Chapter 5
General Discussion
The initiation, propagation and resolution of a thrombus is critically dependent on the efficacy of endogenous fibrinolysis1, 2. This rely on the presence of a healthy endothelium, which plays a vital role in the control of blood flow, inflammation, coagulation and fibrinolysis as well3. The healthy endothelium dynamically release t-PA 4, which promotes the breakdown of fibrin clot via plasmin activation and, therefore, participates to the dynamic modulation of endogenous fibrinolysis5.
It is well documented that a dysfunctional endothelium is associated with atherosclerosis and its associated risk factors 3. Moreover, endothelial dysfunction independently predicts cardiovascular events 6. To date, clinical studies have mainly focused on the assessment of endothelium-dependent vasodilation as a surrogate measure of endothelial function3. Indeed, recent findings suggest that the assessment of endothelium-dependent vasodilation could not be representative of other important aspects of endothelial function, such as the regulation of endogenous fibrinolysis. Accordingly, the release of t-PA has been recently proposed as a new and distinct marker of endothelial function in humans2.
The results of the present studies show that the release of t-PA is a main property of the healthy endothelium in response to several stimuli. In addition, the results indicate that endothelium-derived NO is the main mediator of t-PA release, although other pathways, including a CYP 2C9-derived hyperpolarizing factor, may have a role. In patients with essential hypertension, a clinical condition characterized by impaired NO availability, t-PA release is significantly reduced and impaired, an alteration which might contribute to promote atherosclerosis and cardiovascular events.
In Chapter 2 we showed that muscarinic stimulation with acetylcholine induces t-PA release from endothelial cell in healthy conditions. This effect was specific and flow-independent since the
infusion of sodium nitroprusside, despite inducing a similar vasodilation to that of acetylcholine, failed to stimulate t-PA release from endothelial cells in the same experimental conditions. Of importance, both stimuli had no effect on PAI-1 release. This is a crucial point, because in the absence of PAI-1 release, the amount of t-PA antigen released by the endothelium reflects the free, unbound t-PA which is effective to activate endogenous fibrinolysis. Indeed, only the t-PA locally released, being incorporated into the growing thrombus, effectively activates plasmin, being protected by its main inhibitor PAI-12, 7. Moreover, the conversion of plasminogen to plasmin by t-PA is accelerated in the presence of fibrin 8 and at the endothelial surface where t-PA bound annexin-29. These bindings ensure an efficient and localized activation of fibrinolysis.
The results presented in Chapter 3, show that, besides cholinergic regulation, also adrenergic stimulation is a main activator of endothelial fibrinolytic capacity. Our results show that epinephrine induced a significant release of t-PA, but not of PAI-1, from the vascular endothelium in a group of normotensive subjects. This effect was a flow-independent endothelial property, as suggested by the lack of effect following ouabain infusion despite inducing a similar vasoconstriction. In this study the adrenoreceptor subtype β was identified as the mediator of the effect of epinephrine on t-PA release. Our results show that while epinephrine-induced the release of t-PA was unaffected by the α-adrenoreceptors blockade with phentolamine, β-receptor blockade with propanonol significantly reduced stimulated t-PA release. These findings were further confirmed by the significant increase of t-PA release obtained following the administration of the β -agonist isoproterenol.
Finally, the results reported in Chapter 4 show that bradykinin is a specific stimulus for the assessment of endothelial t-PA release. Bradykinin is a vasoactive peptide that promotes vasodilation10, exerts antiproliferative effects11, inhibits thrombin induced platelets activation12. Besides these effect, our results confirm that bradykinin also stimulate t-PA release from endothelial cells in healthy conditions13. The findings that sodium nitroprusside do not stimulate
t-PA release under the same experimental conditions suggest that this effect of bradykinin is specific and flow-independent.
A major finding of the present studies is the demonstration that the capacity of t-PA release from endothelial cells is mainly related to a preserved NO pathway.
Results reported in Chapter 2, demonstrate that in the presence of NO-synthase inhibition with the selective inhibitor L-NMMA, both tonic and acetylcholine-induced t-PA release were significantly reduced in a group of normotensive subjects. In these subjects a preserved endothelium-dependent vasodilation was confirmed since in the presence of NO-inhibition, acetylcholine-induced vasodilation was significantly impaired. Taken together these findings demonstrate that NO pathway, not only participates to the modulation of vascular tone but also to the tonic and stimulated capacity of t-PA release by endothelial cells.
The role of NO pathway in the modulation of endothelial t-PA release have been further confirmed by the results of the study presented in Chapter 3 using adrenergic stimuli. Epinephrine-induced t-PA release was significantly blunted in the presence of NO-synthase blockade with L-NMMA in a group of healthy subjects. A final demonstration that the adrenergic-induced t-PA release is mediated by the β-adrenergic/NO pathway activation has been obtained testing the effect of isoproterenol in the presence of NO synthase inhibition. In a group of normotensive subjects isoproterenol-induced t-PA release was significantly blunted in the presence of L-NMMA co-infusion. Of interest, vasodilation to isoproterenol was significantly reduced in the presence of NO synthase inhibition, a finding that further confirm that isoproterenol-induced t-PA is mediated by the activation of β-adrenoreceptors expressed on endothelial cells. Taken together, these data suggest that epinephrine- and possibly adrenergic-induced t-PA release is mediated via the β -adrenergic/NO pathway.
Finally, in Chapter 4 we reported that also bradykinin-induced t-PA release is mediated by the activation of NO-pathway. Our results demonstrate that bradykinin-mediated t-PA release was blunted by NO synthase inhibition. These findings further confirms the modulatory role of NO-pathway in physiological conditions.
Another important novelty of the present studies is the evidence that endothelial t-PA release is impaired in patients with essential hypertension. It is well known that endothelial dysfunction, characterized by impaired NO availability, is an hallmark of essential hypertension3. In line with previous reports14, 15, our findings confirm the presence of a reduced capacity of t-PA release in response to several endothelial stimuli in patients with essential hypertension in the absence of any other cardiovascular risk factors.
In Chapter 2, we reported that t-PA release in response to acetylcholine is impaired in essential hypertensive patients. These findings are in line with a previous report 14 showing that muscarinic stimulation with metacholine failed to significantly increase t-PA release from vascular endothelium in a group of hypertensive patients. The presence of a reduced endothelial capacity of t-PA release in hypertensive patients, as reported in Chapter 3, was confirmed using adrenergic stimuli. In particular, epinephrine-induced t-PA release was impaired in hypertensive patients. A similar alteration was confirmed by using the β-selective agonist isoproterenol in a group of hypertensive patients. Finally, as shown in Chapter 4, an impairment of t-PA release following bradykinin-infusion was confirmed in a group of hypertensive patients.
When considering that NO pathway mediates t-PA release from endothelial cells in healthy conditions, the finding of a reduced capacity of t-PA release in essential hypertension is not surprising. Indeed, the impaired NO availability characterizing essential hypertension16, could account for the reduced t-PA release in this clinical condition. Results reported in Chapter 2 show that the release of t-PA in response to acetylcholine was unaffected by the NO inhibition with
adrenergic stimuli. As reported in Chapter 3, epinephrine- as well as isoproterenol-induced t-PA release were not affected by NO-synthase inhibition, thereby suggesting that the β-adrenergic activation of NO pathway is lacking in activating t-PA release in hypertensive patients.
A final demonstration that the NO-mediated t-PA release is impaired in hypertensive patients was obtained following the administration of bradykinin. As shown in Chapter 4, bradykinin induced t-PA release was again unaffected by the simultaneous NO synthase inhibition with L-NMMA.
Another major novelty of these results concern the demonstration that EDHF-pathway contributes to bradykinin-stimulated t-PA release. It is well documented that besides NO, EDHF pathway causes endothelium-dependent vasodilation via the hyperpolarization and relaxation of vascular smooth muscle cells. Our results indicate that a CYP 2C9 dependent pathway is physiologically able to participate in the modulation of t-PA release from vascular endothelium in humans, in line with previous experimental evidence in vitro. Of particular note was the finding that in the presence of simultaneous inhibition of NO synthase and CYP 2C9, bradykinin-stimulated t-PA release was almost abolished, demonstrating that the two antagonists L-NMMA and sulfaphenazole, respectively, act on different, but complimentary pathways. Moreover, these data suggest that in essential hypertension, the impairment of NO availability leads to a reduction in fibrinolytic capacity. In these patients, the residual t-PA release depends on a CYP 2C9-related pathway, possible an EDHF identified with EETs. Therefore our results demonstrate that in physiological conditions NO- and EDHFs-pathways appears to be equally involved in the modulation of stimulated t-PA release. In essential hypertensive patients, the CYP 2C9-dependent pathway appears to operate as a “residual” mechanism responsible for the modulation of t-PA release in the presence of impaired NO availability.
Conclusions
The results of the present studies demonstrate that t-PA release form the endothelium is a distinct marker of endothelial function. In healthy conditions the stimulated t-PA release in response to muscarinic, adrenergic as well as bradykinin depends on the activation of NO pathways. Moreover, in healthy conditions, bradykinin-induced t-PA release is dependent on the activation of a EDHF-dependent pathways which contributes to the NO-EDHF-dependent t-PA release. In hypertensive patients, the NO-dependent t-PA release in response to different stimuli is reduced. In these patients the EDHF-dependent pathway is preserved and accounts for the residual t-PA release observed following bradykinin stimulation.
The reduced dynamic t-PA release from vascular endothelium could be part of a generalized endothelial dysfunction which characterize essential hypertensive patients. Moreover, this alteration may contribute to the hypofibrinolytic state characterizing this clinical condition and possibly to the increased risk of atherothrombotic events.
The investigation of mechanisms underlying endothelial fibrinolytic function in essential hypertension may provide major new insights into the pathophysiology of cardiovascular disease and to shape future therapeutic interventions. Indeed, the improvement of endothelial fibrinolytic capacity could represent a major target of antihypertensive treatment. Therefore, the development of validated and accurate, but non invasive, methodologies to assess endothelial fibrinolytic function would provide an accessible biomarker for risk of atherothrombotic events in future clinical trials.
References
1. Hrafnkelsdottir T, Gudnason T, Wall U, Jern C, Jern S. Regulation of local availability of active tissue-type plasminogen activator in vivo in man. J Thromb Haemost. 2004;2:1960-1968.
2. Oliver JJ, Webb DJ, Newby DE. Stimulated tissue plasminogen activator release as a marker of endothelial function in humans. Arterioscler Thromb Vasc Biol. 2005;25:2470-2479. 3. Brunner H, Cockcroft JR, Deanfield J, Donald A, Ferrannini E, Halcox J, Kiowski W,
Luscher TF, Mancia G, Natali A, Oliver JJ, Pessina AC, Rizzoni D, Rossi GP, Salvetti A, Spieker LE, Taddei S, Webb DJ. Endothelial function and dysfunction. Part II: Association with cardiovascular risk factors and diseases. A statement by the Working Group on Endothelins and Endothelial Factors of the European Society of Hypertension. J Hypertens. 2005;23:233-246.
4. Emeis JJ. Regulation of the acute release of tissue-type plasminogen activator from the endothelium by coagulation activation products. Ann N Y Acad Sci. 1992;667:249-258. 5. van Zonneveld AJ, Veerman H, Pannekoek H. On the interaction of the finger and the
kringle-2 domain of tissue-type plasminogen activator with fibrin. Inhibition of kringle-2 binding to fibrin by epsilon-amino caproic acid. J Biol Chem. 1986;261:14214-14218. 6. Lerman A, Zeiher AM. Endothelial function: cardiac events. Circulation.
2005;111:363-368.
7. Fox KA, Robison AK, Knabb RM, Rosamond TL, Sobel BE, Bergmann SR. Prevention of coronary thrombosis with subthrombolytic doses of tissue-type plasminogen activator.
Circulation. 1985;72:1346-1354.
8. Mosesson MW. Fibrin polymerization and its regulatory role in hemostasis. J Lab Clin Med. 1990;116:8-17.
9. Brownstein C, Falcone DJ, Jacovina A, Hajjar KA. A mediator of cell surface-specific plasmin generation. Ann N Y Acad Sci. 2001;947:143-155; discussion 155-146.
10. Vanhoutte PM. Endothelium and control of vascular function. State of the Art lecture.
Hypertension. 1989;13:658-667.
11. Linz W, Scholkens BA. A specific B2-bradykinin receptor antagonist HOE 140 abolishes the antihypertrophic effect of ramipril. Br J Pharmacol. 1992;105:771-772.
12. Hasan AA, Amenta S, Schmaier AH. Bradykinin and its metabolite, Arg-Pro-Pro-Gly-Phe, are selective inhibitors of alpha-thrombin-induced platelet activation. Circulation. 1996;94:517-528.
13. Brown NJ, Gainer JV, Stein CM, Vaughan DE. Bradykinin stimulates tissue plasminogen activator release in human vasculature. Hypertension. 1999;33:1431-1435.
14. Hrafnkelsdottir T, Wall U, Jern C, Jern S. Impaired capacity for endogenous fibrinolysis in essential hypertension. Lancet. 1998;352:1597-1598.
15. Ridderstrale W, Ulfhammer E, Jern S, Hrafnkelsdottir T. Impaired capacity for stimulated fibrinolysis in primary hypertension is restored by antihypertensive therapy. Hypertension. 2006;47:686-691.
16. Taddei S, Virdis A, Ghiadoni L, Magagna A, Salvetti A. Vitamin C improves endothelium-dependent vasodilation by restoring nitric oxide activity in essential hypertension.