Inhibition of MMP-9 by a selective gelatinase inhibitor protects neurovasculature from embolic focal cerebral ischemia

Stroke is a devastating cerebrovascular disease with an estimated 800,000 cases each year and mortality data from 2006 indicated that stroke accounted for approximately 1 out of every 18 deaths in the U.S. [1]. Acute ischemic stroke is the most common form of stroke and is majorly caused by thrombosis or embolism in the cerebral arteries. Blockage of blood flow (ischemia) results in oxygen deprivation, glucose deficiency in the affected region, and infarction in the brain. Cerebral ischemia initiates cascades of pathological events, including vasogenic edema, disruption of the blood–brain barrier (BBB), intracerebral hemorrhage (ICH), astroglial activation, and neuronal death [2, 3]. Although the molecular mechanisms underlying these outcomes have not yet been fully addressed, there is considerable evidence supporting the important role of matrix metalloproteinases (MMPs) in mediating ischemia-induced neurovasculature impairment. Increased expression and activation of MMPs, and gelatinases in particular, are likely to play critical roles in excitotoxicity-induced disruption of cell-matrix homeostasis and neuronal cell death [4, 5]. MMPs also play a role in thrombolysis-mediated BBB leakage and edema, resulting in ICH. In clinical settings, MMP-mediated neurotoxicity and hemorrhagic transformation during the acute phase of cerebral ischemia are frequently complicated upon treatment with tissue plasminogen activator (tPA) [6]. In addition, gelatinases function in neurovascular remodeling and microvascular recanalization [2, 7, 8].

MMPs comprise a family of zinc-containing endoproteases that degrade components of the extracellular matrix (ECM) and tight-junctions in the brain. MMPs are also needed for modulating interactions between cells during development and tissue remodeling [5, 7]. Reportedly, unregulated MMP activity contributes to neurological disorders, including stroke and other inflammatory responses. MMP-9 activity, in particular, is significantly elevated in humans after stroke [9‐11]. High plasma MMP-9 concentrations in the acute phase of a cerebral infarct are considered as independent predictors of hemorrhagic transformation in all stroke subtypes [12]. We and others have shown that aberrant MMP-9 proteolytic activity is associated with an increase in BBB permeability, which results in brain edema and hemorrhage, and contributes directly to neuronal injury, apoptosis and brain damage after acute cerebral ischemia [13‐16]. Moreover, cerebral infarct size is reduced in mice deficient in MMP-9, or after treatment with MMP inhibitors [17].

Among the different cell types in the neurovasculature, pericytes are known to play a role in safeguarding the brain against injury, and control key neurovascular functions and neuronal phenotype in the adult and aging brain [18‐20]. Loss of pericytes, associated with accumulation of neurotoxic and vasculotoxic macromolecules in the brain, could reduce brain microcirculation, diminish brain capillary perfusion, and disrupt the BBB, leading to vascular damage. Analysis of pericyte-deficient mice (e.g., with Pdgfb mutants) reveals that pericyte deficiency leads to increased permeability of the BBB, which probably occurs by facilitating endothelial transcytosis [18]. Although MMP-9 inhibition or knockout can attenuate proteolysis of BBB [13, 17], more recent studies suggest its possible role in neurovascular regeneration, especially in the delayed phase of cerebral ischemia [21]. Thus, successful anti-stroke therapies require selective inhibition of aberrant activity without altering the physiological function of MMPs, such as their roles in axonal growth, synaptic plasticity, and vascularization in the central nervous system [22‐26].

Pathologically activated therapy (PAT) is a novel neuroprotective strategy based on the principle that drugs are activated during the pathological state of the target, while sparing normal tissue function [27]. One PAT strategy is the target-induced activation of MMP inhibitors. Specifically, (4-phenoxyphenylsulfonyl)methylthiirane (referred to as SB-3CT) is the first mechanism-based MMP inhibitor selective for gelatinases [28]. Active gelatinases bind to SB-3CT and catalyze the opening of the thiirane ring in the molecule. The resultant species generated within the active site of the enzyme affords tight binding between the inhibitor and the enzyme. That is to say, the activity of the enzyme generates the potent inhibitory species within the active site. This mechanism-based inhibition confers a PAT strategy to abrogate the deleterious activity of gelatinases, making this class of inhibitors potentially suitable for more prolonged treatment, because of the selectivity that it affords. We first reported that SB-3CT prevents proteolysis of the ECM basement membrane laminin and rescues neurons from focal cerebral ischemia [29]. Most importantly, significant therapeutic benefit can be observed for up to 6 hours after initial injury. Recent studies show that SB-3CT abolishes oxygen-glucose deprivation-induced reduction of the tight junction protein occludin as well as decreases Evans blue extravasation and apoptotic cell death after spinal cord injury, subarachnoid hemorrhage [30‐32], and cardiopulmonary resuscitation-induced BBB disruption [33]. These results are consistent with the notion that inhibiting pathological gelatinolytic activity can maintain the brain neurovascular integrity and protect it from ischemia or other neurovascular insults.

In the present study, we use a fibrin-rich blood clot to induce middle cerebral artery (MCA) occlusion in mice. This embolus-induced focal cerebral ischemia model is more physiologically relevant to thromboembolic stroke in humans. We demonstrate the protective effects of SB-3CT on the neurovascular unit, and document that SB-3CT can manifest inhibitory activity even in the presence of an embolus blocking the MCA and its branches. We disclose that repeated-dose administration of SB-3CT over seven days in the embolus-induced “permanent” focal cerebral ischemia model also protects the brain from neurovascular impairment with no apparent cytotoxicity.

The rodent embolus model has distinct advantages in testing anti-thrombolytic and neuroprotectant agents for cerebral ischemia and is recommended by the Stroke Therapy Academic Industry Roundtable (STAIR) [40]. In our embolic model, the MCA supporting the brain region blocked by the embolus shows insufficient perfusion compared to the contralateral hemisphere. This model mimics the pathological conditions of an autologous clot-induced MCA occlusion. Initially, one of our goals was to test the reliability of the embolus model in comparison with the widely used filament model [29, 34‐36]. We also aimed to extend previous studies with the selective gelatinase inhibitor SB-3CT in the embolus model in mice, and further evaluate the suitability of the selective gelatinase inhibitor after two doses on day 1 or repeated-dose administration for 7 days. Results from this study indicate that the embolus model can be used to test thrombolytic or neuroprotective agents.

In the developing and adult brains, MMP proteolytic activity is known to play roles in modulating diverse physiological functions in the central nervous system, such as ECM remodeling, myelin turnover, axon outgrowth, angiogenesis, long-term potentiation, and synaptic plasticity in memory and spatial learning [5, 7, 25, 26]. However, these enzymes also play important roles in mediating pathological processes, especially after stroke or traumatic injuries [5, 7]. Elevated MMP-9 activity is correlated with BBB disruption, brain edema, hemorrhagic transformation, and neuronal apoptosis [2, 4, 12, 13]. There is evidence indicating that specific inhibition of MMP activity during stroke onset or immediately following brain injury likely improves neurological outcomes. Lo and colleagues reported that minocycline, a broad-spectrum MMP inhibitor, could reduce neuronal cell death after ischemia and extend the thrombolytic time window [41]. Moreover, MMP-9 knockout can stabilize the BBB by preventing degradation of the tight-junction protein ZO-1 [17]. During ischemic stroke, MMP-9 inhibition also mitigates BBB disruption and reperfusion injury experimentally induced by the thrombolytic agent tPA, a therapeutic agent that can itself increase the risk of bleeding [6, 14]. A recent clinical trial indicated that minocycline can lower plasma MMP-9 levels, even at 72 hours after stroke, and improve neurological outcomes in acute ischemic stroke patients treated with tPA [11]. In the present study, using the embolus-induced ischemia model, treatment with SB-3CT revealed an unexpected finding as compared to the control group. During ischemia, expression of inactive proMMP-9 increased significantly, a small portion of which was processed to active MMP-9, which manifests the pathological consequences of ischemia. When ischemic animals were treated with SB-3CT, we observed a general down regulation of proMMP-9 expression. This could be the consequence of a feedback process whereby active MMP-9 interacting with SB-3CT via its thiolate acts on an unknown downstream target to modulate the MMP-9 expression by transcription factors such as NF-kappa B and AP-1 [5].

MMPs have been implicated in beneficial roles in recovery following stroke. Shortly after ischemic insult, a cascade of events is initiated in attempt to repair the damage, a process similar to that found in wound healing [5, 7]. Following injury, blood vessels are dependent on the plasminogen-activator system and on MMPs for their regeneration [42]. For example, Lo and colleagues showed that delayed inhibition of MMPs by broad-spectrum inhibitors is detrimental during the ischemic recovery stage seven days after cerebral ischemia, hindering brain repair in mice, and attenuating neurovascular regeneration in the penumbra [21]. Perhaps, a suitable balanced level of MMP-9 activity is important for vascular remodeling after spinal cord injury [22]. Therefore, extended inhibition of MMPs, especially through the use of broad-spectrum inhibitors, might prove deleterious [2, 5].

In the present study, we observed that the mNSS scores of both motor and sensory functions after two-doses of SB-3CT on day 1 are significantly lower, compared to the vehicle-treated group (Figure 3C). Moreover, repeated-dose administration of SB-3CT for seven days appears to offer beneficial effects after embolic ischemic injury, as demonstrated by its ability to attenuate degradation of neuronal laminin, protect neurons from ischemia shown on cresyl violet stained brain sections and ameliorate neurobehavioral outcomes (Figure 7). These data are in line with the decrease in infarct volume and the ability of SB-3CT to protect the brain from ischemic damage. Measurement of the sensory functions showed significant improvement at the end of the seven-day SB-3CT treatment, but no further change in motor function (Fig. 7C). However, we observed that mice in the vehicle-treated group had significantly improved motor functions by seven days after ischemia, as indicated by a diminution in their motor function mNSS from a score of 5 at day 1 to a score of 2 at day 7. It has been shown that rodents are able to recover gradually in motor function after ischemic insult despite persistence of the infarct area [43, 44]. These observations for the improvement in motor function might be due to compensatory effects of the endogenous neurogenic response and/or subsequent retrieval of interhemispheric functional connectivity within the sensorimotor system in rodents. Nevertheless, repeated-dose administration of SB-3CT for seven days offered beneficial effects, such as protecting neurons and ameliorating neurobehavioral functions in stroke outcomes relative to vehicle-treated mice. On the other hand, delayed treatment with the broad-spectrum MMP inhibitors FN-439 (day 3 or 7) and BB-94 (day 7) significantly worsened infarct volumes [21]. Since these broad-spectrum MMP inhibitors are peptidomimetics and they were administered intraventricularly [21], they are unlikely to cross the BBB. In contrast, SB-3CT crosses the BBB in healthy animals (unpublished data). These data suggest the benefit of selective gelatinase inhibitors over broad-spectrum MMP inhibitors [27].

Using the embolic model for “permanent” MCA occlusion in mice, we documented the protective effects of SB-3CT on the neurovascular unit. Since SB-3CT is a small molecule that distributes to the brain, it enters the brain via the unaffected vasculature, and diffuses to the infarct regions to manifest its inhibitory activity. In this study, we demonstrated the protective effects of SB-3CT despite the fact that the embolus remained intact in the brain during the course of treatment. As such, this inhibitor is considered a PAT agent. The basis of the selectivity of SB-3CT is the ability of gelatinases to catalyze a requisite thiirane-ring opening of the SB-3CT structure [45]. The thiolate, formed only within the active site of gelatinase, is capable of coordinating with the active-site zinc ion, and precipitates potent “slow-binding” inhibition, a hallmark of this class of inhibitors. Inhibition inherent to this class of compounds suppresses pathological activity of the target gelatinase, which is activated during the diseased state [27]. Under this condition, the inhibitory effect of SB-3CT is limited to gelatinases. In addition to stroke, this mechanism-based, selective gelatinase inhibitor can exhibit promising efficacy on other diseases. For example, recent studies demonstrated that SB-3CT attenuates BBB damage by decreasing oxygen-glucose deprivation-induced occludin degradation in early ischemic stroke, protects against oxidative stress-mediated apoptosis after spinal cord injury, and increases proliferation of NG2 progenitor cells after spinal cord injury [30, 32, 46]. Another study showed that SB-3CT could prevent laminin degradation and ameliorate neuronal death in a rat model of subarachnoid hemorrhage [31]. Taken together, these studies along with the recent findings of SB-3CT on neurovasculature [8, 20], indicate that SB-3CT has promise in the treatment of gelatinase-mediated diseases. The inhibitor also exhibits significant efficacy in maintaining brain neurovascular integrity and protective effects on neuroinflammation against degradation of ECM components, pericyte lumen contraction, BBB disruption, neuronal apoptosis, and hemorrhagic transformation in brain and spinal cord injuries.

Brain function and neuronal viability are dictated by the adequate delivery of oxygen and glucose through cerebral microvessels. Because of their small diameter and relatively low flow velocity, microvessels are prone to occlusion by thromboembolism. Substantial evidence indicates that pathological MMP-9 activity after cerebral ischemia degrades components of the ECM and tight-junctions, thus contributing to microvessel and BBB leakage [13, 17]. We previously showed that MMP-9 activation is essential for degradation of laminin after focal cerebral ischemia [29]. Pericytes are known to play a role in modulating capillary diameter by constricting the vascular wall, a process that can obstruct capillary blood flow during ischemia in the adult brain [20]. In this study, we observed a significant decrease in laminin-positive pericytes, and an increase in pericyte lumen contraction in the ischemic cortex (Figure 5). Because oxidative and nitrosative stress might promote capillary constriction after ischemia, and S-nitrosylation contributes to MMP-9 activation after stroke [16], it is reasonable to consider pericytes as critical targets for MMP-9 proteolytic activity after cerebral ischemia. The ability of SB-3CT to protect laminin-positive pericytes, and inhibit lumen contraction after embolic MCA occlusion is critical for its efficacy in restoring microvascular patency and protecting against neurovascular impairment. Moreover, the ability of SB-3CT to attenuate gelatinase-mediated damage of endothelial cells and BBB tight-junction ZO-1 and to protect against endothelial deformation is important in its ability to decrease ICH in ischemic mice.