The role of the platelet pool of Plasminogen Activator Inhibitor-1 in well-controlled type 2 diabetes patients.

BACKGROUND: The main inhibitor of the fibrinolytic system, Plasminogen Activator Inhibitor -1 (PAI-1), irreversibly binds tissue-type Plasminogen Activator (t-PA) and thereby inhibits the protective action of tPA against thrombus formation. Elevated levels of plasma PAI-1 are associated with an increased risk of cardiovascular events and are observed in subjects with type 2 diabetes (T2D) and obesity. Platelets contain the majority of PAI-1 present in blood and exhibit the ability to synthesis active PAI-1. Diabetic platelets are known to be hyper-reactive and larger in size; however, whether these features affect their contribution to the elevated levels of plasma PAI-1 in T2D is not established. OBJECTIVES: To characterize the PAI-1 antigen content and the mRNA expression in platelets from T2D subjects compared to obese and lean control subjects, in order to elucidate the role of platelet PAI-1 in T2D. METHODS: Nine subjects with T2D and obesity were recruited from Primary Care Centers together with 15 healthy control subjects (8 lean subjects and 7 with obesity). PAI-1 antigen levels in plasma, serum and platelets were determined by ELISA, and PAI-1 mRNA expression was analyzed by qPCR. RESULTS: There was no significant difference in PAI-1 mRNA expression or PAI-1 antigen in platelets in T2D subject in comparison to obese and lean control subjects. An elevated level of plasma PAI-1 was seen in both T2D and obese subjects. PAI-1 gene expression was significantly higher in both obese groups compared to lean. CONCLUSION: Similar levels of protein and mRNA expression of PAI-1 in platelets from T2D, obese and lean subjects indicate a limited role of platelets for the elevated plasma PAI-1 levels. However, an increased synthesis rate of mRNA transcripts in platelets from T2D and an increased release of PAI-1 could also result in similar mRNA and protein levels. Hence, synthesis and release rates of PAI-1 from platelets in T2D and obesity need to be investigated to further elucidate the role of platelets in obesity and T2D.

Pulmonary embolism and deep vein thrombosis-comorbidities and temporary provoking factors in a register-based study of 1.48 million people.

Background: Knowledge on differences in patients who present with deep vein thrombosis (DVT) and those with pulmonary embolism (PE) is incomplete. Objective: To determine comorbidities and temporary provoking factors in patients with a first-time PE or DVT. Methods: This was a nationwide Swedish registry-based, retrospective, case-control study including 298 172 patients with first-time venous thromboembolism (VTE) and 1 185 079 controls matched for age, sex, and county of residence, free of VTE at the time of matching. Results: Patients with PE were older than those with DVT (mean age, 69 vs 66 years) and included slightly more women (PE, 53.4% vs DVT, 52.1%). After multivariable adjustment for comorbidities (within 7 years) and temporary provoking factors (within 3 months), heart failure (PE: adjusted odds ratio [aOR], 2.64 [99% confidence interval [CI], 2.55-2.73]; DVT: aOR, 1.66 [99% CI, 1.60-1.72]), ischemic heart disease (PE: aOR, 1.51 [99% CI, 1.47-1.56]; DVT: aOR, 1.01 [99% CI, 0.98-1.04]), and chronic obstructive pulmonary disease (PE: aOR, 2.51 [99% CI, 2.40-2.63]; DVT, 1.54 [99% CI, 1.47-1.62]) were among diseases that showed higher odds ratios in patients with PE than in those with DVT, compared with controls. Comorbidities registered within 6 months were associated with higher aORs than those within 7 years. The highest population attributable risks for PE were for cancer (13.0%) and heart failure (11.7%). Conclusion: Cardiopulmonary diseases, particularly with recent onset, imply a higher risk for PE, whereas orthopedic surgery and lower-extremity fractures carry a higher risk of DVT.

Disturbed Laminar Blood Flow Causes Impaired Fibrinolysis and Endothelial Fibrin Deposition In Vivo.

Endothelial expression of tissue-type plasminogen activator (t-PA) is crucial for maintaining an adequate endogenous fibrinolysis. It is unknown how endothelial t-PA expression and fibrinolysis are affected by blood flow in vivo. In this study, we investigated the impact of different blood flow profiles on endothelial t-PA expression and fibrinolysis in the arterial vasculature. Induction of disturbed laminar blood flow (D-flow) in the mouse carotid artery potently reduced endothelial t-PA messenger ribonucleic acid and protein expression, and caused fibrin deposition. En face immunohistochemistry demonstrated that arterial areas naturally exposed to D-flow had markedly lower endothelial t-PA levels than areas with sustained laminar blood flow (S-flow), and displayed pronounced fibrin deposition despite an intact endothelium. In t-PA and plasminogen-deficient mice, fibrin deposition did not extend into S-flow areas, indicating that areas of D-flow and S-flow differ, not only in fibrinolytic capacity, but also in coagulation. Furthermore, plasminogen accumulation was found at D-flow areas, and infusion of recombinant t-PA activated fibrinolysis and significantly reduced the fibrin deposits. In conclusion, D-flow potently impairs the fibrinolytic capacity and causes endothelial fibrin deposition in vivo. Our data also indicate that t-PA is the limiting factor for efficient fibrinolysis at the thrombosis-prone D-flow areas in the arterial vasculature.

Rapid and specific hypomethylation of enhancers in endothelial cells during adaptation to cell culturing.

Epigenetics, including DNA methylation, is one way for a cell to respond to the surrounding environment. Traditionally, DNA methylation has been perceived as a quite stable modification; however, lately, there have been reports of a more dynamic CpG methylation that can be affected by, for example, long-term culturing. We recently reported that methylation in the enhancer of the gene encoding the key fibrinolytic enzyme tissue-type plasminogen activator (t-PA) was rapidly erased during cell culturing. In the present study we used sub-culturing of human umbilical vein endothelial cells (HUVECs) as a model of environmental challenge to examine how fast genome-wide methylation changes can arise. To assess genome-wide DNA methylation, the Infinium HumanMethylation450 BeadChip was used on primary, passage 0, and passage 4 HUVECs. Almost 2% of the analyzed sites changed methylation status to passage 4, predominantly displaying hypomethylation. Sites annotated as enhancers were overrepresented among the differentially methylated sites (DMSs). We further showed that half of the corresponding genes concomitantly altered their expression, most of them increasing in expression. Interestingly, the stroke-related gene HDAC9 increased its expression several hundredfold. This study reveals a rapid hypomethylation of CpG sites in enhancer elements during the early stages of cell culturing. As many methods for methylation analysis are biased toward CpG rich promoter regions, we suggest that such methods may not always be appropriate for the study of methylation dynamics. In addition, we found that significant changes in expression arose in genes with enhancer DMSs. HDAC9 displayed the most prominent increase in expression, indicating, for the first time, that dynamic enhancer methylation may be central in regulating this important stroke-associated gene.

Normalization of qPCR in platelets - YWHAE a potential genericreference gene.

The mRNA of human platelets has been extensively studied and it is generally appreciated that platelets contain mRNA transcripts derived from the megakaryocytes, and they have the ability to translate it into proteins. Additionally, platelets contain microRNA (miRNA) that has been shown to potentially regulate the translation of certain proteins. When quantifying gene expression by quantitative real-time polymerase chain reaction (qPCR), a valid normalization method is required and the use of reference genes is a common and robust approach. It is recommended to perform a proper validation of potential reference genes for each individual experimental setup. Previous studies have mainly been performed using commonly used reference genes for nucleated cells, and to our knowledge there are no global evaluations of the stability of transcripts in platelets. Finding a stable transcript would be valuable for inter-study comparisons, and the aim of this study was to identify one or more stable mRNA transcripts suitable as generic reference genes for mRNA gene expression studies in platelets. Platelets were incubated for 24 h and microarray of platelet mRNA revealed that the levels of YWHAE, B2M, ITM2B, H3F3A, PF4V1 remained similar between 0 and 24 h. Further validation of the stability of these genes together with GAPDH, RN18S1, and PPIA, genes frequently used as reference genes in platelet studies, was performed using qPCR after different in vitro conditions. In addition, inter-individual stability of the genes was analyzed in diabetic patients compared with healthy matched controls. Analysis of gene stability by the software RefFinder revealed that YWHAE, PF4V1, and B2M were the most stable genes in platelets from healthy donors. In addition, YWHAE was stable between subjects. Furthermore, the potential influence of miRNA on the selected genes was investigated by knockdown of Dicer1 in the megakaryocytic cell line MEG01. YWHAE, H3F3A, B2M, and GAPDH remained unchanged over time in MEG01 cells indicating that these genes are not regulated by miRNA and hence are more stably expressed. In conclusion, YWHAE is a stable transcript in platelets and we suggest the use of YWHAE as a generic reference gene in mRNA gene expression studies.

Dependence of Proximal GC Boxes and Binding Transcription Factors in the Regulation of Basal and Valproic Acid-Induced Expression of t-PA.

Objective. Endothelial tissue-type plasminogen activator (t-PA) release is a pivotal response to protect the circulation from occluding thrombosis. We have shown that the t-PA gene is epigenetically regulated and greatly induced by the histone deacetylase (HDAC) inhibitor valproic acid (VPA). We now investigated involvement of known t-PA promoter regulatory elements and evaluated dependence of potential interacting transcription factors/cofactors. Methods. A reporter vector with an insert, separately mutated at either the t-PA promoter CRE or GC box II or GC box III elements, was transfected into HT-1080 and HUVECs and challenged with VPA. HUVECs were targeted with siRNA against histone acetyl transferases (HAT) and selected transcription factors from the Sp/KLF family. Results. An intact VPA-response was observed with CRE mutated constructs, whereas mutation of GC boxes II and III reduced the magnitude of the induction by 54 and 79% in HT-1080 and 49 and 50% in HUVECs, respectively. An attenuated induction of t-PA mRNA was observed after Sp2, Sp4, and KLF5 depletion. KLF2 and p300 (HAT) were identified as positive regulators of basal t-PA expression and Sp4 and KLF9 as repressors. Conclusion. VPA-induced t-PA expression is dependent on the proximal GC boxes in the t-PA promoter and may involve interactions with Sp2, Sp4, and KLF5.

Dynamic Enhancer Methylation--A Previously Unrecognized Switch for Tissue-Type Plasminogen Activator Expression.

Tissue-type plasminogen activator (t-PA), which is synthesized in the endothelial cells lining the blood vessel walls, is a key player in the fibrinolytic system protecting the circulation against occluding thrombus formation. Although classical gene regulation has been quite extensively studied in order to understand the mechanisms behind t-PA regulation, epigenetics, including DNA methylation, still is a largely unexplored field. The aim of this study was to establish the methylation pattern in the t-PA promoter and enhancer in non-cultured compared to cultured human umbilical vein endothelial cells (HUVECs), and to simultaneously examine the level of t-PA gene expression. Bisulphite sequencing was used to evaluate the methylation status, and real-time RT-PCR to determine the gene expression level. While the t-PA promoter was stably unmethylated, we surprisingly observed a rapid reduction in the amount of methylation in the enhancer during cell culturing. This demethylation was in strong negative correlation with a pronounced (by a factor of approximately 25) increase in t-PA gene expression levels. In this study, we show that the methylation level in the t-PA enhancer appears to act as a previously unrecognized switch controlling t-PA expression. Our findings, which suggest that DNA methylation is quite dynamic, have implications also for the interpretation of cell culture experiments in general, as well as in a wider biological context.

Histone deacetylase inhibition enhances tissue plasminogen activator release capacity in atherosclerotic man.

UNLABELLED: The expression of the tissue plasminogen activator (t-PA) gene appears to be under epigenetic control and can be affected by histone deacetylation inhibition. The study aimed to test if histone deacetalyase inhibitor treatment lead to increased t-PA release or reduced exhaustion in t-PA release in response to stimulation, as well as change in plasminogen activator inhibitor-1 (PAI-1) in subjects with coronary disease. In this clinical study, 16 post-myocardial infarction subjects, the perfused forearm model was used with isoprenaline provocation during 20 minutes, to stimulate local t-PA release. Each subject was measured twice on the same day (repeated stimuli sequences) as well as on two different occasions, without treatment and after four weeks of treatment with valproic acid (500 mg, twice daily). Net forearm release for t-PA in response to isoprenaline at minutes 1.5, 3, 6, 9, 12, 15 and 18 was measured, allowing assessment of cumulative t-PA release. There was a reduction in the exhaustion of cumulative t-PA release during repeated and prolonged stimulation with valproic acid treatment compared to non-treatment. Plasma PAI-1 antigen was decreased following treatment compared to non-treatment (18.4 ± 10.0 vs. 11.0 ± 7.1 nanograms/ml respectively, mean with 95% confidence interval). These findings demonstrate that histone deacetylation inhibition increases the capacity for endogenous t-PA release in subjects with vascular disease. Furthermore, the fibrinolytic balance is favored with suppressed PAI-1 levels. More studies are needed to establish the clinical relevance of these findings. TRIAL REGISTRATION: EU Clinical Trials Register 2012-004950-27.

Profibrinolytic effect of the epigenetic modifier valproic acid in man.

AIMS: The aim of the study was to test if pharmacological intervention by valproic acid (VPA) treatment can modulate the fibrinolytic system in man, by means of increased acute release capacity of tissue plasminogen activator (t-PA) as well as an altered t-PA/Plasminogen activator inhibitor -1 (PAI-1) balance. Recent data from in vitro research demonstrate that the fibrinolytic system is epigenetically regulated mainly by histone deacetylase (HDAC) inhibitors. HDAC inhibitors, including VPA markedly upregulate t-PA gene expression in vitro. METHODS AND RESULTS: The trial had a cross-over design where healthy men (n = 10), were treated with VPA (Ergenyl Retard) 500 mg depot tablets twice daily for 2 weeks. Capacity for stimulated t-PA release was assessed in the perfused-forearm model using intra-brachial Substance P infusion and venous occlusion plethysmography. Each subject was investigated twice, untreated and after VPA treatment, with 5 weeks wash-out in-between. VPA treatment resulted in considerably decreased levels of circulating PAI-1 antigen from 22.2 (4.6) to 10.8 (2.1) ng/ml (p<0.05). It slightly decreased the levels of circulating venous t-PA antigen (p<0.05), and the t-PA:PAI-1 antigen ratio increased (p<0.01). Substance P infusion resulted in an increase in forearm blood flow (FBF) on both occasions (p<0.0001 for both). The acute t-PA release in response to Substance P was not affected by VPA (p = ns). CONCLUSION: Valproic acid treatment lowers plasma PAI-1 antigen levels and changes the fibrinolytic balance measured as t-PA/PAI-1 ratio in a profibrinolytic direction. This may in part explain the reduction in incidence of myocardial infarctions by VPA treatment observed in recent pharmacoepidemiological studies. TRIAL REGISTRATION: The EU Clinical Trials Register 2009-011723-31.

Histone deacetylase inhibitor treatment increases coronary t-PA release in a porcine ischemia model.

BACKGROUND: The expression of the tissue plasminogen activator gene can be affected by histone deacetylation inhibition and thus appears to be under epigenetic control. OBJECTIVES: The study aimed to test if in vivo pharmacological intervention by valproic acid treatment would lead to increase in tissue plasminogen activator release capacity. METHODS: In an anaesthetized pig model, a controlled transient coronary occlusion was used to stimulate coronary tissue plasminogen activator release in a valproic acid treated (one week) and a non-treated group. Coronary venous blood samples from the ischemic region were collected, great cardiac vein thermodilution flow measurements were performed, and trans-coronary tissue plasminogen activator fluxes were calculated. Plasminogen activator inhibitor-1 was also measured. RESULTS: Adequate sampling from the affected area after the 10 minute ischemic period was confirmed by lactate measurements. Fluxes for tissue plasminogen activator at minutes 1, 3, 5, 7 and 10 were measured and then used to present cumulative net tissue plasminogen activator release for the whole measurement period for both groups. Area under the curve was higher for the valproic acid treated group at 10 minutes; 932 ± 173 nanograms (n = 12) compared to the non-treated group, 451 ± 78 nanograms (n = 10, p = 0.023). There was no difference in levels of plasminogen activator inhibitor-1 between groups. CONCLUSIONS: These findings support a proof of concept for histone deacetylation inhibition positive effect on tissue plasminogen activator expression in an in vivo setting. Further studies are needed to find an optimal way to implement histone deacetylation inhibition to achieve desired clinical changes in tissue plasminogen activator expression.

Histone deacetylase inhibitors stimulate tissue-type plasminogen activator production in vascular endothelial cells.

A reduced capacity for acute tissue-type plasminogen activator (t-PA) release is likely to be associated with an impaired endogenous defense against intravascular thrombosis. Efficient approaches to pharmacologically restore a defective t-PA release have been lacking, but recent observations suggest that histone deacetylase inhibitors (HDACis) enhance t-PA production in vitro. HDACis have diverse chemical structures and different HDAC-enzyme sub-class targeting. We here compared the effects of several clinically used HDACis on t-PA production in endothelial cells. Human umbilical vein endothelial cells were exposed to a panel of 11 different HDACis and t-PA mRNA and protein levels were quantified. All HDACis dose-dependently stimulated t-PA mRNA and protein expression with similar maximal efficacy but with different potencies. Already at low concentrations, the majority of inhibitors caused significant and sustained effects on t-PA production. In addition, selected HDACis were capable of normalizing t-PA production when suppressed by the inflammatory cytokine TNF-α. We conclude that HDACis targeting classical HDAC enzymes are powerful inducers of t-PA expression in cultured endothelial cells and could be promising candidates for pharmacological modulation of endogenous fibrinolysis in man.

Role of histone acetylation in the stimulatory effect of valproic acid on vascular endothelial tissue-type plasminogen activator expression.

AIMS: Stimulated release of tissue-type plasminogen activator (t-PA) is pivotal for an intravascular fibrinolytic response and protects the circulation from occluding thrombosis. Hence, an impaired t-PA production is associated with increased risk for atherothrombotic events. A pharmacological means to stimulate the production of this enzyme may thus be desirable. We investigated if the anti-epileptic drug valproic acid (VPA) is capable of enhancing t-PA expression in vitro in vascular endothelial cells, and further examined if its histone deacetylase (HDAC)-inhibitory activity is of importance for regulating t-PA expression. METHODS AND RESULTS: Human endothelial cells were exposed to valproic acid and t-PA mRNA and protein levels were quantified. Potential changes in histone acetylation status globally and at the t-PA promoter were examined by western blot and chromatin immunoprecipitation. Valproic acid dose-dependently stimulated t-PA mRNA and protein expression in endothelial cells reaching a 2-4-fold increase at clinically relevant concentrations and 10-fold increase at maximal concentrations. Transcription profiling analysis revealed that t-PA is selectively targeted by this agent. Augmented histone acetylation was detected at the t-PA transcription start site, and an attenuated VPA-response was observed with siRNA knock of HDAC3, HDAC5 and HDAC7. CONCLUSIONS: Valproic acid induces t-PA expression in cultured endothelial cells, and this is associated with increased histone acetylation at the t-PA promoter. Given the apparent potency of valproic acid in stimulating t-PA expression in vitro this substance may be a candidate for pharmacological modulation of endogenous fibrinolysis in man.

Platelets retain high levels of active plasminogen activator inhibitor 1.

The vascular fibrinolytic system is crucial for spontaneous lysis of blood clots. Plasminogen activator inhibitor 1 (PAI-1), the principal inhibitor of the key fibrinolytic enzyme tissue-type plasminogen activator (tPA), is present in platelets at high concentrations. However, the majority of PAI-1 stored in platelets has been considered to be inactive. Our recent finding (Brogren H, et al. Blood 2004) that PAI-1 de novo synthesized in platelets remained active for over 24 h, suggested that PAI-1 stored in the α-granules might be active to a larger extent than previously reported. To re-evaluate this issue, we performed experiments where the fraction of active PAI-1 was estimated by analyzing the tPA-PAI-1 complex formation. In these experiments platelets were lysed with Triton X-100 in the presence of serial dilutions of tPA and subsequently the tPA-PAI-1 complex was evaluated by Western blot. Also, using a non-immunologic assay, tPA was labeled with (125)I, and (125)I-tPA and (125)I-tPA-PAI-1 was quantified by scintigraphy. Interestingly, both methods demonstrated that the majority (>50%) of platelet PAI-1 is active. Further analyses suggested that pre-analytical procedures used in previous studies (sonication or freezing/thawing) may have substantially reduced the activity of platelet PAI-1, which has lead to an underestimation of the proportion of active PAI-1. Our in vitro results are more compatible with the role of PAI-1 in clot stabilization as demonstrated in physiological and pathophysiological studies.

The impaired fibrinolytic capacity in hypertension is unaffected by acute blood pressure lowering.

The endogenous fibrinolytic system and the ability of the endothelium to release tissue-plasminogen activator (t-PA) play a pivotal role to protect humans from atherothrombotic events. We have recently reported that the decreased capacity for t-PA release in hypertension is restored with chronic blood pressure lowering. Thus, we explored if acute blood pressure lowering has the same effect. The capacity for acute t-PA release was investigated in the perfused-forearm model during stimulation by intra-arterial substance P 8 pmol/min in hypertensive subjects. The procedure was then repeated during acute blood pressure lowering (n = 9) induced by sodium nitroprusside (SNP) infusion or during placebo infusion (n = 3). SNP lowered mean arterial pressure from 108.6 (2.6) to 83.0 (2.6) (mean and SEM) mmHg (P < 0.001). Substance P induced significant increase in t-PA release during both high- and low-pressure conditions (P < 0.01, ANOVA). Peak t-PA release rate was 199 (77) and 167 (41) (mean and SEM) ng/min/l tissue, and accumulated t-PA release was 2,395 (750) and 2,394 (473) ng, during high- and low-pressure conditions, respectively. t-PA release and hemodynamic responses were almost identical during high- and low-pressure conditions (P = ns, for all). Acute blood pressure lowering does not restore stimulated t-PA release from the endothelium in hypertensive subjects. These findings are in contrast to previously described effects of chronic blood pressure treatment. Although data need to be confirmed in a larger study, they suggest that high blood pressure decreases the cellular t-PA pool rather than interferes with release mechanisms of the protein.

Hypothermia in cardiogenic shock reduces systemic t-PA release.

Therapeutic hypothermia has been found to improve hemodynamic and metabolic parameters in cardiogenic shock. Tissue plasminogen activator (t-PA) is a pro-thrombolytic enzyme, which also possesses pro-inflammatory properties. Interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α) are pro-inflammatory cytokines; interleukin 10 (IL-10) and transforming growth factor beta 1 (TGF-ß1) are anti-inflammatory cytokines. The aim of this experiment was to investigate the mechanism behind the protective effect of therapeutic hypothermia in cardiogenic shock. This was done by studying the effect of hypothermia on basal t-PA levels, peripheral t-PA release, and on the inflammatory response. Cardiogenic shock was induced by inflation of an angioplasty balloon in the proximal left anterior descending artery for 40 min in 16 pigs, followed by 110 min of reperfusion. The animals were randomized to hypothermia (33°C, n = 8), or normothermia (n = 8) at reperfusion. Hemodynamic parameters were continuously monitored. Plasma was sampled every 30 min for analysis of blood-gases and t-PA, and for analysis of inflammatory markers at baseline and at the end of the experiment. t-PA, IL-6 and TGF-ß1 increased during cardiogenic shock. Apart from favourably affecting hemodynamic and metabolic variables, hypothermia was found to reduce basal arterial and venous t-PA levels, and to inhibit the release of t-PA from the peripheral vascular bed. Hypothermia did not alter the inflammatory response. In conclusion, mild hypothermia improves hemodynamic and metabolic parameters in cardiogenic shock. This is associated with a reduction in basal t-PA levels and t-PA release from the peripheral vascular bed, but not with an altered inflammatory response.

Transcutaneous electrical nerve stimulation induces vasodilation in healthy controls but not in refractory angina patients.

CONTEXT: Transcutaneous electrical nerve stimulation (TENS) is an effective treatment option to relieve ischemic pain in refractory angina pectoris (RAP). In healthy persons, TENS enhances local blood flow, but the mechanism responsible for the anti-ischemic effect in RAP seems to be different. OBJECTIVE: The aim of the present investigation was to compare the difference in blood flow and vasodilatory response to TENS between angina patients and healthy controls and evaluate how vascular response in these groups is affected by amperage dosage above and below motor threshold levels. METHODS: Our study evaluated upper limb vascular responses to low- and high-dose TENS (below and above motor threshold) in RAP patients compared with healthy controls. TENS was applied on the nondominating forearm. Forearm blood flow (FBF) was measured by venous occlusion plethysmography. Forearm vascular resistance (FVR) was determined (mean arterial pressure [MAP]/FBF). Measurements were done during baseline, low-dose TENS, high-dose TENS, and during recovery. RESULTS: A significant dose-dependent increase in FBF in response to TENS stimulation was seen in controls (n=18) but not in RAP (n=23) (P=0.008). There was no significant difference in FVR ratio (FVR(stim)/FVR(ctrl)) between control (n=7) and RAP (n=23) groups at low dose (controls, 5.7+/-21%; RAP, 9.7+/-20%) or recovery (controls, -4.6+19%; RAP, 5.9+25%). High-dose TENS resulted in a significantly reduced FVR ratio (-16.8+/-11%) in controls (n=7) compared with RAP (1.6+/-32%, n=23) (P=0.02). CONCLUSION: High-dose TENS induces forearm vasodilation in healthy subjects but not in patients with RAP. These findings suggest that TENS has different vascular effects in patients with severe coronary artery disease compared with healthy controls.

Mild hypothermia markedly reduces ischemia related coronary t-PA release.

In experimentally induced myocardial ischemia, mild hypothermia (33-35 degrees C) has a robust cardioprotective effect. Tissue plasminogen activator (t-PA) is a profibrinolytic enzyme that is released from the vascular endothelial cells in response to ischemia and other injurious stimuli. t-PA has also been found to have proinflammatory properties that could contribute to reperfusion injury. We postulated that hypothermia could attenuate t-PA release in the setting of myocardial ischemia. Sixteen 25-30 kg pigs were anesthetized and a temperature of 37 degrees C was established using an intravascular cooling/warming catheter. The pigs were then randomized to hypothermia (34 degrees C) or control (37 degrees C). A doppler flow wire was placed distal to a percutaneous coronary intervention balloon positioned immediately distal to the first diagonal branch of the left anterior descending artery (LAD). The LAD was then occluded for 10 min in all pigs. Coronary blood flow and t-PA was measured before, during and after ischemia/reperfusion. t-PA was measured in peripheral arterial blood and locally in the venous blood from the coronary sinus. Net t-PA release over the coronary bed was calculated by subtraction of arterial values from coronary sinus values. An estimate of differences in total t-PA release was calculated by multiplying net t-PA release with the relative increase in flow compared to baseline, measured in relative units consisting of ((ng/ml - ng/ml) x (cm/s/cm/s)). There was no observed difference in t-PA levels in peripheral arterial samples. As shown previously, net t-PA release increased during reperfusion. Hypothermia significantly inhibited the increase in t-PA release during reperfusion (peak value 9.44 +/- 4.34 ng/ml vs. 0.79 +/- 0.45 ng/ml, P = 0.02). The effect was even more prominent when an estimation of total t-PA release was performed with mean peak value in the control group 26-fold higher than in the hypothermia group (69.74 +/- 33.86 units vs. 2.62 +/- 1.10 units, P = 0.01). Mild hypothermia markedly reduces ischemia related coronary tissue plasminogen activator release. The reduction of t-PA release may contribute to the cardioprotective effect of hypothermia.

Suppression of endothelial t-PA expression by prolonged high laminar shear stress.

Primary hypertension is associated with an impaired capacity for acute release of endothelial tissue-type plasminogen activator (t-PA), which is an important local protective response to prevent thrombus extension. As hypertensive vascular remodeling potentially results in increased vascular wall shear stress, we investigated the impact of shear on regulation of t-PA. Cultured human endothelial cells were exposed to low (< or =1.5 dyn/cm(2)) or high (25 dyn/cm(2)) laminar shear stress for up to 48 h in two different experimental models. Using real-time RT-PCR and ELISA, shear stress was observed to time and magnitude-dependently suppress t-PA transcript and protein secretion to approximately 30% of basal levels. Mechanistic experiments revealed reduced nuclear protein binding to the t-PA specific CRE element (EMSA) and an almost completely abrogated shear response with pharmacologic JNK inhibition. We conclude that prolonged high laminar shear stress suppresses endothelial t-PA expression and may therefore contribute to the enhanced risk of arterial thrombosis in hypertensive disease.

Heterogeneous glycosylation patterns of human PAI-1 may reveal its cellular origin.

The main inhibitor of intravascular fibrinolysis is plasminogen activator inhibitor 1 (PAI-1) which binds to and irreversibly inhibits tissue plasminogen activator (tPA). PAI-1 is present in blood, both in platelets and in plasma, and PAI-1 levels are associated with risk of atherothrombosis. Several tissues express PAI-1 but the source of plasma PAI-1 is not known. We recently found that platelets can de novo synthesize PAI-1 and the amount synthesized in vitro in 24 hours is 35-fold higher than required to maintain normal plasma levels. Recombinant human PAI-1 expressed in different cell types or secreted naturally by human cell lines, exhibit heterogeneous glycosylation patterns. The aim of this study was to investigate the hypothesis that platelets might be the source of plasma PAI-1 and that the cellular source of PAI-1 can be determined by its tissue-specific glycosylation pattern. PAI-1 was isolated from platelets, macrophages, endothelial cells, adipose tissue, as well as plasma from lean and obese subjects. The glycosylation was analyzed by nanoLC-MS/MS. PAI-1 isolated from cell lysates and conditioned media from macrophages, endothelial cells, and adipose tissue expressed heterogeneous glycosylation patterns. By contrast, no glycans were detected on PAI-1 isolated from plasma or platelets from healthy lean individuals. Hence, our data suggest that platelets may be the main source of plasma PAI-1 in lean individuals. Interestingly, plasma PAI-1 from obese subjects had a glycan composition similar to that of adipose tissue suggesting that obese subjects with elevated PAI-1 levels may have a major contribution from other tissues.