49
Chapter 3
t-PA RELEASE IN HEALTHY SUBJECTS AND HYPERTENSIVE PATIENTS: RELATIONSHIP WITH ββββ-ADRENERGIC RECEPTORS AND THE NO PATHWAY
Introduction
The endogenous fibrinolytic system contributes to the maintenance of vessel patency via the cleavage of insoluble fibrin by plasmin. The activation of plasmin by tissue-type plasminogen activator (t-PA) is a physiological process that, clearing inappropriate intravascular fibrin deposition, prevents vascular atherothrombotic events 1, 2.
The coagulation factors thrombin and activated factor X, acting as counter-regulatory mechanisms during fibrin deposition, are considered the main stimuli for acute t-PA release from endothelial cells 2. Thus, the vascular endothelium plays a major regulatory role in endogenous fibrinolysis 3. One of the main mechanisms by which a healthy endothelium regulates acute release of t-PA involves the nitric oxide (NO) pathway 4. Certain pathological conditions characterized by reduced NO availability, such as essential hypertension, in which an impaired endothelium-dependent vasodilation occurs, 5 a concurrent impairment in t-PA release is observed 4, 6.Worth of note t-PA release and endothelium-dependent vasodilation can predict the risk of cardiovascular events 7, 8. Another important stimulus involved in modulating the fibrinolytic system 1 is adrenergic activation
9
. As the sympathetic nervous system plays a major role in cardiovascular homeostasis and is involved in the determination of cardiovascular risk 10, the relationship between adrenergic activation and the fibrinolytic system could be relevant in elucidating the pathophysiology of atherothrombosis. Previous human studies have measured circulating t-PA concentration after systemic stimuli, such as mental stress 11, exercise or intravenous drug infusions 12, 13, which do not necessarily reflect the local amount of t-PA released by endothelial cells 14. This is a crucial issue, since effective fibrinolysis must be sustained by local t-PA release which cannot be measured by systemic assessment 15, 16. The relationship between the adrenergic system and endothelial fibrinolysis is currently not fully characterized and local t-PA release following adrenergic stimulation represents a major issue requiring further investigation 14.
In the present study we hypothesized that the (adrenergic-mediated) t-PA release in response to adrenergic stimulus is mediated by NO pathway. Therefore, we have assessed t-PA release in the
forearm microcirculation of normotensive subjects and in patients with essential hypertension under specific adrenergic stimuli. In particular, we investigated which adrenergic receptor subtype (i.e. α- or β- adrenoceptors) is involved and the contribution of NO in modulating t-PA release in healthy conditions.
Material and Methods Subjects
The study population included 58 healthy male volunteers and 44 patients with essential hypertension. Patients were recruited among newly diagnosed cases in our outpatient clinic. Inclusion criteria were age between 30 and 65 years, and sitting blood pressure values (after 10 minutes of rest) between 140-90 and 160-99 mmHg, confirmed on two separate occasions within a period of one month according to European guidelines 17. Exclusion criteria were dyslipidemia, diabetes, smoking, body mass index (BMI)> 30 Kg/m2, renal or liver impairment, and established cardiovascular diseases other than essential hypertension. Secondary forms of hypertension were excluded by routine diagnostic procedures. Patients either were never treated for hypertension or they did not receive any medications for ≥1 month prior to enrolment in the study.
The study protocol was approved by the local ethics committee and performed according to the guidelines of our institution. All patients were aware of the nature, purpose and potential risks of the study and gave their written consent to it.
Experimental Procedure
The perfused-forearm model used in this study has been previously described in detail 5. Briefly, intravenous catheters were placed into the deep antecubital vein of each arm (experimental and contralateral forearm as control) and the brachial artery of the non-dominant arm cannulated for drug infusion at systemically ineffective rates, and for the intra-arterial blood pressure and heart rate monitoring. Forearm blood flow (FBF) was measured in both forearms by strain gauge venous plethysmography (EC-6, D.E. Hokanson Inc., Bellevue, WA, USA). Prior to FBF measurement, simultaneous arterial and venous samples for t-PA and plasminogen activator inhibitor-1 (PAI-1) antigen concentration measurements were collected before and after each infusion of study drug. Each dose was infused for 15 minutes. Infusions were interrupted during arterial sampling.
Plasma concentrations of t-PA and PAI-1 antigen were determined by enzyme-linked immunosorbent assay (ELISA, Tecnoclone GmbH, Vien, Austria). All samples were assayed in duplicate on the same test plate. Intra-assay and inter-assay coefficients of variation were <10%. Coefficient of variation for FBF repeated measurements in our laboratory is <10% 5.
Experimental Design
Relationship between adrenergic stimuli and acute t-PA and PAI-1 release in normotensive subjects and essential hypertensive patients
t-PA and PAI-1 release in response to intra-arterial administration of 2 doses of epinephrine (0.1 and 0.3 µ g/100mL/min) were measured in 18 NT and 16HT subjects. Intra-arterial ouabain (0.3 and 0.7 µg/100mL/min), a vasoconstrictor compound which acts via direct hyperpolarization of vascular smooth cells 18, was used as control. Epinephrine and ouabain were infused in random order and each dose for a duration of 15 minutes.
NO contribution to adrenergic-mediated t-PA release in normotensive subjects and essential hypertensive patients.
To evaluate the contribution of NO to adrenergic-mediated t-PA release, another group of 12 normotensive subjects and 12 hypertensive patients was subjected to infusion of epinephrine (0.1 µg/100mL/min) both in absence and presence of the nitric oxide synthase (NOS) inhibitor NG -monomethyl-L-arginine (L-NMMA; 100 µg/100mL/ min). Since L-NMMA reduces blood flow, the effect of epinephrine was evaluated in presence of the NO-clamp, which allows assessment of endothelial agonists in the presence of NOS blockade without change in basal blood flow, thus avoiding any perturbation that could alter net-forearm t-PA release. Briefly, following 10 minutes of L-NMMA infusion, sodium nitroprusside was co-infused at an adjusted dose (0.3 and 0.4 µg/100mL/min) to restore the FBF reduced by L-NMMA infusion to baseline, as previously described in detail 19. Since sodium nitroprusside is an exogenous NO donor acting directly on vascular smooth muscle cells 20, it does not stimulates t-PA release from endothelial cells in vivo 4,
21
Receptor characterization of adrenergic-mediated t-PA release in normotensive subjects. To characterize the receptor subtype involved in the adrenergic-induced t-PA release, in another group of 12 normotensive subjects epinephrine infusion (0.1 µ g/100mL/min) either in the presence of phentolamine (8 µg/100mL/min) or of propanolol (10 µg/100mL/min), an α− and β-receptor blocker respectively. Each drug was pre-infused for 10 minutes in random order. The effectiveness of these infusion rates was validated in previous studies 22, 23.
Effect of selective ββββ-receptor stimulation and NO contribution on t-PA release in normotensive subjects and essential hypertensive patients
To confirm the role of β-receptors in endothelial t-PA release, in another group of 16 normotensive subjects and 16 hypertensive patients isoproterenol (0.03 µg/100mL/ min), a β-selective agonist, was infused in the absence and presence of L-NMMA, using the NO-clamp technique.
Isoproterenol was infused for 15 minutes. To exclude the possible confounding effect of flow-increase, a dose response curve to intra-arterial sodium nitroprusside (0.5-1.0 µg/100mL/min), a direct smooth muscle cell relaxant, was performed.
Data analysis
Forearm plasma flow was determined by FBF and hematocrit. Net forearm release or uptake rates for t-PA and PAI-1 were calculated by the following formula: net release=(Cv-Ca) x [FBF x (101-hematocrit)/100], where Cv and Ca are the venous and arterial concentrations, respectively. A positive value indicates a net release, while a negative value indicates an up-take of the fibrinolytic component. The total amount of t-PA released was calculated using the area under curve formula for repeated measures 24. Study population characteristics, basal venous and arterial concentrations and t-PA balance at baseline were compared using the Student t-test or non-parametric test, depending on the results of test for normality. Responses to epinephrine, isoproterenol and ouabain were analyzed by one-way (infusion) and two-way (group and infusion) analysis of variance (ANOVA) for repeated measures, applying the Bonferroni post-hoc analysis. Results were expressed as mean±SD. Findings were considered statistically significant at p<0.05. Computations for the power calculation and for the statistical methods were performed using SPSS 15.0® statistics software. Thepresent study was designed to have the 80% power at the 5% levelto detect a 30% modification in fibrinolytic components release after drug infusion.
Drugs
Epinephrine (Biologici Italia), isoproterenol (Abbott), L-NMMA (Clinalfa AG), sodium nitroprusside (Malesci SpA), ouabain (Clinalfa AG), propanolol (Astra Zeneca), phentolamine (Novartis) were obtained from commercially available sources and diluted to the desired concentration by addition of normal saline. Sodium nitroprusside was dissolved in 5% glucose solution and protected from light by aluminum foil.
Results
Clinical characteristics of the study population are shown in Table 1. Groups were similar in characteristics except for systolic and diastolic blood pressure values which were significantly higher in the hypertensive patients.
No changes in intra-arterial blood pressure or heart rate were observed during intra-brachial drug infusion (data not shown).
Table 1. Clinical Characteristics of Study Group
Normotensive Subjects (n=58) Hypertensive Patients (n=44) Age (years) 47±9 48±11 Gender (male/female) 60/0 32/0 Smokers (yes/no) No No SBP (mmHg) 131±7 153±8* DBP (mmHg) 83±6 93±5* BMI (Kg/m2) 25±2 25±3 Total cholesterol (mg/dl) 197±28 210±27 HDL cholesterol (mg/dl) 50±10 51±11 LDL cholesterol (mg/dl) 124±26 118±30 Plasma glucose (mg/dl) 91±8 90±9 Plasma homocysteine (µmol/l) 9.8±2.7 9.8±4.0 Plasma folate (ng/ml) 8.1±4.6 7.9±3.8 Plasma vitamin B12 (pg/ml) 396.7±50.1 299.1±60.4
CRP (mg/dl) 3.1±0.9 2.8±0.8
Data are presented as mean value ± SD or number of subjects. SBP: systolic blood pressure; DBP: diastolic
blood pressure; BMI: body mass index; HDL: high density lipoprotein; LDL: low density lipoprotein; CRP: C Reactive Protein. *p<0.01 vs. healthy subjects.
Adrenergic stimuli and acute t-PA and PAI-1 release in normotensive subjects and essential hypertensive patients
Normotensive subjects showed a dose-dependent vasoconstriction in response to epinephrine that was significantly lower than that observed in the hypertensive patients (Figure 1A). In contrast, vasoconstriction to ouabain was similar in the two groups (Figure 1B).
Figure 1: Vasoconstriction and t-PA release to Epinephrine and Ouabain in normotensive subjects and essential hypertensive patients.
Epinephrine (A) and Ouabain-induced (B) a reduction in FBF in controls (solid circles) and essential hypertensive patients (open circles). Effect of Epinephrine (C) and Ouabain (D) on t-PA release in normotensive subjects (solid circles) and essential hypertensive patients (open circles). Data are shown as mean±SD. *p<0.05; † p<0.01 vs baseline.
At baseline, arterial and venous concentrations of t-PA were significantly (p<0.05) lower in hypertensive patients than in the normotensive subjects (Arterial: 0.5±0.1 vs. 3.7±0.3 ng/mL; Venous: 0.5±0.1 vs. 3.8±0.2 ng/mL). In the normotensive group, net t-PA release significantly
increased during epinephrine infusion, a maximal effect being observed with the lower dose of the agonist (Figure 1C). By contrast, in hypertensive patients t-PA release was reduced, reaching a peak effect with the higher dose of epinephrine (Figure 1C). Accordingly, the total amount of t-PA released was significantly lower in hypertensive patients as compared to controls (1.2±0.4 vs. 4.3±1.7 ng/100mL of forearm tissue; p<0.01). No significant difference in net t-PA release was recorded in either group after ouabain infusion (Figure 1D). At baseline arterial and venous concentrations of PAI-1 antigen were similar in normotensive subjects and hypertensive patients (Arterial: 29.8±4.8 vs. 27.6±5.2 ng/mL; Venous: 30.0±5.0 vs. 27.7±5.7 ng/mL). No significant difference in net PAI-1 release after epinephrine infusion was noted in either the normotensive subjects (from 0.5±0.2 to 0.7±0.3 ng/100mL/min) or the hypertensive patients (from 0.7±0.3 to 0.6±0.2 ng/100mL/min). Similarly, no significant changes in t-PA release were observed following ouabain infusions in both normotensive (from 0.8±0.3 to 0.9±0.4 ng/100mL/min) and hypertensive (from 0.8±0.3 to 1.0±0.5 ng/100mL/min) groups.
NO contribution to adrenergic-mediated t-PA release in normotensive subjects and essential hypertensive patients.
In normotensive subjects, a lower vasoconstriction to intra-brachial epinephrine as compared to hypertensive patients was observed (Figure 2A, 2B). Vasoconstriction in response to epinephrine in normotensive subjects was increased by the NO synthase blockade with L-NMMA, which significantly reduced basal FBF (Figure 2A). In hypertensive patients L-NMMA infusion, which reduced basal FBF insignificantly, did not modify epinephrine-induced vasoconstriction (Figure 2B).
In normotensive subjects the lower dose of epinephrine induced a significant increase in t-PA release (Figure 2C). Moreover, the NO synthase blockade with L-NMMA decreased basal t-PA release (from 0.2±0.1 to -0.1±0.0 ng/min/100mL of forearm tissue; p<0.05) and significantly blunted epinephrine-induced t-PA release (Figure 2C). In contrast, epinephrine-induced t-PA release in hypertensive patients was significantly lower as compared to normotensive subjects
(p<0.01) and L-NMMA co-infusion failed to affect either constitutive (from 0.1±0.0 to 0.1±0.1 ng/min/100mL of forearm tissue) or stimulated t-PA release (Figure 2D).
Figure 2: Vasoconstriction and release of t-PA following intra-brachial Epinephrine either in the absence or in the presence of NOS inhibition in normotensive subjects and essential hypertensive patients.
Epinephrine (Epi)-induced decrease in FBF during saline (open circles) or L-NMMA (solid circles) in normotensive subjects (A) and hypertensive patients (B). t-PA release before (empty bars) and after (solid bars) Epi infusion during saline or L-NMMA in normotensive subjects (C) and hypertensive patients (D). * p<0.05 vs saline infusion; † p<0.01 vs baseline.
Receptor characterization of adrenergic-mediated t-PA release in normotensive subjects. In the group of normotensive subjects, a non-significant vasoconstriction in response to the lower dose of epinephrine was observed (Figure 3A). Phentolamine pre-infusion significantly increased FBF (Figure 3A). In the presence of α-blockade by phentolamine, epinephrine induced a significant vasodilation (Figure 3A). By contrast, in the presence of β-blockade with propanolol, which failed
to significantly affect FBF when pre-infused, epinephrine induced a significant vasoconstrictor effect (Figure 3B).
Figure 3: Effect of Epinephrine on FBF and t-PA balance either in the absence or in the presence of αααα-adrenergic (Phentolamine) and ββββ-adrenergic (Propanolol) blockade in normotensive subjects.
Effect of Phentolamine (solid circles) or saline (open circles) on FBF (A) and t-PA release (C) following Epinephrine infusion. Effect of Propanolol (solid circles) or saline (open circles) on FBF (B) and t-PA release (D) during Epinephrine infusion. In figure B the range of values of FBF is different to allow clearer
understanding of results. Data are expressed as mean value ± SD. *p<0.01;† p<0.01 vs baseline.
Intra-brachial infusion of the lower dose of epinephrine significantly increased net t-PA release, a response not affected by phentolamine co-infusion (Figure 3C). In contrast, epinephrine-induced t-PA release was abolished in the presence of propanolol (Figure 3D).
Effect of selective ββββ-receptor stimulation and NO contribution on t-PA release in normotensive subjects and essential hypertensive patients
Normotensive subjects exhibited vasodilation to isoproterenol similar to that observed in hypertensive patients (Figure 4A, 4B). In healthy subjects, vasodilation to isoproterenol was significantly reduced by L-NMMA infusion (Figure 4A), while in hypertensive patients L-NMMA infusion only caused not significant reduction in vasodilation to isoproterenol (Figure 4B). Vascular response to sodium nitroprusside was found to be similar in the two groups (normotensive subjects: FBF from 3.5±0.2 to 18.2±0.9 mL/ min/100 mL of forearm tissue; hypertensive patients: FBF from 3.5±0.3 to 18.4±0.6 mL/ min/100 mL of forearm tissue; p=N.S.).
Figure 4: Effect of Isoproterenol on FBF and t-PA release either in the absence or in the presence of NOS inhibition.
Vasodilation to Isoproterenol in normotensive subjects (A) and in essential hypertensive patients (B) during saline (open circles) or L-NMMA (solid circles). Release of t-PA before (empty bars) and after (solid bars) the infusion of isoproterenol during saline or L-NMMA in normotensive subjects (C) and hypertensive patients (D). * p<0.05 vs Isoproterenol plus saline; † p<0.05 vs baseline.
In normotensive subjects isoproterenol induced a significant increase in net t-PA release versus baseline (Figure 4C). L-NMMA infusion, which reduced constitutive t-PA release (from 0.2±0.1 to -0.2±0.0 ng/min/100mL of forearm tissue; p<0.05), significantly blunted isoproterenol-induced t-PA release (Figure 4C). By contrast, in hypertensive patients isoproterenol-induced t-t-PA release was significantly reduced as compared to normotensive subjects and L-NMMA infusion failed to significantly affect both constitutive (0.2±0.0 to 0.1±0.0 ng/min/100mL of forearm tissue) and stimulated t-PA release (Figure 4D). No significant increase in t-PA release was observed during sodium nitroprusside infusion (from 0.2±0.1 to 0.3±0.1 ng/min/100mL of forearm tissue).
In both groups contra-lateral FBF and venous-arterial concentrations of t-PA remained unchanged throughout each protocol (data not shown)
Discussion
The main finding of the present study is that in physiological conditions adrenergic stimulation induces t-PA release via the activation of β-adrenergic mediated NO-pathway. Contrastingly, in hypertensive patients β -adrenergic activation of the t-PA/NO pathway is impaired, an alteration potentially involved in the hypofibrinolytic state characterizing this clinical condition 4, 6.
In normotensive subjects, epinephrine infusion caused a dose-dependent vasoconstriction and a net release of t-PA but not of its inhibitor PAI-1. This effect of epinephrine is specific since the infusion of ouabain in the same experimental conditions, despite inducing a similar vasoconstriction, failed to increase the release of t-PA and PAI-1. Taken together, these results indicate that adrenergic-mediated t-PA release in healthy subjects is a specific and flow-independent endothelial property.
In normotensive subjects vasoconstriction to epinephrine was significantly lower as compared to hypertensive patients, in line with the previous findings of an imbalance of adrenoreceptors in essential hypertension 25, 26. Indeed, although epinephrine is a potent non-selective adrenergic agonist with a higher affinity for β-adrenoreceptors, it induces vasoconstriction when infused locally 27, due to the higher density of α-receptors in peripheral arterioles 27. The hemostatic effect of epinephrine also depends on platelets aggregation and the increase of several clotting factors 9. The present results show that epinephrine also participates in the modulation of local fibrinolysis via the activation of t-PA release in the microcirculation of healthy subjects.
Epinephrine-induced t-PA release was blunted in hypertensive patients, a finding which strongly reinforces the concept of impaired t-PA release in essential hypertension, as previously reported with different stimuli, such as desmopressin 6, substance P 28, and acetylcholine 4.
This is a crucial issue since only t-PA acutely released and incorporated into the growing thrombus effectively activates plasminogen to plasmin 29, being protected from its main circulating inhibitor PAI-1 6, 28.
The major novel finding of the present study is the demonstration that epinephrine-induced t-PA release is mediated by the activation of NO-pathway. The inhibition of NO with L-NMMA significantly reduced basal and epinephrine-induced t-PA release in normotensive subjects, thereby confirming a positive modulating effect of NO on both tonic and stimulated t-PA release in healthy conditions 4, 30.
NO pathway also plays a crucial role in modulating the vascular effects of epinephrine in healthy conditions. In normotensive subjects, L-NMMA, a NO-synthase inhibitor, decreased basal flow and accentuated the vasoconstriction in response to epinephrine. The latter effect is probably related to the NO activation induced by the β-adrenoceptor component of the epinephrine response 31
.
By contrast, no significant modification in either tonic or epinephrine-induced t-PA release was observed during L-NMMA co-infusion in hypertensive patients. Moreover, in hypertensive patients, basal vasoconstriction to L-NMMA was reduced and the inhibition of NO synthase did not have any effect on the vascular response to epinephrine. Taken together, these findings confirm the presence of impaired NO-availability in essential hypertension18, 32-34 which accounts for the reduced endothelial t-PA release capacity in this clinical condition, as already documented with different stimuli 4, 6
To elucidate the receptor subtype involved in the modulation of fibrinolysis by adrenergic stimuli we assessed the effect of epinephrine on t-PA release in the presence of either phentolamine, an α-antagonist, or propanolol a β- α-antagonist, in a group of normotensive subjects. The results show that α-blockade, which induced significant vasodilation to epinephrine, failed to affect t-PA release. In contrast, the blockade of β-adrenoceptors with propanolol, which potentiated vasoconstriction to epinephrine, significantly blunted t-PA release following epinephrine infusion. These findings support the possibility that epinephrine-induced t-PA release across the forearm microcirculation is mediated by β-adrenoreceptors 21
. A final demonstration is provided by the study with the selective β-adrenergic agonist isoproterenol. In normotensive subjects isoproterenol caused vasodilation and
a parallel t-PA release. Note that both effects were blunted by L-NMMA. In hypertensive patients, while vasodilation to isoproterenol was similar to that of controls, but unaffected by L-NMMA co-infusion, β-adrenoceptor-induced t-PA release was still present, but significantly reduced.
The results of the present study are in contrast with those of a previous study in which a lacking effect of NO synthase inhibition on bradykinin-induced t-PA release was reported 35. Indeed, in this study L-NMMA infusion failed to significantly affect vasodilation to bradykinin in healthy subjects. Therefore an incomplete endothelial NOS inhibition, which may account for the lacking effect on t-PA release, cannot be excluded.
The discrepancy between the vascular and fibrinolytic effects of isoproterenol observed in hypertensive patients requires further explanation. It is conceivable that while mechanisms compensating for the reduced NO availability, e.g. hyperpolarization 36, may account for the similar degree of vasodilation, a preserved NO pathway is crucial to maintain adequate t-PA release. Recent experimental findings support the hypothesis that the reduced, but still present, t-PA release observed in our hypertensive patients might be sustained by a non-compensatory endothelial-derived hyperpolarizing factor (EDHF)-dependent pathway37. Muldowney et al37, reported that the EDHF 5,6 epoxyeicosatrienoic acid (EET) induces the exocitosis of t-PA from storage granules via calcium- and cAMP-dependent pathways, in line with previous experimental findings 38,
39
.According to our findings EET-mediated t-PA release was not affected by the ouabain-dependent part of the EDHF pathway 37.
The major limitation of the present in vivo clinical study concern the assessment of dynamic endothelial t-PA release in the forearm. Indeed, although this vascular district is less susceptible to atherothrombosis, it is less invasive and is considered a valid surrogate of the coronary circulation1. Moreover, the dynamic assessment of t-PA release across the entire vascular forearm bed does not necessarily reflect the local capacity for t-PA release at the site of developing thrombus.
In the present study we didn’t measure t-PA and PAI-1 activity or fibrin degradation products to estimate the activity of the fibrinolytic system. However, several findings 14, 40indicate that in the
presence of no demonstrable release of PAI-1 antigen across the forearm, t-PA antigen concentration increases in parallel with t-PA activity1.
Perspectives
The results of the present study demonstrate that adrenergic-induced t-PA release is mediated by β-adrenoreceptors, via a mechanism which involves the NO pathway. In essential hypertension, the reduced NO availability impairs adrenergic-mediated t-PA release, possibly playing a crucial role in determining the hypofibrinolytic state characterizing this clinical condition. The reduced dynamic endothelial t-PA release could be part of a generalized endothelial dysfunction which characterizes essential hypertensive patients and contributes to the risk of atherothrombotic events 7, 41. Thus, understanding the mechanisms underlying stimulated t-PA release could lead to specific therapeutic strategies to improve endothelial fibrinolytic function and possibly reduce cardiovascular risk in essential hypertension.
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