The endothelium plays a crucial role in the control of flow, coagulation, fibrinolysis and inflammation. To date, several clinical studies have focused on the assessment of endothelium-dependent vasomotion as a surrogate measure of endothelial function. However, endothelium-dependent vasodilation may not be representative of other important aspects of endothelial function, such as the regulation of fibrinolytic system. This is a crucial aspect to investigate since the initiation, progression and resolution of thrombus associated with eroded or unstable coronary plaque are critically dependent on the efficacy of endogenous fibrinolysis. In particular, the acute release of tissue-type plasminogen activator (t-PA), the main activator of fibrinolysis, from the endothelium strongly contributes to the defense against intravascular thrombosis.
The aim of the following studies was to investigate the role of endothelial pathways in the regulation of endothelial t-PA release in physiological condition and in patients with essential hypertension.
The first aim was to determine the possible regulatory role of nitric oxide (NO)-synthase pathway in modulating endothelial t-PA release in healthy conditions and in a clinical condition characterized by impaired NO availability and increased cardiovascular risk, such as essential hypertension. In healthy volunteers and essential hypertensive patients, we studied local t-PA release and forearm blood flow changes (strain-gauge plethysmography) induced by intrabrachial administration of acetylcholine (0.45-1.5 µg/100mL/min), and of sodium nitroprusside (0.5-1.0 µg/100 mL/min), an endothelium-dependent and –independent agonist, respectively. Acetylcholine was also repeated in the presence of intra-arterial infusion of the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) (100 µg/100mL/ min). In normotensive subjects, vasodilation to acetylcholine was blunted by L-NMMA. In these subjects, acetylcholine infusion induced a significant, dose-dependent increase in net forearm t-PA release. The inhibition of NO synthase with L-NMMA significantly reduced basal as well as acetylcholine-induced t-PA release. In essential hypertensive patients, vasodilation to acetylcholine was reduced as compared to controls and resistant to L-NMMA. In contrast to what observed in healthy controls, in hypertensive patients acetylcholine had no effect on t-PA release and L-NMMA failed to affect either tonic or agonist-induced t-PA release.
In conclusion, both tonic and agonist-induced release of NO are directly involved on t-PA release by endothelial cells. Essential hypertension, characterized by a reduction in both tonic and stimulated NO availability, is also associated with impaired capacity of t-PA release. Taken together these findings suggest major role of impaired nitric oxide availability in worsening not only endothelium-dependent vasodilation but also t-PA release from endothelial cells.
The second aim of our studies was to determine the relationship between adrenergic stimuli and NO in modulating t-PA release from endothelial cells in the forearm microcirculation of healthy subjects and essential hypertensive patients. Sympathetic activation is a well known stimulus for fibrinolysis and it is also involved in the determination of cardiovascular risk in essential hypertension. However, whether a cross-talk between adrenergic stimuli and NO pathway in modulating t-PA release exists is still unknown.
In this study we assessed the release of t-PA in the forearm microcirculation of healthy subjects and essential hypertensive patients under specific intra-arterial adrenergic stimuli. Adrenaline induced t-PA release was significantly higher in healthy subjects as compared to hypertensive patients. L-NMMA infusion blunted adrenaline-induced t-PA release in healthy subjects but not in hypertensive patients. In healthy controls, t-PA release by adrenaline, not affected by the beta-blocker phentolamine co-infusion, was abolished in the presence of the beta-blocker propanolol. The beta-agonist isoproterenol induced a significant increase in t-PA release, an effect blunted by L-NMMA co-infusion. In hypertensive patients, response to isoproterenol was impaired and unaffected by L-NMMA. In conclusion, adrenergic-induced t-PA release is mediated by beta-adrenoreceptors via a mechanism which involves the activation of NO pathway. The reduced NO availability characterizing essential hypertension impairs adrenergic-stimulated t-PA release, thereby reducing endothelial fibrinolytic capacity in this clinical condition.
Finally, in the third study we sough to evaluate the role of cytochrome P450 2C9 (CYP 2C9)-derived endothelium-derived hyperpolarizing factor (EDHF) in the modulation of local t-PA release in healthy subjects and in essential hypertensive patients by utilizing sulfaphenazole, a selective CYP 2C9 inhibitor. The vasoactive role of CYP 2C9-derived EDHF and its compensatory role in the presence of impaired NO availability are well documented. CYP 2C9-derived EDHF also modulates t-PA expression and release from culture endothelial cells. However, no regulatory role on endogenous fibrinolysis has been documented in humans. The release of t-PA was measured in the forearm microcirculation of 42 healthy subjects and 44 essential hypertensive patients following intra-brachial bradykinin (0.015 µg/100mL/min), either in the absence or in the presence of sulfaphenazole (0.03 µg/100 ml/min). The infusion of bradykinin was repeated in the presence of NG-monomethyl-L-arginine (L-NMMA) (100 µg/100mL/ min) and/or sulfaphenazole (0.03 µg/100 ml/min). Bradykinin-induced t-PA release was significantly (p<0.05) higher in healthy subjects as compared to hypertensive patients. Sulfaphenazole significantly (p<0.01) blunted t-PA release in both groups. In healthy subjects, L-NMMA infusion, significantly (p<0.01) reduced t-PA release. When L-NMMA was co-infused with sulfaphenazole stimulated t-PA release was further reduced (p<0.01). In hypertensive patients, while t-PA release was unaffected by L-NMMA, the co-infusion of sulfaphenazole alone or sulfaphenazole plus L-NMMA significantly (p<001) reduced bradykinin-induced t-PA release. These findings show that CYP 2C9-derived EDHF modulates bradykinin-induced t-PA release in humans. In essential hypertension, t-PA release depends exclusively on EDHF pathway, which is an ineffective compensatory mechanism in the presence of impaired NO availability
