13.6 ng · min−1 · 100 mL−1 in whites). Thus, ethnicity significantly influenced the relationship between the flow and tPA release responses to BK (P =0.037). These data suggest that the BK-dependent alterations in vascular fibrinolytic function are preserved in black Americans compared with white Americans.

Acute tissue-type plasminogen activator release in human microvascular endothelial cells: The roles of Gαq, PLC-β, IP3 and 5,6-epoxyeicosatrienoic acid

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 Gαq,-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 Gαq,- 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 Gαq inhibitory peptide, the PLC-β antagonist U73122, and the IP3 receptor antagonist 2-aminoethoxyphenylborane, while the Gαq 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 μM 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 Gαq,-, PLC-β-, IP3-, and 5,6-EET-dependent while being prostacyclin-, NO- and K+ channel-independent in HMECs.

Tissue- and agonist-specific regulation of human and murine plasminogen activator inhibitor-1 promoters in transgenic mice

Summary.  Numerous studies have described regulatory factors and sequences that control transcriptional responses in vitro. However, there is a paucity of information on the qualitative and quantitative regulation of heterologous promoters using transgenic strategies. In order to investigate the physiological regulation of human plasminogen activator inhibitor type‐1 (hPAI‐1) expression in vivo compared to murine PAI‐1 (mPAI‐1) and to test the physiological relevance of regulatory mechanisms described in vitro, we generated transgenic mice expressing enhanced green fluorescent protein (EGFP) driven by the proximal −2.9 kb of the hPAI‐1 promoter. Transgenic animals were treated with Ang II, TGF‐β1 and lipopolysaccharide (LPS) to compare the relative activation of the human and murine PAI‐1 promoters. Ang II increased EGFP expression most effectively in brain, kidney and spleen, while mPAI‐1 expression was quantitatively enhanced most prominently in heart and spleen. TGF‐β1 failed to induce activation of the hPAI‐1 promoter but potently stimulated mPAI‐1 in kidney and spleen. LPS administration triggered robust expression of mPAI‐1 in liver, kidney, pancreas, spleen and lung, while EGFP was induced only modestly in heart and kidney. These results indicate that the transcriptional response of the endogenous mPAI‐1 promoter varies widely in terms of location and magnitude of response to specific stimuli. Moreover, the physiological regulation of PAI‐1 expression likely involves a complex interaction of transcription factors and DNA sequences that are not adequately replicated by in vitro functional studies focused on the proximal −2.9 kb promoter.