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Immunotherapeutic targeting of surface regulatory proteins and pharmacologic inhibition of critical signaling pathways has dramatically shifted our approach to the care of individuals with B cell malignancies. This evolution in therapy reflects the central role of the B cell receptor (BCR) signaling complex and its co-receptors in the pathogenesis of B lineage leukemias and lymphomas. Members of the Fc receptor-like gene family ( FCRL1-6 ) encode cell surface receptors with complex tyrosine-based regulation that are preferentially expressed by B cells. Among them, FCRL1 expression peaks on naïve and memory B cells and is unique in terms of its intracellular co-activation potential. Recent studies in human and mouse models indicate that FCRL1 contributes to the formation of the BCR signalosome, modulates B cell signaling, and promotes humoral responses. Progress in understanding its regulatory properties, along with evidence for its over-expression by mature B cell leukemias and lymphomas, collectively imply important yet unmet opportunities for FCRL1 in B cell development and transformation. Here we review recent advances in FCRL1 biology and highlight its emerging significance as a promising biomarker and therapeutic target in B cell lymphoproliferative disorders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mamidi, Huang, Honjo, Li, Tabengwa, Neeli, Randall, Ponnuchetty, Radic, Leu and Davis.)

Convalescent plasma (CP) is widely used to treat COVID-19, but without formal evidence of efficacy. Here, we report the beneficial effects of CP in a severely ill COVID-19 patient with prolonged pneumonia and advanced chronic lymphocytic leukemia (CLL), who was unable to generate an antiviral antibody response of her own. On day 33 after becoming symptomatic, the patient received CP containing high-titer (ID 50 > 5,000) neutralizing antibodies (NAbs), defervesced, and improved clinically within 48 h and was discharged on day 37. Hence, when present in sufficient quantities, NAbs to SARS-CoV-2 have clinical benefit even if administered relatively late in the disease course. However, analysis of additional CP units revealed widely varying NAb titers, with many recipients exhibiting endogenous NAb responses far exceeding those of the administered units. To obtain the full therapeutic benefits of CP immunotherapy, it will thus be important to determine the neutralizing activity in both CP units and transfusion candidates., Competing Interests: R.S.D. and K.H. have submitted an intellectual property disclosure related to the ACE2/RBD assay., (© 2020 The Author(s).)

Human apolipoprotein A-I (apoA-I) mimetic L-4F inhibits acute inflammation in endotoxemic animals. Since neutrophils play a crucial role in septic inflammation, we examined the effects of L-4F, compared to apoA-I, on lipopolysaccharide (LPS)-mediated activation of human neutrophils. We performed bioassays in human blood, isolated human neutrophils (incubated in 50 % donor plasma), and isolated human leukocytes (incubated in 5 and 50 % plasma) in vitro. In whole blood, both L-4F and apoA-I inhibited LPS-mediated elevation of TNF-α and IL-6. In LPS-stimulated neutrophils, L-4F and apoA-I (40 μg/ml) also decreased myeloperoxidase and TNF-α levels; however, L-4F tended to be superior in inhibiting LPS-mediated increase in IL-6 levels, membrane lipid rafts abundance and CD11b expression. In parallel experiments, when TNF-α and IL-8, instead of LPS, was used for cell stimulation, L-4F and/or apoA-I revealed only limited efficacy. In LPS-stimulated leukocytes, L-4F was as effective as apoA-I in reducing superoxide formation in 50 % donor plasma, and more effective in 5 % donor plasma (P<0.05). Limulus ambocyte lysate (LAL) and surface plasmon resonance assays showed that L-4F neutralizes LAL endotoxin activity more effectively than apoA-I (P<0.05) likely due to avid binding to LPS. We conclude that (1) direct binding/neutralization of LPS is a major mechanism of L-4F in vitro; (2) while L-4F has similar efficacy to apoA-I in anti-endotoxin effects in whole blood, it demonstrates superior efficacy to apoA-I in aqueous solutions and fluids with limited plasma components. This study rationalizes the utility of L-4F in the treatment of inflammation that is mediated by endotoxin-activated neutrophils.

Members of the extended Fc receptor-like (FCRL) family in humans and mice are preferentially expressed by B cells and possess tyrosine-based immunoregulatory function. Although the majority of these proteins repress B cell receptor-mediated activation, there is an emerging evidence for their bifunctionality and capacity to counter-regulate adaptive and innate signaling pathways. In light of these findings, the recent discovery of ligands for several of these molecules has begun to reveal exciting potential for them in normal lymphocyte biology and is launching a new phase of FCRL investigation. Importantly, these fundamental developments are also setting the stage for defining their altered roles in the pathogenesis of a growing number of immune-mediated diseases. Here we review recent advances in the FCRL field and highlight the significance of these intriguing receptors in normal and perturbed immunobiology.

Background: Wine polyphenol quercetin upregulates tissue-type plasminogen activator (t-PA) transcription in cultured human umbilical cord vein endothelial cells (HUVECs). However, the regulatory elements and signaling pathways involved in this regulation are unknown., Objectives: We aimed to localize quercetin-responsive t-PA promoter elements, identify the proteins that bind these elements, and decipher signaling pathways involved in the regulation of t-PA., Methods: To localize quercetin-responsive elements, HUVECs were transiently transfected with various t-PA promoter-reporter constructs. Element functionality was evaluated by mutational analysis. Nuclear protein-t-PA element interactions were evaluated by electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP) analysis. Mitogen-activated protein kinase (MAPK) inhibitors were used to determine the signaling pathways involved in t-PA regulation. MAPK inhibition effects were evaluated by real-time PCR, immunoblotting analysis, and transfections. Coimmunoprecipitation was used to evaluate MAPK and transcription factor interaction., Results: Deletion of the t-PA promoter region - 288 to - 250 resulted in loss of quercetin responsiveness. This region contains putative Sp1-binding elements, which we termed Sp1a and Sp1b. Sp1b mutation abolished the quercetin-inducible response, whereas Sp1a mutation had no effect. EMSA and ChIP analysis demonstrated quercetin-enhanced Sp1 binding to Sp1b. Inhibition of p38 MAPK abrogated basal and quercetin-induced t-PA expression and promoter activity, as well as quercetin-induced Sp1 binding to Sp1b. Quercetin enhanced p38 MAPK and Sp1 physical association, which was similarly diminished by p38 MAPK inhibition., Conclusions: We showed, for the first time, the presence of a functional Sp1-binding element in the t-PA promoter controlling quercetin induction via the p38 MAPK pathway. Understanding these mechanisms may provide new insights into polyphenol cardioprotective effects.

The reduction in coronary heart disease (CHD) from moderate alcohol intake may be mediated, in part, by increased fibrinolysis; endothelial cell (EC)-mediated fibrinolysis should decrease acute atherothrombotic consequences (eg, plaque rupture) of myocardial infarction (MI). We have shown that alcohol and individual polyphenols modulate EC fibrinolytic protein (t-PA, u-PA, PAI-1, u-PAR and Annexin-II) expression at the cellular, molecular, and gene levels to sustain increased fibrinolytic activity. Herein we describe the sequence of molecular events by which EC t-PA expression is increased through common activation of p38 MAPK signaling. Up-regulation of t-PA gene transcription, through specific alcohol and polyphenol transcription factor binding sites in the t-PA promoter, results in increased in vitro fibrinolysis and in vivo clot lytic activity (using real-time fluorescence [Fl] imaging of Cy5.5-labeled fibrin clot lysis in a mouse model). Fl-labeled fibrin clots injected into untreated C56Bl/6 wild-type control mice are lysed in approximately 2 hours and clot lytic rates significantly increased in mice treated with either alcohol, catechins, or quercetin (4-6 weeks). Fl-labeled clot lysis in ApoE knock-out mice (atherosclerosis model) showed impaired in vivo clot lysis that was "normalized" to wild-type control levels by treatment with alcohol, catechin, or quercetin for 6 to 8 weeks.

Epidemiologic data have indicated that the intake of polyphenols is inversely associated with mortality from cardiovascular disease. Mitogen-activated protein kinases (MAPKs) are ubiquitous signaling proteins that have been associated with gene regulation. This study determined whether polyphenols (catechin and quercetin) activated kinase-signaling cascades that suppress PAI-1 expression and whether this suppression is at the transcription level in human coronary artery endothelial cells (ECs) remains unresolved. ECs were incubated in the absence/presence of polyphenols and RNA and protein were analyzed by real-time PCR and Western blot analysis. MAPKs were analyzed using antibodies to active form of p38, JNK, and ERK1/2. ECs were transiently transfected with a 1.1-kb PAI-1 promoter (pPAI110/luc) and promoter activity were assays after treatment with polyphenols. Catechin and quercetin decreased EC PAI-1 mRNA in a time- and dose-dependent manner, reaching a maximum at 4 and 2 h, respectively. These polyphenols activated EC p38 and ERK1/2 within 2.5 and 5 min, respectively, while maximal JNK activation occurred at 10-15 min. An inhibitor of p38 MAPK had no effect on polyphenol-induced repression of PAI-1. Inhibitors of ERK or JNK prevented polyphenol repression of EC PAI-1 gene expression. Exposing ECs transiently transfected with pPAI110/luc to polyphenols decreased promoter activity 50%. Polyphenols repress EC PAI-1 expression, in part, by activating ERK and JNK signaling pathways and this repression is at transcriptional levels. Thus MAPK seem to play an important role in polyphenol-induce repression of PAI-1 expression in ECs.

In bullous pemphigoid (BP), the binding of BP180-specific antibodies to their hemidesmosomal target antigen is not sufficient for blister formation, but must be accompanied by the release of proteases. Using plasminogen activator (PA) knock-out mice, the PA system has previously been shown to be a prerequisite for blister formation in experimental murine BP. Here, we found elevated levels of plasmin and tPA, but not of uPA, in blister fluid from BP patients (n = 7) compared to blisters from patients with toxic epidermal necrolysis (n = 4) and suction blisters in healthy controls (n = 7). Subsequently, we addressed the question whether keratinocytes release PA in response to the binding of anti-BP180 antibodies. Treatment of cultured normal human keratinocytes with BP IgG, but not with control IgG, led to both increased protein and mRNA levels of tPA, but not of uPA, as determined by ELISA and RT-PCR, respectively. The specificity of this finding was confirmed using BP180-deficient keratinocytes from a patient with generalized atrophic benign epidermolysis bullosa, where no tPA release was observed after stimulation with BP IgG. Our results show the elevated expression and release of tPA from normal human keratinocytes upon stimulation with antibodies to human BP180. Keratinocytes, by secreting tPA, may thus play an active role in blister formation of BP.

Background: Moderate alcohol consumption is associated with reduced risk for coronary heart disease. This may due, in part to increased fibrinolysis. Monocytes synthesize fibrinolytic proteins, tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), and their receptors. These studies were carried out to determine the effect of low alcohol on monocyte fibrinolytic activity and PA messenger RNA (mRNA) synthesis., Methods: Peripheral blood monocytes and U937 cells were incubated in absence/presence of low alcohol (0.1%, v/v) for various times (0-1 hr), followed by incubations in the absence of alcohol (0-24 hr) before measurement of fibrinolytic activity and PA mRNA levels (reverse transcriptase polymerase chain reaction)., Results: Brief exposure (15 min, 4 degrees C) of U937 cells to low alcohol resulted in an approximately 2- to 3-fold increase (269.0 +/- 5.6 fmol/1 x 10 cells versus 656.0 +/- 94.0 fmol/1 x 10 cells) in fibrinolytic activity. Preincubation of U937 cells and peripheral blood monocytes in low alcohol (1 hr, 37 degrees C) followed by incubation in the absence of alcohol (24 hr) resulted in a sustained approximately 4- to 5-fold increase (414.0 +/- 174.7 vs. 965.33.0 +/- 104.8 fmol/1 x 10 cells) and an approximately 3- to 4-fold (20.5 +/- 2.14 vs. 74 +/- 2.28 fmol/2 x 10 cells, respectively) increase in fibrinolytic activity. Preincubation of monocytes with low alcohol (1 hr, 37 degrees C) followed by incubation in the absence of alcohol (6 hr) resulted in an approximately 5- to 6-fold (0.06 +/- 0.02 vs. 0.42 +/- 0.02) and an approximately 2- to 3-fold (0.89 +/- 0.04 vs. 2.07 +/- 0.29) increase in t-PA and u-PA mRNA (reverse transcriptase polymerase chain reaction; PA/glyceraldehyde-3-phosphate dehydrogenase ratio), respectively., Conclusions: These data suggest that low alcohol exerts a rapid, direct, and sustained effect on monocyte fibrinolytic activity, which may be, due in part, to increased monocyte t-PA/u-PA expression. These data provide a feasible molecular mechanism by which alcohol effects on monocyte fibrinolysis may contribute to the cardioprotective benefit associated with moderate alcohol consumption.

Background: Moderate alcohol consumption is associated with reduced risk for coronary heart disease and this cardioprotection may be due, in part, to increased fibrinolysis. We have previously demonstrated that low concentrations of ethanol (0.1%, v/v) induce the short-term (<1 hr) and sustained, long-term (24 hr) increase in surface-localized fibrinolytic activity; it up-regulates t-PA, u-PA, and the candidate plasminogen receptor (PmgR), annexin II, and gene transcription in cultured human umbilical vein endothelial cells (HUVECs). These studies describe the short- and long-term effects of low concentrations of ethanol on the kinetics of cell-bound 125I-labeled Glu-plasminogen (Glu-Pmg) activation by receptor (R)-bound t-PA, resulting in increased fibrinolytic activity in cultured HUVECs., Methods: Live cultured HUVECs were incubated with varying concentrations of Glu-Pmg (0.25-2 /M) and ethanol (0.025-0.1%, v/v) (in the presence of Aprotinin and alpha2-antiplasmin) and the direct activation of cell-bound 125I-labeled Glu-Pmg quantitated by measurement of 125I-labeled Mr 20 kDa plasmin light-chain, after reduction/SDS-PAGE. The effects of ethanol on '25I-labeled Glu-Pmg and t-PA ligand binding were determined by Scatchard analysis (Bmax, sites/cell)., Results: Cell-bound t-PA (endogenous/exogenous) activation of cultured HUVEC-bound 125I-labeled Glu-Pmg (short- and long-term) obeyed Michaelis-Menten kinetics, both in the absence/presence of low ethanol, as shown by Lineweaver-Burke plot analysis. In the short-term, ethanol (at 0.1%) increased the Vmax (2.5-fold), kcat (2-fold) and the apparent kcat/Km (4-fold), commensurate with a significant decrease in the apparent Km (6-fold) and increase in '25I-labeled Glu-Pmg ligand binding, Bmax (2-fold). In the long-term, ethanol increased the Vmax (2- to 3-fold), kcat (2.5-fold), apparent kcat/Km (5-fold), and Bmax (2-fold) for 125I-labeled Glu-Pmg ligand binding, without significantly affecting the apparent K ., Conclusions: Low concentrations of ethanol induce the short- versus long-term increase in surface-localized fibrinolytic activity in cultured HUVECs via different mechanisms. Short-term effects may be mediated by ethanol-induced membrane conformational changes that simultaneously facilitate increased surface-localized HUVEC PmgR availability and fibrinolytic protein/receptor interactions, resulting in the increased affinity of t-PA for Glu-Pmg and the accelerated activation of Glu-Pmg (increased Bmax, decreased apparent Km). The long-term effects may be attributed primarily to the ethanol-induced increased availability of both newly synthesized t-PA and PmgR and, hence, the accelerated activation of Glu-Pmg (increased Bmax)

Background: Moderate alcohol consumption has been correlated to reduced coronary artery disease (CAD) risk and mortality. This alcohol effect may be mediated in part by an increased endothelial cell (EC) fibrinolysis. ECs synthesize fibrinolytic proteins, tissue plasminogen activator (t-PA), urokinase type plasminogen activator (u-PA), and plasminogen activator inhibitor type-1(PAI-1). In addition, they synthesize and regulate receptors for fibrinolytic proteins, namely (t-PA and plasminogen receptor) Annexin II and u-PA receptor (u-PAR). These receptors play an important role in the regulated expression of receptor-bound plasminogen activator conversion of receptor-bound plasminogen to receptor-bound plasmin on the EC surface (surface-localized fibrinolytic activity). Therefore, systemic factors, such as ethanol, that affect the level, or activity or interaction of one or more of these components, resulting in the increased expression of surface-localized EC fibrinolytic activity, will be expected to reduce the risk for thrombosis, CAD, and myocardial infarction (MI). We have previously shown that low ethanol up-regulates t-PA and u-PA gene transcription, while it down-regulates PAI-1, hence resulting in increased (sustained, 24 hr) surface-localized EC fibrinolytic activity. The current studies were carried out to determine whether low ethanol increased u-PAR expression in cultured human umbilical cord vein ECs (HUVECs)., Methods: Cultured HUVECs were preincubated (1 hr) in the absence/presence of ethanol (0.025-0.2%, v/v); u-PAR mRNA (RT-PCR), antigen (western blot), and activity (125I-u-PA ligand binding/Scatchard analysis) levels were then measured after 0-24 hr. To determine whether the ethanol-induced changes in the u-PAR expression were transcriptional, transient transfection studies were carried out using a u-PAR/ luciferase promoter construct (pu-PAR120/luc [1.2-kb u-PAR promoter fragment ligated to a promoterless luciferase vector])., Results: uPAR mRNA levels increased 2- to 3-fold and antigen levels (western blot) increased 2- to 4-fold while u-PA binding activity increased 36% (1.25 vs. 1.7 x 10(5) sites/cell, Bmax) without significantly affecting the Kd (1-2 nM). Transient transfection of cultured HUVECs with a pu-PAR120/luc construct resulted in a 2- to 3-fold increase in promoter activity in ethanol-induced cultures, compared with controls., Conclusion: These combined results demonstrate that low ethanol (< or =0.1%, v/v) induces the up-regulation of u-PAR gene transcription, resulting in increased u-PAR ligand binding activity. These results also further identify/define the contribution and role of another fibrinolytic protein in the overall ethanol-induced increase in surface-localized EC fibrinolysis that may underlie and contribute, in part, to the cardioprotection attributed to moderate alcohol consumption.

Background: Moderate red wine consumption has been associated with a reduced risk for coronary heart disease, and this cardioprotection may be mediated, in part, by promoting fibrinolysis. This protection may be attributed to the combined or perhaps synergistic effects of alcohol and other red wine components (i.e., polyphenolics). These studies were carried out to determine whether individual phenolics (i.e., catechin, epicatechin, quercetin, and resveratrol) affect fibrinolytic protein (tissue-type plasminogen activator [t-PA] and urokinase-type PA [u-PA]) expression and surface-localized fibrinolytic activity in cultured human umbilical vein endothelial cells (HUVECs)., Methods: Cultured HUVECs were preincubated (1 hr, 37 degrees C) in the absence or presence of varying concentrations of catechin, epicatechin, quercetin, and resveratrol (0.001-10 microM) and then were washed and incubated for various times in the absence of phenolics. Secreted t-PA/u-PA antigen (24 hr, enzyme-linked immunoadsorbent assay) and mRNA [0-16 hr, reverse transcription-polymerase chain reaction(RT-PCR)] levels and fibrinolytic activity (direct activation of HUVEC-bound 125I-labeled glutamylplasminogen, quantitation of 125I-labeled Mr 20 kDa plasmin light-chain) were measured. Transient transfections of cultured HUVECs were carried out with the pt-PA222/luc and pu-PA236/luc promoter constructs, by using lipofectamine., Results: Each of the phenolics similarly increased t-PA and u-PA antigen (2- to 3-fold) and mRNA (3- to 4-fold) levels, concomitant with an increase (2- to 3-fold) in sustained (24 hr), surface-localized fibrinolytic activity. Transcription inhibitor actinomycin D abolished the induction of t-PA and u-PA mRNA expression by these phenolics. Transfections with the pt-PA222/luc and pu-PA236/luc promoter constructs showed 2- to 3-fold and 2- to 4-fold increases in luciferase activity for t-PA and u-PA, respectively., Conclusions: These results demonstrate that each of these phenolics up-regulates both t-PA and u-PA gene transcription, which results in the sustained increased expression of surface-localized fibrinolytic activity in cultured HUVECs. Wine phenolics increase fibrinolytic activity, independent of ethanol, and it is likely that the overall cardioprotective benefits associated with moderate red wine consumption are attributable to the combined, additive, or perhaps synergistic effects of alcohol and other wine components.

The relationship between tissue plasminogen activator (tPA) levels and the potential regulation by hypertriglyceridemic very low density lipoprotein (HTG-VLDL) was examined in a human umbilical vein endothelial cell (HUVEC) culture model system. HUVEC cultures were incubated in the absence/presence of HTG-VLDL or normal (NTG)-VLDL (0 to 50 microg/mL) at 37 degrees C for various times (0 to 24 hours), followed by analyses of tPA antigen (ELISA), mRNA (reverse transcription-polymerase chain reaction), endothelial cell surface-localized plasmin generation assays, and nuclear transcription run-on assays. Secreted tPA antigen levels decreased approximately 53% (3.3+/-0.14 versus 6.97+/-0.42 microg/mL) and mRNA levels decreased approximately 70% in HTG-VLDL-treated HUVECs compared with NTG-VLDL-treated and culture medium control cells. Decreased tPA antigen and mRNA expression was associated with a concomitant approximately 98% decrease in tPA-mediated plasmin generation in HTG-VLDL-treated HUVEC cultures. Nuclear transcription run-on assays demonstrated that HTG-VLDL decreased tPA gene transcription approximately 73% (tPA mRNA/GAPDH mRNA) in cultured HUVECs. To identify and localize the repressive element(s) in the tPA promoter responsive to HTG-VLDL, a tPA promoter/luciferase construct (ptPA222/luc) was generated. HUVECs transiently transfected with this construct were incubated in the absence/presence of HTG-VLDL or NTG-VLDL (20 microg/mL). HTG-VLDL decreased promoter activity approximately 52% to 57% in the ptPA222/luc-transfected cells compared with NTG-VLDL-treated or buffer control cells. These results indicate that the 2.2-kb fragment of the promoter and 5' flanking region of the tPA gene contains the repressive sequences that direct the transcriptional downregulation of the tPA promoter. Data from these studies suggest that the repression of tPA gene expression by HTG-VLDL may contribute to the impaired fibrinolysis often associated with hypertriglyceridemia.

Introduction: Epidemiological studies indicate that moderate alcohol consumption reduces the risk for coronary heart disease and that this cardioprotective benefit may be mediated, in part, by increased fibrinolysis. Endothelial cells (ECs) synthesize plasminogen activators, tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), receptors for plasminogen activators, and a receptor for plasminogen, annexin II (Ann-II). These receptors localize and facilitate receptor-bound plasminogen activator-mediated conversion of receptor-bound plasminogen to receptor-bound plasmin on the EC surface, which results in the regulated expression of surface-localized EC fibrinolytic activity. Ethanol is a systemic factor that affects these components, which increases EC fibrinolysis and hence reduces the risk for thrombosis, coronary heart disease, and myocardial infarction (MI)., Methods: This study was carried out to determine whether low ethanol (0.1% v/v) increased plasminogen receptor, Ann-II antigen (western blot), messenger ribonucleic acid (mRNA) (reverse transcription polymerase chain reaction; RT-PCR) expression, activity (ligand binding/Scatchard analysis), and hence fibrinolysis (plasmin generation) in cultured human ECs., Results: Plasminogen receptor activity increased approximately 2-fold (2.5 vs. 5.6 x 10(6) sites/cell), as evidenced by increased 125I-labeled Glu-plasminogen ligand binding/Scatchard analysis. In addition, western blot analyses indicated an increase in Ann-II antigen, and mRNA levels increased approximately 2-fold (RT-PCR). This increase in Ann-II expression was concomitant with approximately 2- to 3-fold sustained increase (approximately 24 hr) in surface-localized EC fibrinolytic activity. Nuclear transcription run-on assays showed an approximately 5- to 6-fold increase in new 32P-labeled Ann-II mRNA levels, compared with controls (no ethanol)., Conclusions: These results demonstrated that low ethanol increased Ann-II antigen/mRNA levels and up-regulated Ann-II gene expression at the transcriptional level. The results further identify and define the contribution and role of the plasminogen receptor, Ann-II, in the ethanol-induced mechanism of increased EC fibrinolysis that may underlie and contribute, in part, to the cardioprotective benefit associated with moderate alcohol consumption.

Human endothelial cells are a major site of synthesis for plasminogen activator inhibitor type-1. Elevated plasminogen activator inhibitor type-1 levels in young survivors of myocardial infarction [1] suggest that plasminogen activator inhibitor type-1 may have an important pathologic role in the development of coronary artery disease. Epidemiological studies indicate that moderate alcohol consumption (1-2 drinks/day) reduces the risk for cardiovascular mortality. This cardioprotective benefit has been attributed in part to an increase in fibrinolysis, which decreases fibrin-based thrombosis. The studies described herein were performed to determine whether moderate levels of ethanol affect plasminogen activator inhibitor type-1 gene expression. Cultured human endothelial cells were exposed to 0.1% v/v ethanol for 1 hour. Following incubation in the absence of ethanol plasminogen activator inhibitor type-1, mRNA levels were decreased in a time- and dose-dependent manner, reaching a maximum decrease of 3- to 4-fold at 2 to 4 hours following ethanol challenge. This decline in mRNA occurs at the transcription level; therefore, nuclear transcription run-on assays were performed. A 2.5- to 5-fold decrease in the rate of plasminogen activator inhibitor type-1 gene transcription was measured at 2 and 4 hours following ethanol challenge. Next, a 3.4- and a 1.1-kb fragment from the plasminogen activator inhibitor type-1 promoter region were linked to a luciferase reporter gene, and these constructs were transfected into human endothelial cells. Treatment of these transiently transfected human endothelial cells with ethanol showed a 2- to 3.5-fold decrease in promoter activity, respectively. These results indicate that low doses of ethanol downregulate transcription of the plasminogen activator inhibitor type-1 gene in cultured human endothelial cells. However, the mechanism(s) for this transcriptional decrease is currently unknown.

Epidemiological studies have suggested that moderate alcohol consumption reduces the risk of cardiovascular mortality. This cardioprotective benefit may be mediated, in part, by promoting fibrinolysis through changes in fibrinolytic components and/or activity, resulting in the decreased risk for thrombosis, coronary artery disease, and eventual myocardial infarction. Endothelial cells (ECs) play a pivotal role in maintaining normal hemostasis by regulating fibrinolysis through the synthesis of plasminogen activators (PAs), tissue-type plasminogen activator (t-PA), and urokinase-type plasminogen activator (u-PA). The studies described herein were conducted to determine whether a single brief preincubation (1 hr, 37 degrees C) of cultured human umbilical vein ECs (HUVECs) with low ethanol (0.1%, v/v), will upregulate t-PA and/or u-PA gene expression at the transcriptional level, using a combination of nuclear transcription run-on assays and transient transfections of cultured HUVECs with the pPA/luc promoter constructs. Nuclear run-on assays showed approximately 2- to 3-fold and approximately 6- to 7-fold increase in the transcription of new t-PA and u-PA mRNAs, respectively. In addition, transient transfections of cultured HUVECs with the pt-PA363/luc and pu-PA236/luc promoter constructs, using lipofectamine, demonstrated approximately 4- to 6-fold and approximately 6- to 9-fold increase in luciferase activity for t-PA and u-PA, respectively. These combined results demonstrate that low ethanol transcriptionally upregulates both t-PA and u-PA gene expression in cultured HUVECs and provides a molecular basis for the ethanol-induced increase in EC-mediated fibrinolytic activity that may underlie and contribute, in part, to the cardioprotective benefit associated with moderate alcohol consumption.

Clinical studies suggest that moderate alcohol consumption may decrease the risk for coronary artery disease and myocardial infarction. This effect may be attributed, in part, to the alcohol-mediated increase in endothelial cell (EC)-mediated fibrinolytic activity mediated by the increase in synthesis and/or activity of tissue-type plasminogen activators (t-PAs) and/or urokinase-type PA (u-PAs). To determine whether low alcohol levels (0.01 to 0.1%, v/v) induced the expression of these proteins, cultured human saphenous vein ECs (HSVECs) were preincubated in the absence/presence of ethanol for 5 to 120 min at 37 degrees C, washed, refed, and further incubated for 8 and 24 hr without alcohol. PA mRNA (reverse transcriptase-polymerase chain reaction) and secreted antigen (ELISA) levels were analyzed after incubation for 8 and 24 hr and the net expression of (sustained) endogenous PA-mediated surface-localized HSVEC fibrinolytic activity (plasmin generation) quantitated by activation of 125I-Glu-plasminogen after incubation for 24 hr. A brief 5 to 30 min preincubation (induction) of both t-PA and u-PA antigen increased approximately 3-fold (t-PA control, 14.2 +/- 1.7, plus alcohol, 25.4 +/- 5 ng/ml; u-PA control, 15 +/- 0.8, plus alcohol, 46.4 +/- 1.3 ng/ml) and mRNA levels approximately 2-fold, as compared with controls. Increased PA expression was associated with a significant concomitant approximately 2-fold increase in surface-localized fibrinolytic activity (control, 96 +/- 2.8, plus alcohol, 255 +/- 42 fmol/ well). These combined results indicate that a brief exposure (<30 min) to low levels of alcohol can induce synthesis of EC-produced t-PA and u-PA resulting in an increased expression of HSVEC surface-localized fibrinolytic activity and may account, in part, for the apparent cardioprotective benefit associated with moderate alcohol consumption.

The hypothesized relationships between plasminogen activator inhibitor (PAI-1) genotypes, PAI-1 levels, and their potential regulation by hypertriglyceridemic (HTG) very low density lipoprotein (VLDL) and lipoprotein(a) [Lp(a)] was examined in a PAI-1 genotyped human umbilical vein endothelial cell (HUVEC) culture model system. Individual human umbilical veins were used to obtain cultured ECs and were genotyped for PAI-1 by using the HindIII restriction fragment length polymorphism (RFLP) as a marker for genetic variation. Digested genomic DNA, examined by Southern blot analysis and probed with an [alpha-32P]dCTP-labeled 2.2-kb PAI-1 cDNA, yielded three RFLPs designated 1/1 (22-kb band only), 1/2 (22-plus 18-kb bands), and 2/2 (18-kb band only). Individual PAI-1 genotyped HUVEC cultures were incubated in the absence or presence of HTG-VLDL (0 to 50 micrograms/mL) or Lp(a) (0 to 50 micrograms/mL) at 37 degrees C for various times (4 to 24 hours), followed by analyses of PAI-1 antigen (by ELISA) and mRNA (by ribonuclease protection assay) levels, EC surface-localized plasmin generation assays, and nuclear run-on transcription assays. Secreted PAI-1 antigen levels were increased approximately 2- to 3-fold by HTG-VLDL and approximately 1.6 to 2-fold by Lp(a); mRNA levels were increased approximately 3- to 4.5-fold by HTG-VLDL and approximately 2.5- to 3.2-fold by Lp(a) compared with medium-incubated controls, primarily in the 2/2 PAI-1 genotype HUVEC cultures. Increases in PAI-1 mRNA induced by HTG-VLDL or Lp(a) could be abolished by coincubation with actinomycin D (2 x 10(-6) mol/mL) or puromycin (1 microgram/mL). In addition, nuclear transcription run-on assays typically demonstrated that HTG-VLDL increased PAI-1 gene transcription rates by approximately 5- to 6-fold and approximately 4- to 5-fold, respectively, primarily in the 2/2 PAI-1 genotype HUVEC cultures compared with 1/1 PAI-1 genotype HUVEC cultures or medium-incubated controls. The positive control interleukin-1 increased both 2/2 and 1/1 PAI-1 mRNA levels by approximately 5- to 6-fold. Increased PAI-1 antigen and mRNA expression were associated with a concomitant 50% to 60% decrease in plasmin generation. These combined results demonstrate the genotype-specific regulation of PAI-1 expression by HTG-VLDL and Lp(a) and further indicate that these risk factor-associated components regulate PAI-1 gene expression at the transcriptional level in cultured HUVECs. Results from these studies further suggest that individuals with this responsive 2/2 PAI-1 genotype may reflect the additional inherent potential for later HTG-VLDL- or Lp(a)-induced fibrinolytic dysfunction, resulting in the early initiation of thrombosis, atherogenesis, and coronary artery disease.

Epidemiological studies demonstrated a positive association between moderate alcohol consumption and reduced cardiovascular mortality that may be mediated, in part, through increased fibrinolysis. These studies were conducted to determine whether low concentrations of alcohol (0.025 to 0.1%, v/v) directly affected the surface-localized versus secreted/solution phase fibrinolytic activity in live cultured endothelial cell (EC) types. Confluent live cultured ECs [human umbilical vein ECs (HUVECs), human saphenous vein ECs (HSVECs), and porcine aortic ECs (PAECs)] were preincubated (0 to 20 min, 4 degrees C) in the absence or presence of varying concentrations of alcohol (0 to 0.1%, v/v), in the presence of saturating levels of 125I-labeled Glu-plasminogem (2 microM) and 125I-Plasmin M(r) 20-kDA light-chain formation quantitated by phosphorimaging autoradiography analysis. Endogenous plasminogen activator (PA)-mediated fibrinolytic activity was time- and dose-dependent; reached a maximum approximately 5- to 10-fold increase at 0.05% alcohol in HUVECs, HSVECs, and PAECs; was completely inhibited by anti-t-PA IgG in HUVECs; and partially inhibited by both anti-t-PA (approximately 40%) and anti-u-PA IgG (approximately 60%) in HSVECs. Complete inhibition of alcohol-induced (0.05%) fibrinolytic activity in cultured HUVECs by 2 mM tranexamic acid (an antagonist of plasminogen binding) indicated that the increased fibrinolytic activity was receptor-bound and localized to the EC surface, rather than present in or secreted into the medium (solution phase). Finally, the alcohol-induced increased fibrinolytic activity in cultured HUVECs returned to essentially normal control levels in approximately 1 hr. These studies have demonstrated a direct effect of low alcohol on EC fibrinolytic activity that may contribute, in part, to the decreased risk for thrombosis, coronary artery disease, and myocardial infarction associated with moderate alcohol consumption.

The effect of normo (NTG)- and hypertriglyceridemic (HTG)-VLDL on cultured human umbilical vein endothelial cell (HUVEC) surface-localized fibrinolysis was examined following pre-incubation with NTG-, HTG-VLDL, LDL (1-20 micrograms/mL) or buffer (control). Ligand binding assays, using 125I-labeled tcu-PA, t-PA, or Glu-plasminogen (Glu-Pmg) were carried out in the absence/presence of lipoproteins. Scatchard analyses showed that HTG-VLDL decreased the Bmax for 125I-labeled Glu-Pmg ligand binding approximately 35% [(2.11 +/- 0.39)-(1.40 +/- 0.32) x 10(6) sites/cell, p < 0.005] and increased the Kd, app approximately 5-fold (0.32 +/- 0.03 to 1.74 +/- 0.08 microM, p < 0.01), while NTG-VLDL, LDL, and buffer had no effect. 125I-labeled PA ligand binding was unaffected by these lipoproteins. Receptor-bound PA activation of cell-bound 125I-labeled Glu-Pmg was measured by quantitation of either the M(r) 20 kDa light- or M(r) 60 kDa heavy-chain of 125I-labeled plasmin, following SDS-PAGE. Kinetic analysis of these data (HTG-VLDL vs controls) indicated that HTG-VLDL decreased the V(max) of tcu-PA- and t-PA-mediated activation of plasminogen approximately 2.7-fold (0.317 +/- 0.023 vs 0.869 +/- 0.068 nM s-1, p < 0.01) and approximately 2.9-fold (0.391 +/- 0.098 vs 1.152 +/- 0.265 nM s-1, p < 0.01), respectively. Increasing concentrations of the HTG-VLDL increased 1/V(max), yielding a series of parallel plots, typical for uncompetitive inhibition with a Ki for inhibition of approximately 10 micrograms/mL. The combined ligand binding and kinetic data best fit an uncompetitive inhibition model in which the binding of the large HTG-VLDL particle to the EC surface may directly affect Glu-Pmg binding and activation, thus contributing to early fibrin deposition and the increased thrombotic risk associated with HTG.

The endothelial cell (EC) urokinase receptor plays an important role in the localization and receptor-mediated activation of EC-bound plasminogen and hence surface-localized fibrinolysis. Thrombin induced a rapid (< 5 minute), time- (0 to 30 minutes) and dose- (0.1 to 8 U/mL) dependent decrease in the specific binding of 125I-labeled two-chain urokinase-type plasminogen activator (tcu-PA) or diisopropylfluoro-phosphate-tcu-PA to urokinase-type plasminogen activator receptor (u-PAR) in cultured ECs from various sources (range, 21% to 50%). The thrombin receptor activation peptide but not control peptide showed a similar but reduced decrease in the specific binding of 125I-labeled tcu-PA to u-PAR. Incubation of thrombin-treated cultures (10 to 12 hours) in complete medium restored 125I-labeled tcu-PA ligand binding to normal levels. u-PAR mRNA levels rapidly (1 hour) increased and peaked 10 to 12 hours after thrombin treatment as analyzed by reverse transcriptase-polymerase chain reaction. Decreased thrombin-induced 125I-labeled tcu-PA binding correlated with the time-dependent decrease in surface-localized plasmin generation, as measured by the direct activation of 125I-labeled Glu-plasminogen and quantification of the 20-kD light chains of 125I-labeled plasmin. After incubation with thrombin, plasmin generation was decreased 50% to 56% (125 to 152 fmol/3 to 3.5 x 10(4) cells). Isolation of metabolically labeled 35S-labeled u-PAR from the media of thrombin and phospholipase C-treated human aortic cultures yielded approximately 10- and approximately 12-fold more 55-kD M(r) and approximately 6-fold more 35-kD M(r) 35S-labeled u-PAR forms than control cultures, respectively. The u-PAR antigen forms (M(r), 54 kD) and the glycosyl-phosphatidylinositol-anchored protein CD59 (M(r), 20 kD) were also simultaneously identified by immunoprecipitation in the media of thrombin-treated cultures. This suggests that thrombin may release u-PAR and decrease u-PA ligand binding through a common pathway involving phospholipase C. These results establish a novel interrelation between thrombin and EC fibrinolysis and suggest that thrombin may also have an additional regulatory role in the net expression of surface-localized EC fibrinolytic activity.