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Thrombus formation and degradation is partly due to a complex interplay between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1). There is accumulating evidence that plasma levels of t-PA and PAI-1 may be influenced by an interaction between the fibrinolytic and renin-angiotensin systems. The goal of this study was to conduct an exploratory data analysis to determine whether there is evidence that the relationship (i.e. correlation) between plasma t-PA and PAI-1 is influenced by interactive effects of the angiotensin converting enzyme (ACE) insertion/deletion (I/D) and plasminogen activator inhibitor 1 (PAI-1) 4G/5G polymorphisms in a sample of 50 unrelated African Americans and 117 unrelated Caucasians. In a single-locus analysis, no evidence for heterogeneity of plasma t-PA and PAI-1 correlations among either ACE I/D or PAI-1 4G/5G genotypes was detected. However, using the combinatorial partitioning method for exploratory data analysis, we identified evidence that is suggestive of heterogeneity of plasma t-PA and PAI-1 correlations among multilocus ACE I/D and PAI-1 4G/5G genotypes in African American females, Caucasian females, Caucasian males, but not African American males. From these results, we propose as a working hypothesis that the correlation between plasma t-PA and PAI-1 may be dependent on epistatic effects of the ACE I/D and PAI-1 4G/5G polymorphisms. This study supports the idea that interactions between the fibrinolytic and renin-angiotensin systems play an important role in the genetic architecture of plasma t-PA and PAI-1.

The detection and characterization of epistasis or non-additive gene-gene interactions remains a statistical challenge in genetic epidemiology. The recently developed combinatorial partitioning method (CPM) may overcome some of the limitations of linear regression for the exploratory analysis of non-additive epistatic effects. The goal of this study was to compare CPM with linear regression analysis for the exploratory analysis of non-additive interactive effects of the angiotensin converting enzyme (ACE) insertion/deletion (I/D) and plasminogen activator inhibitor 1 (PAI-1) 4G/5G polymorphisms on plasma PAI-1 levels in a sample of 50 unrelated African Americans and 117 unrelated Caucasians. Using linear regression, we documented the additive effects of the ACE and PAI-1 genes on plasma PAI-1 levels in African American females (R(2) = 0.10), African American males (R(2) = 0.16), Caucasian females (R(2) = 0.11), and Caucasian males (R2 = 0.09). Using CPM, we found evidence for non-additive effects of the ACE and PAI-1 genes in both African American females (R(2) = 0.22) and African American males (R(2) = 0.24) but not in Caucasian females (R(2) = 0.10) or Caucasian males (R(2) = 0.11). The results of this exploratory data analysis support previous experimental, clinical, and epidemiological studies that have proposed as a working hypothesis that the ACE gene mediates interaction effects of the fibrinolytic and renin-angiotensin systems on plasma levels of PAI-1.

Bradykinin stimulates tissue plasminogen activator release from human endothelium through a flow-independent, B 2 receptor–dependent mechanism. The present study tests the hypothesis that smoking impairs bradykinin-stimulated tissue plasminogen activator release. Graded doses of nitroprusside (1.6 to 6.4 μg/min), methacholine (3.2 to 12.8 μg/min), and bradykinin (100 to 400 ng/min) were infused in the brachial artery in random order in 20 smokers and 12 nonsmokers matched for age, gender, and body mass index. Forearm blood flow was measured by strain-gauge plethysmography. All 3 drugs caused a dose-dependent increase in forearm blood flow, with no significant difference between smokers and nonsmokers. Bradykinin ( P =0.001) and methacholine ( P =0.001) caused significant dose-dependent increases in net tissue plasminogen activator release. The tissue plasminogen activator response to bradykinin was significantly greater than the tissue plasminogen activator response to methacholine in the nonsmokers (maximal net tissue plasminogen activator release, 73.2±21.5 versus 27.6±7.2 ng/min per 100 mL; P =0.001) but not in the smokers (maximal net tissue plasminogen activator release, 44.5±10.7 versus 24.8±9.3 ng/min per 100 mL; P =0.154). The effect of bradykinin ( P =0.037), but not methacholine ( P =0.978), on net tissue plasminogen activator release was significantly reduced in smokers compared with nonsmokers. The vascular tissue plasminogen activator response to bradykinin, but not methacholine, is impaired in smokers. Stimulated tissue plasminogen activator release may be a more sensitive measure of endothelial function than vasodilation.

Recent studies have defined a link between the renin-angiotensin-aldosterone system and fibrinolysis. The present study tests the hypothesis that endogenous aldosterone regulates plasminogen activator inhibitor-1 (PAI-1) production in humans. Hemodynamic parameters, PAI-1 and tissue-type plasminogen activator (t-PA) antigen, potassium, PRA, angiotensin II, and aldosterone were measured in nine male hypertensive subjects after a 3-wk washout, after 2 wk of hydrochlorothiazide (HCTZ; 25 mg plus 20 mmol KCl/d), and after 2 wk of spironolactone (100 mg/d plus KCl placebo). Spironolactone (P = 0.04), but not HCTZ (P = 0.57 vs. baseline; P = 0.1 vs. spironolactone), significantly lowered systolic blood pressure. Angiotensin II increased from baseline during both HCTZ (P = 0.02) and spironolactone (P = 0.02 vs. baseline; P = 0.19 vs. HCTZ) treatments. Although both HCTZ (P = 0.004) and spironolactone (P < 0.001 vs. baseline) increased aldosterone, the effect was greater with spironolactone (P < 0.001 vs. HCTZ). HCTZ increased PAI-1 antigen (P = 0.02), but did not alter t-PA antigen. In contrast, there was no effect of spironolactone on PAI-1 antigen (P = 0.28), whereas t-PA antigen was increased (P = 0.01). There was a significant correlation between PAI-1 antigen and serum aldosterone during both baseline and HCTZ study days (r2 = 0.57; P = 0.0003); however, treatment with spironolactone abolished this correlation (r2 = 0.13; P = 0.33). This study provides evidence that endogenous aldosterone influences PAI-1 production in humans.

Rationale: Influenza A virus (IAV) is the leading cause of death from an infectious cause and ranks 8th in the list of attributable annual mortality in the United States. Currently available antiviral treatments are limited and may become ineffective as resistant strains emerge. The coexistence of hemostatic alterations with inflammatory responses to viral infections support the idea that common molecular mechanisms contribute to the regulation of these systems. Previous studies demonstrated that IAV induces the activation of both coagulation and fibrinolytic pathways, and that the impairment of the latter is mainly attributable to high levels of plasminogen activator inhibitor type I (PAI-1), the major inhibitor of tissue-type plasminogen activator (t-PA). Interferon-α (IFN-α) plays a key role in the control of viral replication and overall shaping of the immune response. We sought to determine whether PAI-1, plays a role in the immune response and morbidity and mortality associated with IAV infection. Methods: Wild-type (C57BL/6J), PAI-1-/-, t-PA-/- and PAI-1 stab (transgenic mice expressing an active form of human PAI-1) mice were treated intratracheally with IAV (A/WSN/33 [H1N1] 500 pfu/mouse in 50μl PBS). We assessed the levels of IFN-α in bronchoalveolar lavage fluid (BALF) on day 2 and 4, and influenza A-induced morbidity (weight loss) and mortality. Results: Compared with wild-type mice, PAI-1-/- mice had increased IAV-induced morbidity and mortality. In contrast, both PAI-1 stab and t-PA-/- mice had improved survival. In wild-type mice, IAV induced a significant elevation IFN-α levels in BALF. PAI-1 stab mice had a 2-fold increase, while PAI-1-/-mice had a 69% reduction in IFN-α levels in BALF compared to wild-type mice. Conclusions: Inhibition of fibrinolysis by means of genetic overexpression of PAI-1 or deletion of t-PA augments the antiviral response and attenuates the morbidity and mortality associated with IAV infection. These results suggest an important functional role for PAI-1 and fibrinolysis in the pathogenesis of influenza A infection. Modulation of the fibrinolytic pathway or PAI-1 may potentially be useful as a target for novel therapeutics in the management of IAV-induced lung injury.

Background— Cardiac fibrosis is the pathological consequence of stress-induced fibroblast proliferation and fibroblast-to-myofibroblast transition. MicroRNAs have been shown to play a central role in the pathogenesis of cardiac fibrosis. We identified a novel miRNA-driven mechanism that promotes cardiac fibrosis via regulation of multiple fibrogenic pathways. Methods and Results— Using a combination of in vitro and in vivo studies, we identified that miR-125b is a novel regulator of cardiac fibrosis, proliferation, and activation of cardiac fibroblasts. We demonstrate that miR-125b is induced in both fibrotic human heart and murine models of cardiac fibrosis. In addition, our results indicate that miR-125b is necessary and sufficient for the induction of fibroblast-to-myofibroblast transition by functionally targeting apelin, a critical repressor of fibrogenesis. Furthermore, we observed that miR-125b inhibits p53 to induce fibroblast proliferation. Most importantly, in vivo silencing of miR-125b by systemic delivery of locked nucleic acid rescued angiotensin II–induced perivascular and interstitial fibrosis. Finally, the RNA-sequencing analysis established that miR-125b altered the gene expression profiles of the key fibrosis-related genes and is a core component of fibrogenesis in the heart. Conclusions— In conclusion, miR-125b is critical for induction of cardiac fibrosis and acts as a potent repressor of multiple anti-fibrotic mechanisms. Inhibition of miR-125b may represent a novel therapeutic approach for the treatment of human cardiac fibrosis and other fibrotic diseases.

Background: Apelin is a novel peptide which along with its receptor, APJ, mediates apelinergic signaling. Apelinergic signaling plays a critical role in cardiovascular homeostasis, including regulation of blood pressure. While exogenous apelin blocks Angiotensin II (AngII) mediated nuclear signaling, the role and regulation of endogenous apelin in hypertension (HTN) remains obscure. We hypothesize that apelinergic pathway is downregulated in HTN, which is primarily mediated by aberrant AngII signaling. Approach: To test our hypothesis we utilized two mouse models of HTN, including AngII infusion and oral administration of N (ω)-nitro-l-arginine methyl ester (L-NAME). Blood pressure was monitored via noninvasive tail-cuff device. To determine the signaling involved we investigated the effect of AngII on apelinergic pathway in vitro. Results: Cardiac apelin was decreased significantly in both murine models of HTN. Downregulated apelin also corresponded with increased deposition of collagen, and up-regulation of senescence markers including PAI-1. Meanwhile, APJ levels were unaffected in both these hypertensive models. In our in vitro studies AngII downregulated apelin expression in human aortic endothelial cells (HAECs) and human cardiac fibroblasts (HCFs). Furthermore, our studies in AngII infused mice and in HCFs highlight the role of TGF-β-pSMAD signaling, independent of MEK involvement, in AngII induced apelin downregulation. Conclusion and Significance: Our studies demonstrate that aberrant AngII signaling downregulates apelin in HTN. This downregulation involves canonical Tgf-β1 signaling and affects apelin transcription. Importantly, we propose that AngII mediates its hypertensive pathology by decreasing apelinergic regulation. Since exogenous apelin blocks AngII signaling, further knowledge and negation of AngII induced apelin downregulation could result in the development of novel anti-hypertensive therapies.

Introduction: Earlier, in a first study of its kind, we have demonstrated a novel mechanism that therapeutically significant human CD34+ stem cells secrete membrane bound nano-vesicles called exosomes (CD34Exo). CD34Exo are angiogenic and constitute a critical component of the pro-angiogenic paracrine activity of the cells. Further, when transplanted locally, cell-free CD34Exo induce ischemic tissue repair in a murine hindlimb ischemia model. Here, we hypothesize that exosomes released via paracrine secretion from human CD34+ cells mediate myocardial repair by direct transfer of microRNAs to target cells in the heart. Methods and Results: When injected into mouse ischemic myocardium, cell-free CD34Exo replicated the therapeutic activity of human CD34+ cells by significantly improving ischemia (ejection fraction, 42±4 v 22±6%; capillary density, 113±7 v 66±6/HPF; fibrosis, 27±2 v 48±7%; p Conclusion: Our results reveal a novel molecular and trafficking mechanism of CD34Exo that may be responsible for intercellular transfer of genetic information such as miRNAs from human CD34+ stem cells, selectively to endothelial cells and cardiomyocytes inducing changes in gene expression, angiogenesis and myocardial recovery. Exosomes-shuttled miRNAs may signify amplification of stem cell function and may explain the therapeutic benefits associated with human CD34+ cell therapy.

Cardiac fibrosis is the pathological consequence of fibroblast-to-myofibroblast transition (FMT) within the human myocardium, resulting in heart dysfunction. Transforming growth factor-β (TGF-β) plays a pivotal role in the induction of both FMT and cardiac fibrosis. However, the molecular basis of TGF-β-induced FMT in cardiac fibrosis is not clear. In this study, we propose a novel miRNA-mediated approach to attenuate cardiac fibrosis by blocking TGF-β-induced FMT. We observed that the canonical TGF-β/SMAD pathway, and not the MEK pathway, plays a pivotal role in the induction of FMT in primary cultures of human cardiac fibroblasts. Importantly, we have demonstrated that the specific miRNA is significantly upregulated during cardiac FMT. In addition, we observed significant upregulation of the same miRNA in fibrotic human myocardium and two murine models of cardiac fibrosis (transverse aortic constriction and Angiotensin II). Furthermore, overexpression of the miRNA using mimics augmented TGF-β-induced FMT. Downregulation of the miRNA using an antagomiR approach attenuated TGF-β-induced FMT. Notably, in silico analysis and qRT-PCR analysis revealed that this miRNA directly targets apelin, an anti-fibrotic mediator. Next, efficient delivery of cy3-tagged antagomiRs in the heart, liver and spleen was confirmed by confocal microscopy. In vivo silencing of miRNAs in the heart was achieved by systemic delivery of locked nucleic acid (LNA), both in the presence and absence of Angiotensin II. We conclude that TGF-β-induced specific miRNA is both sufficient and necessary for the induction of cardiac FMT and is a novel repressor of apelin. Our data suggests that the inhibition of miRNAs necessary for FMT may serve as a novel therapeutic strategy to prevent human cardiac fibrosis.

STUDY OBJECTIVES Obstructive sleep apnea (OSA) is strongly associated with cardiovascular disease, including stroke and acute coronary syndromes. Plasminogen activator inhibitor-1 (PAI-1), the principal inhibitor of tissue-type plasminogen activator (t-PA), has a pronounced circadian rhythm and is elevated in both OSA and cardiovascular disease and may be an important link between the two conditions. Endothelial dysfunction is one of the underlying pathophysiological mechanisms of cardiovascular disease, and may be altered in OSA. Our primary aim was to compare circadian variability of PAI-1 and t-PA in patients with OSA and normal controls by determining the amplitude (peak level) and mesor (rhythm adjusted mean) of PAI-1 and t-PA in serial blood samples over a 24-h period. The secondary aim was to measure markers of endothelial function (brachial and radial artery flow) in patients with OSA compared with normal controls. SETTING Cross-sectional cohort study. PATIENTS OR PARTICIPANTS Subjects age 18 y or older, with a body mass index of 25-45 kg/m(2), with or without evidence of untreated OSA. INTERVENTIONS Plasma samples were collected every 2 h, in OSA patients and matched controls, over a 24-h period. PAI-1 and t-PA antigen and activity were measured. The presence or absence of OSA (apnea-hypopnea index of 5 or greater) was confirmed by overnight polysomnography. Endothelial function was measured via brachial artery flow mediated vasodilatation and computerized arterial pulse waveform analysis. MEASUREMENTS AND RESULTS The rhythm-adjusted mean levels of PAI-1 antigen levels in the OSA group (21.8 ng/mL, 95% confidence level [CI], 18 to 25.7) were significantly higher as compared to the non-OSA group (16 ng/mL, 95% CI, 12.2 to 19.8; P = 0.03). The rhythm-adjusted mean levels of PAI-1 activity levels in the OSA group (23.9 IU/mL, 95% CI, 21.4 to 26.5) were also significantly higher than in the non-OSA group (17.2 IU/ mL, 95% CI, 14.6 to 19.9; P < 0.001).There were strong correlations between amplitude of PAI-1 activity and severity of OSA as measured by AHI (P = 0.02), and minimum oxygen levels during sleep (P = 0.04). Endothelial function parameters did not differ significantly between the two groups. CONCLUSION The presence of obstructive sleep apnea adversely affects circadian fibrinolytic balance with higher mean plasminogen activator inhibitor-1 activity and antigen, and significantly lower mean tissue-type plasminogen activator activity compared with controls. This perturbation may be an important mechanism for increased cardiovascular events in patients with obstructive sleep apnea. Intermittent hypoxia and changes in circadian clock gene activity in obstructive sleep apnea may be responsible for these findings and warrant further study. Favorable changes in fibrinolytic balance may underlie the reduction in cardiovascular events observed with the treatment of obstructive sleep apnea.

Background Long-term inhibition of nitric oxide synthase (NOS) is known to induce hypertension and perivascular fibrosis. Recent evidence also suggests that long-term NOS inhibition induces expression of plasminogen activator inhibitor-1 (PAI-1) in vascular tissues and that PAI-1 may contribute to the development of fibrosis after chemical or ionizing injury. On the basis of these observations, we hypothesized that PAI-1 may influence the vascular response to long-term NOS inhibition by N ω -nitro- l -arginine methyl ester (L-NAME). Methods and Results We compared the temporal changes in systolic blood pressure and coronary perivascular fibrosis in PAI-1–deficient (PAI-1 −/− ) and wild-type (WT) male mice (N=6 per group). At baseline, there were no significant differences in blood pressure between groups. After initiation of L-NAME, systolic blood pressure increased in both groups at 2 weeks. Over an 8-week study period, systolic blood pressure increased to 141±3 mm Hg in WT animals versus 112±4 mm Hg in PAI-1 −/− mice ( P P −/− mice). Cardiac type I collagen mRNA expression was greater in control ( P −/− ( P Conclusions These findings suggest that PAI-1 deficiency alone is sufficient to protect against the structural vascular changes that accompany hypertension in the setting of long-term NOS inhibition. Direct inhibition of vascular PAI-1 activity may provide a new therapeutic strategy for the prevention of arteriosclerotic cardiovascular disease.

Abstract —Activation of the renin-angiotensin-aldosterone system (RAAS) is associated with increased circulating PAI-1 antigen and increased risk of thrombotic cardiovascular events. A 4G/5G polymorphism located 675 bp upstream from the transcription start site of the PAI-1 gene affects PAI-1 antigen concentrations. To test the hypothesis that PAI-1 4G/5G genotype influences the effect of activation of the RAAS on PAI-1 expression, we measured morning PAI-1 antigen concentrations in 76 subjects with essential hypertension during low (10 mmol/d) and high (200 mmol/d) salt intake. Low salt intake was associated with activation of the RAAS as measured by plasma renin activity (2.3±0.2 versus 0.5±0.0 ng angiotensin I · mL −1 · h −1 , P P =0.017) and PAI-1 4G/5G genotype (F=7.6, P =0.001) on PAI-1 antigen. More importantly, there was a significant interactive effect (F=7.8, P =0.001) of salt intake and PAI-1 4G/5G genotype on PAI-1 antigen. PAI-1 4G/5G genotype influenced the relationship between serum triglycerides and PAI-1 antigen such that the relationship was significant only in 4G homozygotes during either high ( R 2 =0.31, P =0.014) or low ( R 2 =0.37, P =0.006) salt intake. This study identifies an important gene-by-environment interaction that may influence cardiovascular morbidity and the response to pharmacological therapies that interrupt the RAAS.

Bradykinin is a vasoactive nonapeptide involved in cardiorenal physiology and inflammatory states. It has been linked to the pathophysiology of hypertension and diabetes. Correlating levels of bradykinin with disease states has been hampered by its rapid degradation, artifactual production during blood sampling, and nonspecific radioimmunoassay techniques. We previously identified BK1-5 as the stable in vivo plasma metabolite of systemic bradykinin in humans. We now report a sensitive and specific assay method for BK1-5 in human blood utilizingliquid chromatography–tandem mass spectrometry(MS) with electrospray ionization. [ 13 C 2 , 15 N]Glycine was incorporated into chemically synthesized BK1-5 for use as an internal standard. Blood samples (5 ml) were collected into 15-ml chilled ethanol to prevent artifactual kinin production and degradation. BK1-5 in ethanolic plasma supernatant was purified on a polymeric solid phase extraction cartridge. MS analysis was in the selective reaction monitoring mode. Precision of the assay is ±7.5% and accuracy is 99%. Recovery of BK1-5 through sample preparation was 43% and the lower limit of detection is 4 fmol/ml blood. Concentrations of BK1-5 in 12 normal volunteers were 44.2 ± 7.1 fmol/ml blood (mean ± SE). During blood sampling, no artifactual production of BK1-5 was detected for up to 60 s prior to denaturing the sample. This assay provides the first accurate and precise method using MS to quantify BK1-5 in human blood as a marker for the production of systemic bradykinin in humans.

Rat aortic smooth muscle cells (RASM) express the src suppressed C-kinase substrate (SSeCKS), which is thought to be an integral regulatory component of cytoskeletal dynamics and G-protein coupled-receptor signaling modules. The specific sub-classes of growth factor receptors that regulate the genomic changes in SSeCKS expression in smooth muscle cells have not been characterized. In this study we identify SSeCKS as an angiotensin type 1 (AT(1)) receptor-dependent target gene in RASM cells treated with angiotensin II (Ang II). SSeCKS mRNA levels increase up to three-fold relative to the control within 3.5 h of Ang II treatment and are followed by a slight decrease of mRNA relative to the control levels after 24 h of stimulation. SSeCKS gene expression and plasminogen activator inhibitor-1 (PAI-1) gene expression correlate in RASM cells treated with Ang II. By co-transfecting plasmids bearing recombinant-SSeCKS and a PAI-1-promoter/luciferase reporter into Cos-1 cells, we show that alternative forms of recombinant-SSeCKS protein differentially influence PAI-1 promoter activity. These data indicate a biochemical linkage between SSeCKS activity and one or more of the cytoplasmic signaling pathways that are involved in the control of PAI-1 promoter activity. Finally, we show that the alternative forms of recombinant-SSeCKS protein differentially influence cell-spreading when ectopically expressed in ras -transformed rat kidney (KNRK) fibroblasts. Taken together, our data suggest that SSeCKS interacts with intracellular signaling pathways that control cytoskeletal remodeling and extracellular matrix remodeling following Ang II stimulation of the RASM cell.

Background —Bradykinin stimulates dose-dependent tissue plasminogen activator (tPA) release from human endothelium. Although bradykinin is known to cause vasodilation through B 2 receptor–dependent effects on NO, prostacyclin, and endothelium-derived hyperpolarizing factor production, the mechanism(s) underlying tPA release is unknown. Methods and Results —We measured the effects of intra-arterial bradykinin (100, 200, and 400 ng/min), acetylcholine (15, 30, and 60 μg/min), and nitroprusside (0.8, 1.6, and 3.2 μg/min) on forearm vasodilation and tPA release in healthy volunteers in the presence and absence of (1) the B 2 receptor antagonist HOE 140 (100 μg/kg IV), (2) the NO synthase inhibitor l - N G -monomethyl- l -arginine (L-NMMA, 4 μmol/min intra-arterially), and (3) the cyclooxygenase inhibitor indomethacin (50 mg PO TID). B 2 receptor antagonism attenuated vasodilator ( P =0.004) and tPA ( P =0.043) responses to bradykinin, without attenuating the vasodilator response to nitroprusside ( P =0.36). L-NMMA decreased basal forearm blood flow (from 2.35±0.31 to 1.73±0.22 mL/min per 100 mL, P =0.01) and blunted the vasodilator response to acetylcholine ( P =0.013) and bradykinin ( P =0.07, P =0.038 for forearm vascular resistance) but not that to nitroprusside ( P =0.47). However, there was no effect of L-NMMA on basal ( P =0.7) or bradykinin-stimulated tPA release ( P =0.45). Indomethacin decreased urinary excretion of the prostacyclin metabolite 2,3-dinor-6-keto-prostaglandin F 1α ( P =0.04). The vasodilator response to endothelium-dependent ( P =0.019 for bradykinin) and endothelium-independent ( P =0.019) vasodilators was enhanced during indomethacin administration. In contrast, there was no effect of indomethacin alone ( P =0.99) or indomethacin plus L-NMMA ( P =0.36) on bradykinin-stimulated tPA release. Conclusions —These data indicate that bradykinin stimulates tPA release from human endothelium through a B 2 receptor–dependent, NO synthase–independent, and cyclooxygenase-independent pathway. Bradykinin-stimulated tPA release may represent a marker for the endothelial effects of endothelium-derived hyperpolarizing factor.

Recent data suggest an interaction between the renin-angiotensin-aldosterone system and fibrinolysis. Although previous work has focused on the effect of angiotensin II (Ang II) on plasminogen activator inhibitor (PAI-1) expression, the present study tests the hypothesis that aldosterone contributes to the regulation of PAI-1 expression. To test this hypothesis in vitro, luciferase reporter constructs containing the human PAI-1 promoter were transfected into rat aortic smooth muscle cells. Exposure of the cells to 100 nmol/L Ang II resulted in a 3-fold increase in luciferase activity. Neither 1 μmol/L dexamethasone nor 1 μmol/L aldosterone alone increased PAI-1 expression. However, both dexamethasone and aldosterone enhanced the effect of Ang II in a dose-dependent manner. This effect was abolished by mutation in the region of a putative glucocorticoid-responsive element. A similar interactive effect of Ang II and aldosterone was observed in cultured human umbilical vein endothelial cells. The time course of the effect of aldosterone on Ang II-induced PAI-1 expression was consistent with a classical mineralocorticoid receptor mechanism, and the effect of aldosterone on PAI-1 synthesis was attenuated by spironolactone. To determine whether aldosterone affected PAI-1 expression in vivo, we measured local venous PAI-1 antigen concentrations in six patients with primary hyperaldosteronism undergoing selective adrenal vein sampling. PAI-1 antigen, but not tissue plasminogen activator antigen, concentrations were significantly higher in adrenal venous blood than in peripheral venous blood. Taken together, these data support the hypothesis that aldosterone modulates the effect of Ang II on PAI-1 expression in vitro and in vivo in humans.

Abstract —Bradykinin stimulates tissue plasminogen activator (tPA) release in isolated perfused animal tissues. The present study tests the hypothesis that bradykinin increases tPA release in humans through local effects on the vasculature. Graded doses of sodium nitroprusside (0.8 to 3.2 μg/min), acetylcholine (ACh) (7.5 to 60 μg/min), and bradykinin (100 to 400 ng/min) were administered intra-arterially in random order in 10 salt-depleted (10 mmol/d of Na) normotensive volunteers. None of the drugs altered mean arterial pressure or heart rate. Forearm blood flow (FBF) was measured by strain-gauge plethysmography. All 3 drugs caused a dose-dependent increase in FBF, although ACh was less potent than either nitroprusside or bradykinin (maximum FBF 7.5±2.4 versus 10.0±1.5 and 11.9±2.1 mL · 100 mL −1 · min −1 , respectively). Bradykinin caused a significant, dose-dependent increase in venous (effect of dose F=9.9, P =0.028 by ANOVA), but not arterial (F=0.154, P =0.92) tPA antigen in the infused arm. Thus, net tPA release increased significantly in response to bradykinin (50.6±13.3 at the highest dose versus 0.9±0.4 ng · 100 mL −1 · min −1 at baseline, P =0.014). In contrast, bradykinin did not affect plasminogen activator inhibitor antigen. Neither nitroprusside nor ACh altered plasma levels of tPA or plasminogen activator inhibitor antigen. Bradykinin increased tPA release across the forearm in the absence of systemic effects. This effect could not be attributed to changes in blood flow because doses of equivalent potency of the vasodilator nitroprusside did not increase tPA. These data demonstrate that bradykinin stimulates tPA release in the human vasculature.

SummaryModerate alcohol consumption has been shown to reduce the risk of ischemic heart disease potentially through its effect on specific endothelial-derived compounds. We tested the hypothesis that ethanol increases the expression of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production in bovine aortic endothelial cells (BAEC). Primary cultures of BAEC grown to confluence under standard conditions were treated 3-6 h with 0.1% ethanol in the presence of indomethacin. Ethanol induced a significant increase in both basal and stimulated NO production as determined by chemiluminescence method. This effect was accompanied by a rapid increase of eNOS protein and mRNA expression levels. eNOS mRNA increased two-fold within 3 h and gradually declined, but the increased levels of mRNA persisted for >24 h. A similar increase of eNOS expression was observed in human umbilical endothelial cells exposed to ethanol. These results demonstrate that ethanol augments both basal and stimulated NO production and that this effect is associated with increased eNOS protein and mRNA expression levels. The data are consistent with the hypothesis that the reduced incidence of ischemic heart disease associated with alcohol may be related, at least in part, to the modulation of vascular endothelial cell production of NO

Activation of the renin–angiotensin system increases the risk of atherohrombotic events in hypertensive patients. This relationship may be explained by multiple mechanisms, including effects on platelet function and on fibrinolysis. There is substantial evidence that angiotensin-converting enzyme (ACE) inhibitors may exert antithrombotic effects by enhancing the bradykinin-dependent release of tissue plasminogen activator (t-PA) while reducing the angiotensin-dependent production of its natural inhibitor PAI-1, thus improving vascular fibrinolytic balance. Recent studies suggest that angiotensin (II) type 1 (AT1) receptor antagonists inhibit the effects of prothrombotic prostanoids. The antithrombotic effects of agents that block the RAS may contribute to their vasculoprotective properties.

Thrombin is the ultimate protease in the coagulation cascade whose pleiotropic actions can lead to thrombosis after tissue injury. Thrombin is the key effector of the coagulation cascade and converts fibrinogen to fibrin, which is essential for laying the meshwork for clot formation. In addition

Objectives. This study was designed to determine 1) whether 12-week oral administration of losartan, an angiotensin II receptor antagonist, in patients with heart failure is well tolerated; and 2) whether functional capacity and clinical status of patients with heart failure in whom treatment with an angiotensin-converting enzyme (ACE) inhibitor is replaced with losartan for 12 weeks will remain similar to that noted in patients in whom treatment with an ACE inhibitor is continued. Background. Losartan is a specific, nonpeptide angiotensin II receptor antagonist. Although specific receptor blockade with losartan has certain theoretic advantages over nonspecific ACE inhibition, definitive demonstration of comparable effects in patients with congestive heart failure is lacking. Methods. A double-blind, multicenter, randomized, parallel, enalapril-controlled study was conducted in 116 patients with congestive heart failure (New York Heart Association functional classes II to IV) and left ventricular ejection fraction ≤45% previously treated with stable doses of ACE inhibitors and diuretic agents, with or without concurrent digitalis and other vasodilators. After a baseline exercise period, open-label ACE inhibitors were discontinued, and patients were randomly assigned to 12 weeks of therapy with losartan, 25 mg/day (n = 38); losartan, 50 mg/day (n = 40); or enalapril, 20 mg/day (n = 38). Drug efficacy was evaluated by changes in maximal treadmill exercise time (using a modified Naughton protocol), 6-min walk test, left ventricular ejection fraction and dyspnea-fatigue index. Safety was measured by the incidence of clinical and laboratory adverse experiences. Results. The treadmill exercise time and the 6-min walk test did not change significantly after replacement of ACE inhibitor therapy with losartan. Similarly, a significant change was not observed in either the dyspnea-fatigue index or left ventricular ejection fraction at the end of double-blind period relative to baseline. Conclusions. Losartan was generally well tolerated and comparable to enalapril in terms of exercise tolerance in this short-term (12-week) study of patients with heart failure. The clinical effects of long-term angiotensin II receptor blockade compared with ACE inhibition remain to be studied.

An Alu -repeat polymorphism in the gene coding for tissue-type plasminogen activator has been described recently, and it has been hypothesized that this polymorphism may predict risk of coronary thrombosis. In a prospective cohort of nearly 15,000 apparently healthy men, presence of an Alu -repeat insertion/deletion ( I/D ) polymorphism in the gene coding for tissue-type plasminogen activator was determined among 369 study participants who subsequently suffered a first myocardial infarction (cases) and among a group of 369 ageand smoking-matched study participants who remained free of reported cardiovascular disease during follow-up (controls). The distributions of the II , DI , and DD genotypes of the tissue-type plasminogen activator polymorphism among men who subsequently suffered myocardial infarction (0.30, 0.50, 0.21) were virtually identical to those who remained free of disease (0.29, 0.50, 0.21; P =.9). There was no evidence of association between the Alu insertion polymorphism and risks of future myocardial infarction in models assuming either allelic recessive (relative risk, 1.05; 95% confidence interval, 0.8 to 1.4, P =.8) or allelic dominant (relative risk, 1.04; 95% confidence interval, 0.7 to 1.5, P =.8) modes of inheritance, nor were associations found in analyses stratified by age, family history, hypercholesterolemia, or the presence of other risk factors for premature coronary disease. Multivariate analysis had no important effects on these relationships. In this cohort of middle-aged US men, the presence of the insertion allele of the Alu -repeat polymorphism of the tissue-type plasminogen activator gene is not associated with future risks of myocardial infarction. ( Arterioscler Thromb Vase Biology. 1997;17:1687-1690.)

Background The long-term administration of ACE inhibitors to selected patients with left ventricular dysfunction appears to reduce the incidence of recurrent myocardial infarction (MI) and unstable angina pectoris. The mechanisms responsible for the reduction in ischemic events are unknown, but likely candidates include effects on the atherosclerotic process, thrombosis, and/or vascular tone. Methods and Results The effects of ACE inhibitor therapy with ramipril on plasma fibrinolytic variables were assessed in 120 subjects participating in the Healing and Early Afterload Reduction Therapy (HEART) study, a double-blind, placebo-controlled trial of acute anterior MI patients who were randomly assigned within 24 hours of the onset of symptoms to receive low-dose ramipril (0.625 mg daily), full-dose ramipril (1.25 mg titrated to 10 mg/d), or placebo for 14 days. Plasma levels of plasminogen activator inhibitor-1 (PAI-1) activity and PAI-1 antigen and tissue plasminogen activator (TPA) antigen were measured before randomization and on day 14. Clinical characteristics of the three study groups were similar, as were the prerandomization plasma levels of PAI-1 antigen, PAI-1 activity, and TPA antigen. Compared with the placebo group, PAI-1 antigen levels were 44% lower ( P =.004) at day 14 in the ramipril-treated patients, and PAI-1 activity levels were 22% lower ( P =.02). In contrast, plasma TPA levels were not significantly different between the placebo-treated and ramipril-treated groups. Conclusions Treatment with ramipril has a significant impact on plasma fibrinolytic variables during the recovery phase after acute MI. The renin-angiotensin system appears to play an important role in the regulation of vascular fibrinolysis, and interruption of this regulatory pathway may contribute to the clinical benefits of ACE inhibitors.

Summary Previous studies have identified a correlation between moderate alcohol consumption and plasma fibrinolytic activity. However, experimental studies designed to characterize the effect of alcohol on endothelial fibrinolytic parameters have provided inconsistent results. We investigated the effects of alcohol on the fibrinolytic components in a more simplified tissue culture system. The expression of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) in cultured human endothelial cells exposed to ethanol was studied using Northern blot hybridization. Treatment of endothelial cells with ethanol causes a gradual increase of the level of endothelial t-PA (mRNA) with a concomitant rapid decrease in PAI-1 mRNA levels. These divergent responses at the transcriptional level are associated with an increase in the extracellular t-PA activity in the conditioned medium of cells exposed to ethanol.

SummaryThe successful design of new thrombolytic agents depends on providing these agents with increased clot selectivity. As recently demonstrated (10), entrapment of tissue plasminogen activator into liposomes apparently provided the selective targeting needed to improve the efficacy of this fibrinolytic agent. To test whether liposomal entrapment would benefit streptokinase, a fibrinolytic agent with a different mode of action and inactivation, we compared liposomal streptokinase with free streptokinase in an experimental rabbit model of thrombolysis.First we adapted a new method to produce liposomes of high entrapment efficiency, termed interdigitation-fusion (IF) liposomes, for the encapsulation of streptokinase. This system was then tested in an in vivo rabbit model of thrombolysis where animals with established clots were infused with either free streptokinase (40,000 U/kg), liposomally entrapped streptokinase, free streptokinase + empty liposomes, or the corresponding amount of empty liposomes or saline. Significant differences (p When tested in rabbits immunized against streptokinase, liposomal (33.8 ± 1.5%) but not free streptokinase (29.3 ± 2.1%) showed significant thrombolytic activity compared to saline (22.4 ± 3.3%) (p

Vitiligo is a common skin condition with a complex pathophysiology characterized by the lack of pigmentation due to melanocyte degeneration. In this study, we investigated PAI-1 antigen (Ag) and activity levels in a 34 year old male with extensive vascular disease, alopecia areata and vitiligo. Fasting PAI-1 Ag and activity levels were measured at 9 a.m. in the subject and family members. Both PAI-1 Ag (67 ± 38 vs. 18.6 ± 6.5 ng/ml, P < 0.001) and specific activity (15.8 ± 10.0 vs. 7.6 ± 6.0 IU/pmol, P < 0.04) levels of PAI-1 were moderately elevated in subjects compared to the controls. PAI-1 kinetic studies demonstrated a markedly enhanced stability of plasma PAI-1 activity in the family members. Specific activity at 16 h was significantly higher than expected activity levels (0.078 ± 0.072 vs. 0.001 ± 0.001 IU/ng/ml, P < 0.001). While the exact mechanism of increased stability of PAI-1 activity in vitiligo is not known, it is likely 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 vascular disease and associated melanocyte degeneration. Systemic or local treatment with PAI-1 inhibitors may offer a potential treatment alternative to the near orphan status for vitiligo drug development.

Background Acute coronary syndrome due to acute plaque rupture has been well described and is associated with established risk factors, including hypertension, diabetes mellitus, hyperlipidemia, and smoking. The prevalence of these risk factors in very young patients (aged ≤35 years) is not well known, and they may have other nontraditional risk factors. We hypothesized that acute coronary syndrome in very young patients may represent a thrombotic event independent of underlying atherosclerotic disease. Methods We performed a dual-institution, retrospective study of consecutive patients aged ≤35 years who presented with acute coronary syndrome and underwent coronary angiography from January 2000 to December 2011. Standard demographics, risk factors, and detailed angiographic information were obtained. Results A total of 124 patients met inclusion criteria. The mean age was 31 ± 4 years for both sexes. Approximately half (49%) of the patients were obese (body mass index ≥30 kg/m 2 ); 90% of patients had at least 1 traditional risk factor, most commonly hyperlipidemia (63%) and smoking (60%); 52% of patients underwent re-vascularization, of which 94% were by percutaneous coronary intervention, and 42.9% of patients had intracoronary thrombus, of whom approximately one third had no detectable underlying coronary disease. Conclusions Very young patients with acute coronary syndrome tend to be obese, with a high prevalence of smoking and hyperlipidemia. The presence of thrombus in the absence of underlying coronary disease suggests a thromboembolic event or de novo thrombotic occlusion, which may reflect primary hemostatic dysfunction in a considerable number of these patients.

Introduction: Locally transplanted human CD34 + stem cells have been shown to improve exercise tolerance in patients with myocardial ischemia and promote angiogenesis in animal models. Recently we have demonstrated that CD34 + cells secrete exosomes, membrane bound nano-vesicles, as a major component of their paracrine secretion which induces angiogenesis. We hypothesize that cell-free exosomes from CD34 + cells (CD34 Exo) mimic the beneficial effects of cells, and promote myocardial revascularization and repair via transfer of pro-angiogenic microRNAs, possibly to endothelial cells. Methods and Results: Therapeutic potential of CD34 Exo isolated from equal number of adult human peripheral blood-derived CD34 + cells was evaluated in a murine model of myocardial ischemia (MI). Similar to cells, treatment with CD34 Exo resulted in significant improvement in ischemia compared to treatment with PBS (ejection fraction, 42±4 v 22±6%; capillary density, 113±7 v 66±6/HPF; fibrosis 27±2 v 48±7%, p Conclusion: CD34 + exosomes transfer miR126 to endothelial cells to induce angiogenesis and myocardial repair. The functional benefits associated with CD34 + cell therapy may be mediated by exosomes-mediated transfer of angiogenic microRNAs to endothelial cells.

Fibrosis, a non-physiological wound healing in multiple organs, is associated with end-stage pathological symptoms of a wide variety of vascular injury and inflammation related diseases. In response to chemical, immunological and physical insults, the body’s defense system and matrix synthetic machinery respond to healing the wound and maintain tissue homeostasis. However, uncontrolled wound healing leads to scarring or fibrosis, a pathological condition characterized by excessive synthesis and accumulation of extracellular matrix proteins, loss of tissue homeostasis and organ failure. Understanding the actual cause of pathological wound healing and identification of igniter(s) of fibrogenesis would be helpful to design novel therapeutic approaches to control pathological wound healing and to prevent fibrosis related morbidity and mortality. In this article, we review the significance of a few key cytokines (TGF-β, IFN-γ, IL-10) transcriptional activators (Sp1, Egr-1, Smad3), repressors (Smad7, Fli-1, PPAR-γ, p53, Klotho) and epigenetic modulators (acetyltransferase, methyltransferases, deacetylases, microRNAs) involved in major matrix protein collagen synthesis under pathological stage of wound healing, and the potentiality of these regulators as therapeutic targets for fibrosis treatment. The significance of endothelial to mesenchymal transition (EndMT) and senescence, two newly emerged fields in fibrosis research, has also been discussed.

Fibrosis is defined as an abnormal matrix remodeling due to excessive synthesis and accumulation of extracellular matrix proteins in tissues during wound healing or in response to chemical, mechanical and immunological stresses. At present, there is no effective therapy for organ fibrosis. Previous studies demonstrated that aged plasminogen activator inhibitor-1 (PAI-1) knockout mice develop spontaneously cardiac-selective fibrosis without affecting any other organs. We hypothesized that differential expressions of profibrotic and antifibrotic genes in PAI-1 knockout hearts and unaffected organs lead to cardiac selective fibrosis. In order to address this prediction, we have used a genome-wide gene expression profiling of transcripts derived from aged PAI-1 knockout hearts and kidneys. The variations of global gene expression profiling were compared within four groups: wildtype heart vs. knockout heart; wildtype kidney vs. knockout kidney; knockout heart vs. knockout kidney and wildtype heart vs. wildtype kidney. Analysis of illumina-based microarray data revealed that several genes involved in different biological processes such as immune system processing, response to stress, cytokine signaling, cell proliferation, adhesion, migration, matrix organization and transcriptional regulation were affected in hearts and kidneys by the absence of PAI-1, a potent inhibitor of urokinase and tissue-type plasminogen activator. Importantly, the expressions of a number of genes, involved in profibrotic pathways including Ankrd1, Pi16, Egr1, Scx, Timp1, Timp2, Klf6, Loxl1 and Klotho, were deregulated in PAI-1 knockout hearts compared to wildtype hearts and PAI-1 knockout kidneys. While the levels of Ankrd1, Pi16 and Timp1 proteins were elevated during EndMT, the level of Timp4 protein was decreased. To our knowledge, this is the first comprehensive report on the influence of PAI-1 on global gene expression profiling in the heart and kidney and its implication in fibrogenesis and several other biological processes. The significance of these observations in the light of heart-specific profibrotic signaling and fibrogenesis are discussed.

In vitro and in vivo data provide evidence for an interaction between the renin-angiotensin and fibrinolytic systems. Angiotensin-converting enzyme (ACE) is strategically poised to regulate this interaction. ACE catalyzes the conversion of angiotensin I to angiotensin II (Ang), and Ang II stimulates release of PAI-1, the major inhibitor of tissue-type plasminogen activator (t-PA) and urokinase in the vasculature. Conversely, ACE catalyzes the breakdown of bradykinin, a potent stimulus of t-PA secretion. This interaction between the renin-angiotensin and fibrinolytic systems may partially explain the clinical observation that stimulation or suppression of the renin-angiotensin system can alter the risk of ischemic cardiovascular events. © 1996, Elsevier Science Inc. (Trends Cardiovasc Med 1996;6:239-243).

Recent studies from this laboratory have demonstrated that angiotensin II (Ang II) stimulates the expression of plasminogen activator inhibitor 1 (PAI-1) in cultured endothelial cells. This response does not appear to be mediated via an interaction with either the AT1 or the AT2 receptor subtype. Since a novel angiotensin receptor has been identified in a variety of tissues that specifically binds the hexapeptide Ang IV (Ang II, [3-8]), we therefore examined the effects of Ang IV on the expression of PAI-1 mRNA in bovine aortic endothelial cells. Ang IV stimulated dose- and time-dependent increases in the expression of PAI-1 mRNA. The effect of Ang IV (10 nM) was not inhibited by Dup 753 (1.0 microM), a highly specific antagonist of the AT1 receptor, or by PD123177 (1.0 microM), a highly specific antagonist of the AT2 receptor. In contrast, the AT4 receptor antagonist, WSU1291 (1.0 microM), effectively prevented PAI-1 expression. Although larger forms of angiotensin (i.e., Ang I, Ang II, and Ang III) are capable of inducing PAI-1 expression, this property is lost in the presence of converting enzyme or aminopeptidase inhibitors. These results indicate that the hexapeptide Ang IV is the form of angiotensin that stimulates endothelial expression of PAI-1. This effect appears to be mediated via the stimulation of an endothelial receptor that is specific for Ang IV.

Introduction: Plasminogen activator inhibitor-1 (PAI-1) is an important component of the senescence-associated secretome that contributes directly to cellular senescence. Telomeres, the nucleotide repeat structures located at the ends of chromosomes, shorten progressively with each round of cellular replication and telomere attrition is associated with cellular senescence. In murine models, genetic or pharmacologic inhibition of PAI-1 results in preservation of telomere length. Identification of a unique Amish kindred that harbors a loss-of-function mutation in SERPINE1 (c.699_700dupTA), which encodes PAI-1, provides a novel opportunity to assess the effects of lifelong PAI-1 deficiency on leukocyte telomere length (LTL) in humans. Methods: We conducted an observational study in the Old Order Amish, a founder population who originally settled in Berne, Indiana characterized by a homogeneous diet and lifestyle, and included participants aged 18 years and older. All study participants were genotyped for the c.699_700dupTA frameshift mutation in SERPINE1. For the primary analysis, we excluded participants who were homozygous for the null SERPINE1 mutation (n=7) due to their young age (18-34 years old) and small sample size. Relative LTL was quantified from peripheral blood using Southern blot of the terminal restriction fragments. We tested the association of SERPINE1 mutation status with LTL, after adjustment for age, sex, and relatedness in SOLAR. Results: A total of 170 participants were enrolled with mean age 46±19 years. Forty-three participants were identified as carriers of the null SERPINE1 mutation with an overall minor (null) allele frequency of 16% in the study population. SERPINE1 carriers had a significantly greater LTL compared with noncarriers, after adjustment for age, sex, and family structure (p=0.039; FIGURE). Every 1-year increase in age of study participant was associated with a 30 base pair decrease in LTL (p Conclusions: Heterozygosity for the null SERPINE1 gene, encoding a senescence-associated protein, PAI-1, is associated with longer LTL in a rare loss-of-function cohort. In addition, LTL is strongly and inversely correlated with chronologic age and supports the hypothesis that PAI-1 may contribute to cellular and organismal aging as reflected by LTL. Figure Mean telomere restriction fragments (kilobase pairs) as a function of age and genotype Figure. Mean telomere restriction fragments (kilobase pairs) as a function of age and genotype Disclosures Shapiro: CSL Behring: Other: Clinical research protocols; Kedrion Biopharma: Consultancy; Daiichi Sankyo: Other: Clinical research protocols; OPKO: Other: Clinical research protocols; National Hemophilia Foundation: Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Other: Clinical research protocols; Novo Nordisk Hemophilia Foundation: Membership on an entity's Board of Directors or advisory committees; Baxter/Baxalta/Shire: Membership on an entity's Board of Directors or advisory committees, Other: Clinical research protocols; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Other: Clinical research protocols; Biogen: Membership on an entity's Board of Directors or advisory committees, Other: Clinical research protocols; Genentech: Membership on an entity's Board of Directors or advisory committees; American Thrombosis and Hemostasis Network: Other: Medical Director; ProMetic Life Sciences: Consultancy; Bayer healthcare Pharmaceuticals: Other: International network on pediatric hemophilia; Novartis: Other: Clinical research protocols; Selexys: Other: Clinical research protocols; Octopharma: Other: Clinical research protocols.

Plasminogen activator inhibitor-1 (PAI-1) is the most important inhibitor of tissue-type plasminogen activator (t-PA) in plasma and plays a major role in the regulation of fibrinolysis. Plasma t-PA/PAI-1 complexes are cleared via a receptor-dependent mechanism in hepatocytes, while the fate of complexes formed in the extracellular matrix and in thrombi is less well understood. In this study, the degradation of t-PA/PAI-1 complexes by monocytes was examined. THP-1 monocytoid cells and freshly isolated human monocytes internalize and degrade [125I]t-PA/PAI-1 complexes at rates of 11.4 ± 5.9 (mean ± S.D.) and 44.6 ± 6.3 ng/106 cells/h, respectively. Degradation is blocked by receptor-associated protein (RAP), indicating a member of the low density lipoprotein (LDL) receptor family is involved in the uptake /degradation of t-PA/PAI-1 complexes by monocytes. Degradation of t-PA/PAI-1 complexes is also inhibited by chloroquine and by pepstatin A, suggesting that a lysosomal aspartyl protease is likely involved. SDS-PAGE and Western blotting demonstrated that the purifed. lysosomal aspartyl protease, cathepsin D, is capable of digesting t-PA ( t 1 2 15 min ), active PAI-1 ( t 1 2 2 h ), and t-PA/PAI-1 complex ( t 1 2 30 min ). Cathepsin D sequentially cleaves PAI-1 after hydrophobic amino acids, yielding lower molecular weight fragments. PAI-1 conformation influences the degradative efficiency of cathepsin D, with vitronectin-bound PAI-1 and latent PAM exhibiting resistance to proteolysis and > 10-fold prolongation in t 1 2 . These data provide evidence that t-PA/PAI-1 complexes are internalized by human monocytes via a member of the low density lipoprotein (LDL) receptor family, and identifies cathepsin D-like aspartyl protease activity as largely responsible for the degradation of these complexes. Furthermore, vitronectin-bound PAM and latent PAI-1 are relatively resistant to degradation by cathepsin D, which may be of importance in complex physiological environments.

Plasminogen activator-inhibitor C-1 (PAI-1) plays a critical role in the regulation of fibrinolysis, serving as the primary inhibitor of tissue-type plasminogen activator. Elevated levels of PAI-1 are a risk factor for recurrent myocardial infarction, and locally increased PAI-1 expression has been described in atherosclerotic human arteries. Recent studies have shown that the administration of angiotensin converting enzyme inhibitors reduces the risk of recurrent myocardial infarction in selected patients. Since angiotensin II (Ang II) has been reported to induce PAI-1 production in cultured astrocytes, we have hypothesized that one mechanism that may contribute to the beneficial effect of angiotensin converting enzyme inhibitors is an effect on fibrinolytic balance. In the present study, we examined the interaction of Ang II with cultured bovine aortic endothelial cells (BAECs) and the effects of this peptide on the production of PAI-1. 125I-Ang II was found to bind to BAECs in a saturable and specific manner, with an apparent Kd of 1.4 nM and Bmax of 74 fmol per mg of protein. Exposure of BAECs to Ang II induced dose-dependent increases in PAI-1 antigen in the media and in PAI-1 mRNA levels. Induction of PAI-1 mRNA expression by Ang II was not inhibited by pretreating BAECs with either Dup 753 or [Sar1, Ile8]-Ang II, agents that are known to compete effectively for binding to the two major angiotensin receptor subtypes. These data indicate that Ang II regulates the expression of PAI-1 in cultured endothelial cells and that this response is mediated via a pharmacologically distinct form of the angiotensin receptor.

Rationale Convincing evidence has emerged that impaired alveolar epithelial cell (AEC) injury and repair resulting from ‘exaggerated’ lung aging and mitochondrial dysfunction are critical determinants of the lung fibrogenic potential of toxic agents, including asbestos fibers, but the mechanisms underlying these findings is unknown. We showed that the extent of AEC mitochondrial DNA (mtDNA) damage and apoptosis are critical determinants of asbestos-induced pulmonary fibrosis (Cheresh et al AJRCMB 2014, Kim et al JBC 2014). Klotho is an age-inhibiting gene and Klotho-deficient mice demonstrate a premature aging phenotype that includes a reduced lifespan, arteriosclerosis, and lung oxidative DNA damage, and that Klotho attenuates hyperoxic-induced AEC DNA damage and apoptosis (Ravikumar et al AJP-Lung 2014). We reason that Klotho has an important role in limiting pulmonary fibrosis by protecting the AECs from oxidative stress. Methods Quantitative PCR-based measurement of mtDNA damage was assessed following transient transfection with wild-type Klotho, Klotho siRNA or AKT siRNA in A549 and/or MLE-12 cells for 48 hrs followed by exposure to either amosite asbestos (25 µg/cm2) or H2O2 (200 µM) for 24 hrs. Apoptosis was assessed by cleaved caspase-9/3 levels and DNA fragmentation assay. Murine pulmonary fibrosis was analyzed in male 8–10 week old WT (C3H/C57B6J) mice or Klotho heterozygous knockout (Kl+/−) mice following intratracheal instillation of a single dose of 100 µg crocidolite asbestos or titanium dioxide (negative control) using histology (fibrosis score by Masson9s trichrome staining) and lung collagen (Sircoll assay). Results Compared to control, amosite asbestos or H2O2 reduces Klotho mRNA/protein expression. Notably, silencing of Klotho promotes oxidative stress-induced AEC mtDNA damage and apoptosis whereas Klotho-enforced expression (EE) and Euk-134, a mitochondrial ROS scavenger, are protective. Interestingly, Kl+/− mice have increased asbestos-induced lung fibrosis. Also, we find that inhibition or silencing of AKT augments oxidant-induced AEC mtDNA damage and apoptosis. Conclusions Our data demonstrate a crucial role for AEC AKT signaling in mediating the mtDNA damage protective effects of Klotho. Given the importance of AEC aging and apoptosis in pulmonary fibrosis, we reason that Klotho/AKT axis is an innovative therapeutic target for preventing common lung diseases of aging (i.e. IPF, COPD, lung cancer, etc.) for which more effective management regimens are clearly needed. Funding NIH-RO1 ES020357-01A1 (DK) and VA Merit (DK).

Context While the delivery of cell therapy after ST-segment elevation myocardial infarction (STEMI) has been evaluated in previous clinical trials, the influence of the timing of cell delivery on the effect on left ventricular function has not been analyzed. Objectives To determine the effect of intracoronary autologous bone marrow mononuclear cell (BMC) delivery after STEMI on recovery of global and regional left ventricular function and whether timing of BMC delivery (3 days vs 7 days after reperfusion) influences this effect. Design, Setting, and Patients A randomized, 2 × 2 factorial, double-blind, placebo-controlled trial, Timing In Myocardial infarction Evaluation (TIME) enrolled 120 patients with left ventricular dysfunction (left ventricular ejection fraction [LVEF] ≤ 45%) after successful primary percutaneous coronary intervention (PCI) of anterior STEMI between July 17, 2008, and November 15, 2011, as part of the Cardiovascular Cell Therapy Research Network sponsored by the National Heart, Lung, and Blood Institute. Interventions Intracoronary infusion of 150 × 10 6 BMCs or placebo (randomized 2:1) within 12 hours of aspiration and cell processing administered at day 3 or day 7 (randomized 1:1) after treatment with PCI. Main Outcome Measures The primary end points were change in global (LVEF) and regional (wall motion) left ventricular function in infarct and border zones at 6 months measured by cardiac magnetic resonance imaging and change in left ventricular function as affected by timing of treatment on day 3 vs day 7. The secondary end points included major adverse cardiovascular events as well as changes in left ventricular volumes and infarct size. Results The mean (SD) patient age was 56.9 (10.9) years and 87.5% of participants were male. At 6 months, there was no significant increase in LVEF for the BMC group (45.2% [95% CI, 42.8% to 47.6%] to 48.3% [95% CI, 45.3% to 51.3%) vs the placebo group (44.5% [95% CI, 41.0% to 48.0%] to 47.8% [95% CI, 43.4% to 52.2%]) (P = .96). There was no significant treatment effect on regional left ventricular function observed in either infarct or border zones. There were no significant differences in change in global left ventricular function for patients treated at day 3 (−0.9% [95% CI, −6.6% to 4.9%], P = .76) or day 7 (1.1% [95% CI, −4.7% to 6.9%], P = .70). The timing of treatment had no significant effect on regional left ventricular function recovery. Major adverse events were rare among all treatment groups. Conclusion Among patients with STEMI treated with primary PCI, the administration of intracoronary BMCs at either 3 days or 7 days after the event had no significant effect on recovery of global or regional left ventricular function compared with placebo. Trial Registration clinicaltrials.gov Identifier: NCT00684021

Introduction: Locally transplanted human CD34+ hematopoietic stem cells have stimulated neovascularization in preclinical studies and have been associated with functional improvements in phase I and II clinical trials of patients with ischemic cardiovascular diseases. Recently, we have demonstrated that membrane-bound nano-vesicles called exosomes are one of the primary pro-angiogenic components of human CD34+ cell paracrine secretion that induced angiogenesis independently of the cells. Here, we hypothesize that the functional benefits associated with CD34+ cell therapy are primarily mediated by the secretion of exosomes (Exo), and that CD34+ Exo transfer pro-angiogenic microRNAs to promote angiogenesis and ischemic tissue repair. Methods and Results: Therapeutic potential of CD34+ Exo isolated from adult human peripheral blood-derived CD34+ cells was evaluated in a mouse model of hind limb ischemia (HLI). Similar to CD34+ cells, administration of CD34+ Exo from equal number of cells induced angiogenesis and tissue repair; it significantly improved perfusion (ratio: 1.01±0.04 v 0.57±0.1, P Conclusion: CD34+ exosomes transfer the pro-angiogenic miR-126 to endothelial cells in the ischemic tissues to induce angiogenesis and ischemic tissue repair. Our study illustrates a mechanism of stem cell communication involving intercellular traffic of miRNAs via exosomes to induce functional recovery.

The renin angiotensin-aldosterone system has a diurnal variation, proceeding morning increases in blood pressure and plasminogen activator inhibitor-1 (PAI-1) levels. The RAAS is particularly active in individuals with metabolic syndrome (MetS) resulting in increased blood pressure and elevated PAI-1 levels. Eplerenone is a mineralocorticoid antagonist that is approved for the treatment of hypertension with a half-life of 4-6 hours but can be prescribed once or twice daily. This is a double-blind crossover study that examines the effects of morning (AM) vs. nighttime (PM) administration of eplerenone on blood pressure, PAI-1, plasma renin activity (PRA), and plasma aldosterone levels. Twenty subjects with MetS were studied on three 24-hour inpatient study days on hydrochlorothiazide (25mg) alone and with concomitant morning or evening dosing of eplerenone (100mg). Vital signes were taken and blood was drawn every three hours. There was a statistically significant increase in aldosterone levels with both AM (266±21 ng/dL) and PM (320±31 ng/dL) dosing of eplerenone compared to baseline (194±11 ng/dL) (both p < 0.001) with no significant difference between the two dosing strategies. There was a trend suggesting a daytime (8AM-5PM) systolic blood pressure reduction of 4.9±1.4 mmHg (p = 0.06) with AM dosing. Furthermore, diastolic blood pressure appeared less volatile with AM dosing (range = 38 mmHg vs. 47 mmHg for baseline and PM). There was no difference between baseline, AM and PM dosing with regard to PRA, PAI-1, serum potassium or creatinine. These findings suggest that there may be improved overall control of blood pressure with AM dosing of eplerenone, without obvious deleterious effects such as elevated creatinine or potassium levels. Furthermore, increases in aldosterone did not result in increases in PAI-1. Consequently rational dosing strategies such as this merit further investigation.

Fibrosis is defined as a fibroproliferative or abnormal fibroblast activation–related disease., Deregulation of wound healing leads to hyperactivation of fibroblasts and excessive accumulation of extracellular matrix (ECM) proteins in the wound area, the pathological manifestation of fibrosis. The accumulation of excessive levels of collagen in the extracellular matrix depends on two factors: an increased rate of collagen synthesis and or decreased rate of collagen degradation by cellular proteolytic activities. The urokinase-type/tissue-type plasminogen activator (uPA/tPA) and plasmin play significant roles in the cellular proteolytic degradation of ECM proteins and the maintenance of tissue homeostasis. The activities of uPA/tPA/plasmin and plasmin-dependent MMPs rely mostly on the activity of a potent inhibitor of uPA/tPA, plasminogen activator inhibitor-1 (PAI-1). Under normal physiologic conditions, PAI-1 controls the activities of uPA/tPA/plasmin/MMP proteolytic activities and thus maintains the tissue homeostasis. During wound healing, elevated levels of PAI-1 inhibit uPA/tPA/plasmin and plasmin-dependent MMP activities and thus help expedite wound healing. In contrast to this scenario, under pathologic conditions, excessive PAI-1 contributes to excessive accumulation of collagen and other ECM protein in the wound area and thus preserves scarring. While the level of PAI-1 is significantly elevated in fibrotic tissues, lack of PAI-1 protects different organs from fibrosis in response to injury-related profibrotic signals. Thus PAI-1 is implicated in the pathology of fibrosis in different organs including the heart, lung, kidney, liver and skin. Paradoxically, PAI-1 deficiency promotes spontaneous cardiac-selective fibrosis. In this review we discuss the significance of PAI-1 in the pathogenesis of fibrosis in multiple organs.

Segmental alterations in left ventricular function are generally present in patients who suffer an acute myocardial infarction. Regional wall motion abnormalities in left ventricular systolic function can be identified in the hyperacute period and generally persist in patients who complete a myocardial infarction. Through the process of infarct expansion, the infarcted territory may thin and lengthen in the short term following a myocardial infarction. Some infarct survivors are also prone to further progressive alterations in the shape and size of the left ventricle, a process that has been termed postinfarction ventricular remodeling. Although left ventricular remodeling appears to represent an adaptive process serving to preserve stroke volume (and cardiac output) following myocardial injury, the enlargement process may have undesirable long-term effects on global left ventricular function and on clinical prognosis. Fortunately, recent experimental and clinical evidence demonstrates that ventricular remodeling and its deleterious consequences may be preventable.