Therapeutic efficacy of matrix metalloproteinase-12 suppression on neurological recovery after ischemic stroke: Optimal treatment timing and duration

Well then, where is the protocol for this located and how are you distributing it to all 10 million yearly stroke survivors now and into the future?

Therapeutic efficacy of matrix metalloproteinase-12 suppression on neurological recovery after ischemic stroke: Optimal treatment timing and duration

Siva Reddy Challa^1,2†, Koteswara Rao Nalamolu^1†, Casimir A. Fornal¹, Billy C. Wang^1,3,4, Ryan C. Martin¹, Elsa A. Olson¹, Ammar L. Ujjainwala¹, David M. Pinson⁵, Jeffrey D. Klopfenstein^1,6,7 and Krishna Kumar Veeravalli^1,3,6,8*

• ¹Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL, United States
• ²Department of Pharmacology, KVSR Siddhartha College of Pharmaceutical Sciences, Vijayawada, India
• ³Department of Pediatrics, University of Illinois College of Medicine at Peoria, Peoria, IL, United States
• ⁴Children’s Hospital of Illinois, OSF HealthCare Saint Francis Medical Center, Peoria, IL, United States
• ⁵Department of Health Sciences Education and Pathology, University of Illinois College of Medicine at Peoria, Peoria, IL, United States
• ⁶Department of Neurosurgery, University of Illinois College of Medicine at Peoria, Peoria, IL, United States
• ⁷OSF HealthCare Saint Francis Medical Center, Illinois Neurological Institute, Peoria, IL, United States
• ⁸Department of Neurology, University of Illinois College of Medicine at Peoria, Peoria, IL, United States

We recently showed that the post-ischemic induction of matrix metalloproteinase-12 (MMP-12) in the brain degrades tight junction proteins, increases MMP-9 and TNFα expression, and contributes to the blood-brain barrier (BBB) disruption, apoptosis, demyelination, and infarct volume development. The objectives of this study were to (1) determine the effect of MMP-12 suppression by shRNA-mediated gene silencing on neurological/functional recovery, (2) establish the optimal timing of MMP-12shRNA treatment that provides maximum therapeutic benefit, (3) compare the effectiveness of acute versus chronic MMP-12 suppression, and (4) evaluate potential sex-related differences in treatment outcomes. Young male and female Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion and reperfusion. Cohorts of rats were administered either MMP-12shRNA or scrambled shRNA sequence (control) expressing plasmids (1 mg/kg; i.v.) formulated as nanoparticles. At designated time points after reperfusion, rats from various groups were subjected to a battery of neurological tests to assess their reflex, balance, sensory, and motor functions. Suppression of MMP-12 promoted the neurological recovery of stroke-induced male and female rats, although the effect was less apparent in females. Immediate treatment after reperfusion resulted in a better recovery of sensory and motor function than delayed treatments. Chronic MMP-12 suppression neither enhanced nor diminished the therapeutic effects of acute MMP-12 suppression, indicating that a single dose of plasmid may be sufficient. We conclude that suppressing MMP-12 after an ischemic stroke is a promising therapeutic strategy for promoting the recovery of neurological function.

Introduction

Ischemic stroke remains the most prevalent form, accounting for about 87% of all strokes. The only two FDA-approved recanalization treatments for ischemic stroke are thrombolysis drug therapy with tissue-type plasminogen activator and endovascular thrombectomy. Following the recanalization treatments, blood flow is restored, ending ischemia. Nonetheless, both the residual brain damage and that resulting from recanalization (reperfusion injury) contribute to the development of severe and long-lasting secondary effects, including brain injury and neurologic functional deficits. Consequently, it is of utmost clinical importance to discover new treatments that prevent brain damage and promote the recovery of sensory and motor function. Despite years of intensive research, drug treatments are not available to alleviate progressive brain damage or enhance neurologic functional recovery after an acute ischemic stroke.

Recently, we demonstrated the upregulation of matrix metalloproteinase-12 (MMP-12) in the ischemic brain of young rodents during both the acute and chronic phases following an ischemic stroke (Chelluboina et al., 2015b; Nalamolu et al., 2018). We discovered that the upregulation of MMP-12 in the ischemic brain was approximately 8–200 times greater than any other MMP. MMP-12 expression increased gradually during the first week following transient focal cerebral ischemia and reperfusion, and remained elevated for 14 days, the longest post-reperfusion duration tested in our study (Chelluboina et al., 2015b). MMP-12 possesses autoproteolytic properties and can activate other MMPs (Chen, 2004). It can activate pro-MMP-2 and pro-MMP-3, which can then activate pro-MMP-1 and pro-MMP-9 (Matsumoto et al., 1998). MMP-2 and MMP-9 are the primary mediators of blood-brain barrier (BBB) disruption because they degrade various components of the microvascular basal lamina and BBB tight junction proteins (Rosenberg, 2002; Zhao et al., 2006; Yang et al., 2007; del Zoppo, 2009). In addition, there is evidence that MMP-9 plays a significant role in vasogenic brain edema and secondary brain damage (Montaner et al., 2001). MMP-12 activation has been reported to induce myelin basic protein degradation (Chandler et al., 1996). Demyelination is a major component of white matter injury. It significantly contributes to long-term sensorimotor and cognitive deficits. MMP-12 can induce the release of TNFα from macrophages, thereby initiating the inflammatory cascade (Churg et al., 2003). Animals lacking MMP-12 have defective TNFα release, and MMP-12 can act as a converting enzyme to convert pro-TNFα to active TNFα (Chandler et al., 1996; Churg et al., 2003). The intracellular levels of MMP-12 contribute to the secretion of IFN-α, a master cytokine capable of boosting the production of other pro-inflammatory cytokines, such as IL-1, IL-2, IL-6, TNFα, and IFN-γ, whereas the extracellular MMP-12 cleaves the IFN-α receptor 2–binding site of systemic IFN-α (Marchant et al., 2014). Progranulin (PGRN) is a growth factor and a source of various inflammatory mediators after undergoing proteolysis to granulins. MMP-12 has been identified as a proteolytic enzyme of PGRN, which is widely expressed in mammalian tissues (Bhandari et al., 1992; Daniel et al., 2000, 2003; Mackenzie et al., 2006; Matsubara et al., 2012). In conclusion, the plausible molecular interactions discussed above in relation to elevated MMP-12 levels in the brain may significantly contribute to the pathophysiology of ischemic stroke and poor stroke recovery.

Reducing MMP-12 expression in the brain via shRNA-mediated gene silencing mitigated ischemic brain damage, at least in part, by reducing the degradation of tight junction proteins, MMP-9 elevation, BBB disruption, apoptosis, myelin basic protein degradation, TNFα upregulation, and infarct volume (Chelluboina et al., 2015a,b). In light of these promising findings, we investigated whether suppressing MMP-12 in the brain after an ischemic stroke might have a beneficial effect on stroke recovery and therefore be a potential stroke treatment.

This study had the following objectives: (1) determine the effect of MMP-12 suppression on neurological and functional recovery in stroke-induced male and female animals; (2) determine the optimal timing of MMP-12shRNA treatment for maximum therapeutic benefit; (3) compare the therapeutic efficacy of acute versus chronic MMP-12 suppression; and (4) identify any treatment-related sex differences on functional outcomes.

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