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Studies into the interaction between the plasminogen activating system and the blood-brain barrier

The serine protease tissue-type plasminogen activator (t-PA) is best recognised for its role in haemostasis, where it is converts the zymogen plasminogen into plasmin – a potent and broad-spectrum protease responsible for degradation of fibrin and removal of blood clots. Next to their vascular roles, t-PA and plasmin also possess endogenous functions in the brain, both as modulators of neuronal activity and t-PA also as a regulator of the blood-brain barrier (BBB). While the action of t-PA and plasmin is associated with normal brain function, it often contributes deleteriously to a variety of brain-related pathophysiological or pathological conditions. This basic fibrinolytic function of t-PA has led to its development for treatment of ischaemic stroke. Since 1995, t-PA is the only FDA-approved therapy for this acute condition; however, t-PA can only be administered within 3-4.5h post stroke onset, as later use has reduced clinical benefits. Unfortunately, t-PA treatment is accompanied by an increased risk for development of symptomatic intracerebral haemorrhage (sICH), a life-threatening complication which impairs the safety of the treatment and prevents any attempts for its implementation in longer therapeutic windows. In recent years, it was postulated that the elevated incidence of sICH associated with t-PA-induced thrombolysis may be directly linked to the ability of t-PA to modulate the BBB and enhance its permeability. According to this rationale, blockade of direct interactions between t-PA and the BBB may reduce the haemorrhagic side-effects of t-PA treatment without affecting its capacity to restore blood-flow. My PhD project examined this concept in vitro and aimed to uncover central mechanisms linking t-PA to BBB modulation with the hope that their antagonism may ultimately improve the safety of t-PA-induced thrombolysis in stroke. During the project both human and mouse in vitro BBB models were developed to serve as authentic research tools (chapter 4). Our human BBB system was established based on immortalised human astrocytes and high-passaged human brain endothelial cells (BECs) co-cultured in contact conditions, allowing rapid accumulation of human-relevant information. To confirm these data and assist in mechanistic determination we also generated mouse primary cultures of well-differentiated astrocytes and BECs and used them for assembly of a non-contact mouse BBB model. Together, our human and mouse in vitro BBB models provided us with potent tools to reliably examine the effect of t-PA and plasminogen on the neurovascular unit. The effect of t-PA, without or with plasminogen, was then tested in the intact BBB models (chapter 5). t-PA caused a concentration-dependent increase in permeability which relied on its proteolytic activity. This effect was fully dependent on plasmin formation and was strongly potentiated in the presence of plasminogen. Strikingly, solution-applied plasmin or other plasminogen activators (PAs; reteplase, desmoteplase but most importantly urokinase (u-PA), with or without plasminogen) could not substitute for in situ t-PA-dependent plasminogen activation, while the close t-PA derivative tenecteplase was only partially effective. Of particular interest was the effect of u-PA, which in fact reduced plasminogen-dependent increase in BBB permeability, possibly by competition with endogenous t-PA over plasminogen. Inhibition of matrix metalloproteinases (MMPs) and receptor tyrosine kinases by broad-spectrum inhibitors (GM6001 and imatinib, respectively) was ineffective at blocking the effect of t-PA, while t-PA:PAI-1 complex could not open the BBB, suggesting that other known mechanisms for BBB opening by t-PA are less likely to play a role in our system. Finally, when t-PA and plasminogen were employed together, the BBB opening effect occurred, and was even potentiated, under oxygen-glucose deprivation (OGD), indicating the applicability of the primary event also during brain ischaemia. Surprisingly, the sensitivity of the BBB to high levels of t-PA alone was diminished under OGD, suggesting that the overall response of the BBB to t-PA during stroke may be of much greater complexity. Collectively, these observations implied that a unique action of t-PA, by localisation and activation of plasminogen at selective sites on the surface of the BBB, synergised with plasmin to create a t-PA-specific opening of the BBB. Lastly, to better understand the mechanism underlying this effect, we stimulated isolated cultures of BECs and astrocytes with t-PA and plasminogen and followed morphological, signalling and functional aspects associated with this stimulation (chapter 6). Both BECs and astrocytes dramatically reacted by morphology changes to t-PA-generated, cell-surface-associated plasmin. The response of BECs to t-PA/plasmin seemed rather complex. It did not involve LDLRs, but potentially included proteolysis of tight junctions and basement membrane as well as initiation of Rho/Rho-kinase (ROCK) signalling, promoting together morphological changes, loss of adhesion and barrier compromise. In astrocytes, morphological changes exerted by t-PA and plasmin fully mirrored the proteases action on the in vitro BBB, strongly suggesting that they played a functional role in BBB opening. The principal event triggered by t-PA, via plasmin, in astrocytes was activation of the Rho/ROCK signalling pathway; changes in astrocyte morphology coincided with increased formation of actin stress-fibres, enlargement of focal adhesion size and induction of phosphorylated myosin and actomyosin-dependent contractility, all being hallmarks of Rho/ROCK activation. Inhibition of ROCK with selective ROCK inhibitors, and especially with the clinically-utilised HA1077 (fasudil), blocked the astrocytic morphology changes and importantly reduced t-PA/plasminogen-mediated increases in permeability both in the human and the mouse BBB models. We conclude that during t-PA-driven thrombolysis in acute ischaemic stroke substantial levels of plasmin, selectively generated by t-PA in situ, may indeed interact with both ischaemic and healthy brain microvessels, leading to alterations in the function of BEC but especially to modulation of the astrocytic cytoskeleton by ROCK activation. The subsequent changes in astrocyte morphology increase the permeability of the BBB, potentially contributing to formation of sICH. Blockade of the Rho/ROCK pathway in astrocytes may effectively antagonise t-PA-induced BBB opening during thrombolysis, reducing the incidence of sICH and increasing the safety of the treatment without affecting its fibrinolytic capacity. Rock inhibition could thus have beneficial consequences during thrombolytic therapy in patients with ischaemic stroke.