Nucleic Acid Therapies for Ischemic Stroke

Abstract

Stroke remains a leading cause of disability and death worldwide despite significant scientific and therapeutic advances. Therefore, there is a critical need to improve stroke prevention and treatment. In this review, we describe several examples that leverage nucleic acid therapeutics to improve stroke care through prevention, acute treatment, and recovery. Aptamer systems are under development to increase the safety and efficacy of antithrombotic and thrombolytic treatment, which represent the mainstay of medical stroke therapy. Antisense oligonucleotide therapy has shown some promise in treating stroke causes that are genetically determined and resistant to classic prevention approaches such as elevated lipoprotein (a) and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Targeting microRNAs may be attractive because they regulate factors involved in neuronal cell death and reperfusion-associated injury, as well as neurorestorative pathways. Lastly, microRNAs may aid reliable etiologic classification of stroke subtypes, which is important for effective secondary stroke prevention.

Epidemiology of Stroke

The latest estimates from the Global Burden of Diseases, Injuries and Risk Factors Study 2016 (the most comprehensive source of comparable summary population health measures) have demonstrated a shift from communicable diseases to noncommunicable diseases as the leading causes for reduced disability-adjusted life-years (DALY; i.e., the sum of years of life lost due to poor health or disability) over the last 2 decades [1]. Among these, cerebrovascular diseases are now the second leading cause for DALYs [1]. These changes are driven by an increase in the incidence and prevalence of stroke in low- and middle-income countries resulting in a significantly higher stroke burden in these countries as compared to high-income countries [1]. Concerningly, stroke burden has particularly worsened among younger patients, with a startling 25% increase in the incidence of stroke among adults aged 20 to 64 years representing 31% of all people with incident stroke [2]. In 2010, there were 16.9 million incident cases of stroke (~ 70% ischemic strokes and ~ 30% hemorrhagic strokes) worldwide [2] and approximately 800,000 people suffer a stroke in the USA each year [3, 4]. Despite significant scientific and therapeutic advances, stroke remains the fifth leading cause of death in the USA and there has been a recent flattening, and even increase, in death rates among all age groups [4, 5]. Stroke-associated socioeconomic costs are immense, and given our aging society, it has been estimated that the total direct medical stroke-related costs will more than double from $36.7 billion to $94.3 billion from 2015 to 2035 [4]. Hence, there is a critical need to improve stroke prevention and treatment. In the following pages, we will provide a narrative overview of select, promising strategies that leverage nucleic acid therapeutics to improve stroke care through the 3 main pillars prevention, acute treatment, and recovery (Table 1 summarizes key aspects of the discussed agents) [6, 7]. In this chapter, we will focus on ischemic stroke because the majority of nucleic acid therapies have been tested in this stroke type and because ischemic stroke represents the most common stroke form. For details regarding nomenclature, chemistry, and challenges in delivering nucleic acid therapeutics to the central nervous system, we refer the interested reader to the companion chapters in this special edition.

Table 1 Summary of discussed nucleic acid therapies

Stroke Prevention

Antithrombotic Therapy for Primary and Secondary Stroke Prevention

The 3 major ischemic stroke mechanisms include cardioembolism, large artery atherosclerosis, and cerebral small vessel disease related pathology [8, 9]. The majority of all ischemic strokes are caused by arterial thromboembolism. Hence, prophylaxis with antithrombotics (i.e., anticoagulants and antiplatelet agents) is the mainstay of medical therapy to reduce stroke risk. However, despite the proven benefit of antithrombotic therapy, many recurrences are not prevented. Additionally, the use of antiplatelets and anticoagulants carries the risk of bleeding complications particularly if used in combination [10,11,12,13]. Accordingly, there is a need to refine antithrombotic regimens and to develop novel agents to increase benefit while attenuating hemorrhage risk.

One promising approach for inhibiting platelet function is to block the interaction between von Willebrand factor (vWF) and the platelet receptor glycoprotein 1b (GP1b) to minimize recruitment, activation, and aggregation of platelets at an injured arterial wall. Preclinical studies found that inhibition of GPIb or absence of vWF confers profound antithrombotic effects as well as attenuates infarct size in a mouse transient middle cerebral artery occlusion stroke model without increasing the risk for hemorrhagic transformation of the brain infarct [14,15,16,17]. Under pathological conditions (that cause high shear force in the arterial circulation), vWF is activated through physical deformation that exposes its A1 domain and enables binding to the GP1b receptor resulting in thrombosis [18].

The first aptamer against vWF was ARC1172, a DNA oligonucleotide that bound to the A1-domain of vWF [19]. Subsequently, the anti vWF RNA/DNA hybrid aptamer ARC1779 was developed. ARC1779 binds to the A1 domain of activated vWF, blocking the interaction of vWF with GP1b on platelets, inhibiting the vWF-mediated pathological thrombosis, and leaving the coagulation system and other pathways of platelet activation intact [20]. A clinical phase 1 study in healthy volunteers demonstrated that ARC1779 dose-dependently reduced vWF activity and platelet function. A subsequent small randomized, double-blind, placebo-controlled phase 2 trial in patients undergoing carotid endarterectomy (CEA) demonstrated that patients treated with ARC1779 had significantly later occurrence of postoperative embolic signals as detected by transcranial Doppler during the 3 h of monitoring. Moreover, the number of patients without any embolic signals was significantly lower in the treatment group and there was a trend towards overall fewer embolic signals in ARC1779 patients. Also, none of the 8 subjects in the ARC1779 group who underwent brain MRI had evidence of postoperative ischemic stroke as compared to 2 of 5 patients in the placebo group. Lastly, the number of clinical overt strokes was similar between groups (one each) [21]. Bleeding events were more common in ARC1779-treated subjects [21] for which reason phase II and III trials will be required to establish safety and efficacy of ARC1779 for ischemic stroke prevention particularly in the perioperative setting. In this respect, a possible approach to mitigate hemorrhage risk in the surgical setting as well as to control bleeding complications in patients treated with aptamers targeting vWF is to use an aptamer inhibitor. It has been shown that the RNA aptamer Ch-9.14-T10 maintained arterial patency in the mouse ferric chloride-induced carotid artery thrombosis model [22]. The authors demonstrated that surgically challenged mice (tail transsection) treated with Ch-9.14-T10 dose-dependently exhibited significantly enhanced bleeding as compared with control mice. However, by using a complementary antidote oligonucleotide based on the sequence of aptamer Ch-9.14-T10, they were able to reverse vWF aptamer activity both in vitro and in vivo, resulting in substantial attenuation of bleeding in surgically challenged mice (with similar blood loss as in control animals) [22].

Long-term oral anticoagulation remains the mainstay for preventing ischemic stroke in patients at high risk for cardioembolism such as in the setting of atrial fibrillation, the most common pathological arrhythmia [23,24,25]. Although stroke prevention with oral anticoagulation is key to AF treatment [26,27,28,29], up to 40% of treatment-eligible older atrial fibrillation patients are untreated due to complex decision-making [30,31,32,33,34,35,36,37,38,39,40]. It is particularly challenging for clinicians to advise frail patients about anticoagulation given their high risk for both ischemic stroke and anticoagulation-related bleeding [41,42,43,44]. Currently available oral anticoagulants include vitamin K antagonists (e.g., warfarin) and non-vitamin K oral anticoagulants (NOAC) including the direct thrombin inhibitor dabigatran and factor Xa inhibitors such as apixaban, rivaroxaban, and edoxaban [45]. Aptamer–antidote systems have been developed as a fundamentally attractive regimen to achieve rapid and selective anticoagulation with the ability for graded reversal in patients requiring safe and effective anticoagulation including in the setting of procedures. One of these systems is REG1, which consists of an active anticoagulant (pegnivacogin, RB006) and a complementary oligonucleotide antidote (anivamersen, RB007) that neutralizes the anticoagulant effect as needed, serving as a molecular “on–off” switch [46, 47]. Because anivamersen restores hemostatic capacity by preventing the association of pegnivacogin with factor IXa, the maximal generated levels of factor IXa are limited to pre-existing levels of native factor IX/IXa assuaging concerns that reversal results in exceeding intrinsic factor IXa activity and trigger thrombosis [46, 48]. Following a comprehensive preclinical development program, REG1 was tested as the first-in-human aptamer-based direct factor IXa inhibitor [47,48,49,50]. To achieve prolonged duration of effect, pegnivacogin was chemically stabilized by conjugation to a 40-kDa polyethylene glycol (PEG) carrier. Pegnivacogin selectively blocks the conversion of factor X to factor Xa. In the first phase 1a study that enrolled 85 healthy volunteer subjects, REG1 was overall well tolerated and adverse bleeding events were similar to placebo (mostly consisting of minor bleeding and ecchymoses at the intravenous access site) [47]. Notably, one pegnivacogin-treated patient developed transient speech impairment, mood alteration, confusion, and ptosis that spontaneously resolved. However, given the patient’s personal history (drug abuse), circumstances of symptom occurrence (emotional exchange with study staff), and absent overt mechanistic link, it was uncertain whether the event was caused by the study drug [47]. In a second phase 1 trial, testing repeat dosing of REG1 components in 39 healthy volunteers, no significant adverse events were observed [51]. In anticipation of phase 2 trials of revascularization therapy, the subsequent phase 1b study enrolled 50 subjects with stable coronary artery disease on maintenance single or dual antiplatelet therapy to determine the clinical safety and pharmacodynamic profiles of REG1 [46]. Similar to the phase 1 studies in healthy volunteers, REG1 was overall well tolerated without major bleeding or other serious adverse events or signs of acute encephalopathy [46]. Transient cutaneous reactions (flushing and/or pruritus) were noted in 2 subjects within a few minutes after drug injection. Pegnivacogin resulted in dose-dependent, rapid-onset, and durable anticoagulation that was rapidly reversed with anivamersen. The RADAR phase IIb trial tested different levels of reversal of pegnivacogin by anivamersen compared with heparin in 640 patients with acute coronary syndrome undergoing cardiac catheterization. This trial found that with at least 50% reversal of pegnivacogin, bleeding rates were similar to heparin (enrollment in the 25% reversal arm was stopped due to excess bleeding) and there was a trend towards less frequent acute ischemic complications in REG1 as compared to heparin anticoagulation [52]. However, 3 patients developed serious unexplained allergic reactions and study enrollment was terminated after the third event. Based on the encouraging results of a reduction in the incidence of ischemic events to 3.0% compared with 5.7% in the heparin arm REGULATE-PCI, a large clinical phase III trial commenced with the goal to determine the efficacy of REG1 versus bivalirudin for percutaneous coronary intervention in more than 13,000 patients [53]. In light of the observed allergic reactions in the prior trials, specific guidelines were provided to investigators and patients. The data and safety monitoring board reviewed in real time and periodically all serious allergic events. Although stent thrombosis by day 30 occurred less frequently with REG1, there was no difference in the primary efficacy endpoint (death, myocardial infarction, stroke, or unplanned target lesion revascularization by day 3). Secondary composite or individual efficacy endpoints and bleeding were more frequent among patients receiving REG1. However, this study was halted following enrollment of approximately 3200 participants after 10 serious allergic reactions to pegnivacogin occurred (including 1 fatality) [53]. This observation prompted additional post hoc studies to determine the cause of these adverse events. These found a strong correlation between the incidence of allergic reactions and the presence of pre-existing circulating anti-PEG antibodies. Moreover, patients from REGULATE-PCI who experienced the most severe reaction had the highest levels of pre-existing anti-PEG antibodies [54, 55]. It was concluded that the PEG moiety and not the aptamer component of pegnivacogin was responsible for the severe allergic reactions [54, 55]. Although further clinical development of pegnivacogin has been discontinued, the overall results gained from the clinical trials provided highly insightful information for future drug and trial design. It also showed that aptamer-based inhibition of factor IXa can provide effective anticoagulation providing the rational for ongoing efforts to develop novel aptamer-based anticoagulant strategies [56].

Atherogenic Stroke Prevention

Prospective longitudinal studies have established the importance of major atherogenic risk factors including hypertension, diabetes mellitus, obesity, obstructive sleep apnea, and smoking [57]. Dyslipidemia is another well-established modifiable risk factor that contributes to the development of cerebrovascular disease and stroke. The use of HMG-CoA reductase inhibitors (statins) is recommended by the American Heart Association to reduce the risk of stroke and cardiovascular events particularly in patients where the stroke was related to atherosclerotic disease [57, 58]. These recommendations are based on the SPARCL trial that demonstrated a 16% relative risk reduction of recurrent strokes in patients treated with 80 mg atorvastatin [59]. However, it is interesting to note that a meta-analysis of 45 prospective studies including ~ 450,000 subjects did not find a significant association between total serum cholesterol level and stroke incidence [60]. Similarly, observations from the Framingham Heart Study indicated that a low high-density lipoprotein (HDL) but not total serum cholesterol and low-density lipoprotein (LDL) cholesterol relate to stroke risk [61]. Nevertheless, total serum cholesterol and LDL cholesterol have been shown to directly contribute to extracranial carotid artery atherosclerosis whereas HDL cholesterol has been found to exert protective effects [62,63,64]. Accordingly, it remains presently unclear whether the beneficial effects of statins are mediated through its proven LDL-lowering properties, through anti-inflammatory, neuroprotective, and neurorestorative attributes, or by targeting other (clinically less frequently assessed) lipoprotein fractions.

For example, a meta-analysis suggested that atorvastatin may lower lipoprotein (a) (Lp(a)) [65]. Lp(a) is a unique LDL-like particle that is comprised of a moiety essentially identical to LDL, which is covalently linked to the distinguishing protein component apolipoprotein(a) [66,67,68,69]. Lp(a) is an attractive target for reducing stroke risk through nucleic acid therapies because (1) Lp(a) concentrations in the atherogenic range are highly prevalent, affecting an estimated 20 to 30% of the worldwide population; (2) Lp(a) has a proven causal association with cardiovascular diseases and ischemic stroke [70,71,72,73,74,75,76,77]; and (3) plasma Lp(a) concentrations are largely determined by genetic factors confined to the apo(a) encoding gene LPA [78], which renders it resistant to dietary and other lifestyle modifications as well as treatment with classic lipid-lowering agents (such as niacin and statins) [66, 79]. Furthermore, though several trials demonstrated feasibility to modestly lower Lp(a) concentrations using classic lipid-lowering agents, none have provided evidence that the achieved degree of Lp(a) reduction leads to reduced cardiovascular events and stroke [76, 79, 80]. Yet, proof of principle that Lp(a) lowering in maximally treated patients may improve outcome stems from a prospective observational multicenter study demonstrating that lipid apheresis effectively reduced the frequency of cardiovascular and cerebrovascular events over a follow-up period of 2 years [81].

Antisense oligonucleotide (ASO) therapy has shown some promise in treating elevated Lp(a). For example, plasma Lp(a) levels have been consistently and significantly reduced with mipomersen (ISIS 301012). Mipomersen is a second-generation antisense oligonucleotide that specifically binds to the apolipoprotein B-100 mRNA, which was the first agent to enter clinical trials utilizing an antisense mechanism for reducing the production of apolipoprotein B. By inhibition of messenger ribonucleic acid translation, mipomersen blocks the hepatic protein synthesis resulting in dose-dependent lowering of the concentration of the apoB-100 containing atherogenic lipoproteins including Lp(a) in patients with varying extents of hyperlipidemia who are at high risk for cerebrovascular events (such as in patients with familial hypercholesterolemia and on the background of treatment with statins and other conventional lipid-lowering drugs) [82,83,84,85,86,87,88,89]. Specifically, several phase III clinical trials have shown that mipomersen lowers Lp(a) by approximately 17 to 30% [85,86,87,88,89]. Importantly, an individual patient analysis of subjects that had participated in one of the 3 phase III trials of the mipomersen program [85,86,87] found a significant reduction in major adverse cardiac events from 25.7 of 1000 patient-months of follow-up before mipomersen treatment to 3.9 of 1000 patient-months of follow-up during approximately 24 months of mipomersen treatment (odds ratio 0.053 [95% CI, 0.016-0.168], P < 0.0001) [88]. Nevertheless, despite the compelling 84% relative reduction in major cardiovascular events, it needs to be emphasized that mipomersen treatment related to a significant reduction in all atherogenic lipid fractions. Thus, it remains uncertain to what extent the lowering of Lp(a) contributed to the risk reduction. Moreover, major predictors of mipomersen-associated Lp(a) reduction were white race and lower baseline values, which is of clinical importance because approximately 30% of whites, yet 60% to 70% of blacks, have elevated Lp(a) levels of > 30 mg/dL [90]. Lastly, despite the proven efficacy in lowering Lp(a) and other atherogenic lipids, mipomersen possesses a significant side effect profile including risk for hepatotoxicity, for which reason it is unlikely to find wide acceptance for the specific treatment of isolated elevations of Lp(a) (and for which reason it is restricted for use in homozygous familial hypercholesterolemia through a risk evaluation and mitigation strategy program in the USA and was rejected by European Medicines Agency) [79, 91]. Nevertheless, subsequent investigations into optimized ASOs for treatment of increased Lp(a) identified IONIS-APO(a)Rx, which achieved a dose-dependent mean Lp(a) reduction of 78% in healthy volunteers [70]. In this first phase I study, IONIS-APO(a)Rx did not cause any serious or severe adverse events and there were no significant changes in liver function assays. Common less severe events included mild injection site reactions as well as flu-like symptoms [70]. The results from this study provide the rationale for future clinical trials to determine whether lowering Lp(a) plasma concentrations reduces cardiovascular events including ischemic stroke.

Prevention in Inherited Stroke Syndromes

The majority of ischemic strokes are multifactorial in nature, and any genetic contributions are likely the result from multiple risk alleles each with small effects [92]. Nevertheless, a small subset of ischemic strokes have monogenic causes, posing a substantial challenge for clinicians because standard approaches to risk factor modification and secondary prevention measures, while partially beneficial, are not sufficient in preventing disease progression [92]. A prominent example is cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). It is caused by mutations in the NOTCH3 gene on chromosome 19 first reported in European families. Today, CADASIL has been reported from all continents and in hundreds of families of European, American, African, and Asian descent. It is estimated to inflict 1 in 25,000 to 50,000 people. Patients with the disease tend to be middle aged who classically develop migraine with aura as the earliest clinical manifestation. While migraine with aura may be the prominent symptom in some families, stroke is the most frequent clinical manifestation over a patient’s lifetime, with approximately two thirds of symptomatic subjects having had a stroke/transient ischemic attack. Additional symptoms include mood disturbances as well as dementia relating to chronic, progressive brain injury due to white matter disease and strokes. Cognitive disturbances are typically in multiple domains including visuospatial and speech as well as memory. Specific imaging findings include MRI hyperintensities in the bilateral temporal horns, the external capsule, and the corpus callosum indicating extensive cerebral small-vessel disease-related pathology. Whereas imaging findings and clinical presentation are suggestive, diagnosis is made through genetic testing or skin biopsy, revealing loss of the media tunica and fibrosis of the adventitia, with cytoplasmic inclusions in the vascular smooth muscles. It has been shown that mutations in the NOTCH3 gene lead to cell surface aggregates, yet the intracellular cytoskeleton is affected. Ultimately, the vascular smooth muscles are unable to contract causing aberrancies in autoregulation of small vessels in the central nervous system [93]. To date, there are no specific treatments for this devastating disease and therapy rests on supportive measures [94,95,96]. CADASIL treatment has been met with difficulties as NOTCH3 is ubiquitously expressed and functions in multiple organ systems, even at early stages of embryogenesis [97, 98]. Several ongoing trials testing the use of antibodies in order to alter the activation of notch-3 cascade have been promising [98, 99]. Silencing the gene through shRNA, transfected via a lentivirus, causes similar pathology [93]. In a proof-of-concept experiment, smooth muscle cells from CADASIL patients were treated using ASOs to alter the pre-mRNA splicing and correct the even number of cystein molecules at the extracellular epidermal growth factor-like repeat (EGFr) domain. The remaining notch-3 protein was functional and did not form pathologic aggregates [100]. Although in vivo validation of this approach is warranted, nucleic acid-based therapies may be an exciting novel approach to treating this as well as other monogenic stroke causes and related sequelae.

Acute Ischemic Stroke Therapy

The brain relies on a constant supply of oxygen and high-energy substrates (predominantly glucose) to satisfy its high metabolic demands to maintain functional and structural integrity. Simplistically, focal brain ischemia results from occlusion or stenosis of a brain supplying vessel through embolism of material originating elsewhere in the vasculature or from in situ vascular pathology such as atherothrombosis [101]. Vascular occlusion and thus interrupted delivery of substrates to a vascular territory of the brain quickly result in ischemia that progresses to irreversible infarction if blood flow is not reinstated in a timely fashion [102]. It has been estimated that with each minute of ischemia, 1.9 million neurons are permanently lost [103]. Accordingly, rapid reperfusion of the ischemic but not yet infarcted tissue is paramount to mitigate brain injury. Indeed, the only proven efficacious acute stroke treatment strategies are based on this principle. Although there are a multitude of preclinical studies that have utilized nucleic acid therapeutics to investigate pathophysiology and establish proof of principle therapeutic targets, there are presently no well-developed clinical nucleic acid therapeutic programs similar to those presented in the preceding sections on stroke prevention. Therefore, we will focus on general principles of acute stroke therapy and opportunities for intervention supported by discussion of select targets of potential interest.

Thrombolysis

The most commonly used therapy that is effective and safe for acute ischemic stroke therapy is intravenous recombinant tissue plasminogen activator (tPA). Since its initial approval for treatment of patients within a narrow time window of 3 h from symptom onset [104,105,106], its indication has been safely expanded for use in the 4.5-h time window in patients selected on additional clinical criteria [107], and more recent studies demonstrate the possibility to treat even longer after symptom onset by using advanced neuroimaging criteria [108]. Nevertheless, despite proven benefit and overall safety, thrombolysis with tPA is only partially effective in many patients and it increases the risk for intracranial hemorrhage [109,110,111]. Furthermore, preclinical studies indicated that tPA may exert neurotoxic properties and exacerbate ischemic lesions via several distinct pathways [112,113,114]. Hence, minimizing tPA-mediated toxicity is a potential strategy to increase the benefit-to-risk ratio. A recently proposed strategy is to target the interaction of tPA with the low density lipoprotein receptor related protein-1 (LRP-1) [115], which is a transmembrane receptor expressed on several cell types including neurons, vascular endothelial cells, pericytes, smooth muscle cells, and astrocytes. The LRP-1 interaction with tPA appears to be an important mediator of adverse effects after tPA-mediated thrombolysis. For example, LRP-1-dependent blood–brain barrier (BBB) disruption as well as hemorrhagic transformation has been shown after tPA administration [114, 116]. Indeed, tPA-binding RNA aptamers have been developed that inhibit the tPA/LRP-1 complex formation and subsequent receptor-mediated endocytosis of tPA without substantially affecting the fibrinolytic properties of tPA in vitro [115]. These observations provide proof of principle that aptamer technology can be leveraged to attenuate potential tPA-mediated tissue toxicity while preserving its beneficial thrombolytic properties. This observation opens the door to developing novel strategies to increase the safety of thrombolytic agents.

Neuroprotection

After the onset of focal brain ischemia, the brain region with impaired cerebral blood flow contains subregions that progress to irreversible infarction at differing amounts of time depending primarily upon the severity of the initial cerebral blood flow decline, but metabolic factors and temperature can affect the rapidity of infarct development [117]. The ischemic region that is already infarcted at any given time point after the onset of ischemic stroke is the ischemic core, whereas the ischemic region at risk for becoming infarcted over time is known as the ischemic penumbra. The ischemic penumbra is the tissue target of acute stroke therapies, mediated either by reperfusion or neuroprotection, because therapeutic intervention can salvage ischemic tissue destined to become infarcted and thereby preserve functional capacity, leading to a better clinical outcome [118]. The cellular consequences of reduced or absent blood flow to the brain are manifold and referred to as the ischemic cascade [119].

However, in contrast to the increasingly positive outcomes with pharmacological (i.e., tPA-treatment) or mechanical thrombolysis in ischemic stroke [104,105,106,107,108, 120,121,122,123,124,125,126,127,128], clinical trials testing putative neuroprotective drugs targeting the various key factors in the ischemic cascade have been disappointing. Although well over 1000 compounds have shown promise in preclinical studies as neuroprotective agents, none of them were found to be effective in phase III clinical trials in which they were compared to placebo [129, 130]. Many reasons were identified for the failure of translation of monotherapy neuroprotection from successful animal models in clinical trials, which led to a number of suggestions as to how to improve neuroprotective drug testing [131,132,133,134,135,136]. In addition, the major advances in reperfusion therapy for acute ischemic stroke require reassessment of neuroprotection. This should now be viewed as an adjunctive therapy to be employed before, during, or after systemic thrombolysis and mechanical thrombectomy rather than standalone treatment because the convincing efficacy of reperfusion therapy would make it unethical to withhold these proven therapies. Several scenarios for using neuroprotection in conjunction with reperfusion can be envisioned including the expansion of the time window for definite reperfusion (“penumbral freezing”). Proof of principle for extending penumbral survival, and thus the therapeutic time window for tPA-mediated reperfusion, has been shown with several pharmacological interventions as well as with inhaled nitric oxide and high-flow oxygen therapy to function as neuroprotective gas treatments [137,138,139]. Such observations raise the intriguing possibility of testing neuroprotective drugs as a way to keep the ischemic core from expanding and the ischemic penumbra from shrinking prior to definite reperfusion therapy, for example, as patients are being transported to tertiary stroke centers for treatment.

In this respect, nucleic acid therapies targeting microRNA (miRNA) may be attractive because distinct miRNA expression patterns have been found in patients with ischemic stroke with both up- and downregulation of miRNAs as assessed in the blood [140], and also because miRNAs have been implicated in the regulation of factors involved in neuronal cell death as well as reperfusion-associated injury [141,142,143]. For example, miR-215 has been found upregulated in preclinical models of neuronal ischemic injury and overexpression of miR-215 inhibited apoptosis and autophagy in vitro, as well as attenuated the infarct volume and improved functional deficits in a mouse ischemic stroke model [144]. It has been posited that decreased levels of miR-215 in ischemic conditions lead to upregulation of nuclear factor (NF)-κB activator (Act)1 based on bioinformatics modeling, which ultimately leads to activation of interleukin 17 (Il-17) causing enhanced apoptosis and autophagy.

Targeting Inflammation

Inflammation plays a pivotal role at all stages of cerebral ischemia and is initiated via activation of platelets, complement, and endothelial cells. Leukocytes are subsequently activated by the release of cytokines and adhesion molecules, which includes tumor necrosis factor alpha among others. The humoral response is subsequently propagated by all cell types in the neurovascular unit, including endothelial cells, glia (astrocytes, microglia, oligodendrocytes), and neurons. With breakdown of the BBB due to the release of proteases including matrix metalloproteinases (MMPs), an influx of immune cells occurs, in turn leading to exacerbation of the initial insult, and formation of vasogenic edema and reactive oxygen species, which further compounds brain injury [145,146,147]. Although aspects of the inflammatory cascade, particularly in the early phase, can mediate detrimental effects, inflammation in the restoration phase is important to further tissue repair. The release of anti-inflammatory cytokines such as interleukin 10 is crucial in halting the inflammatory process, and thence the release of growth factors by immune cells [145]. Preclinical studies demonstrated that ischemic cells release molecules designated as danger-associated molecular patterns, which act in promoting an inflammatory response via pattern recognition receptors, of which toll-like receptor-4 (TLR-4) is a member. Indeed, mice lacking TLR-4 as well as animals treated with a TLR-4 antagonist have smaller infarct volumes and improved neurological function as compared to wild-type controls [148, 149].

In a mouse middle cerebral artery occlusion (MCAO) stroke model, differential expression of miRNAs in the infarct zone compared to the peri-infarct zone has been observed. This lead to the notion that miRNA regulation of gene expression and/or protein translation in the peri-infarct zone could decrease the inflammatory cascade and improve outcomes. Several groups identified differential miRNA expression showing a proinflammatory array in the infarct zone including miR-181a, and an anti-inflammatory array in the peri-infarct zone including miR-1906, which suppresses TLR-4 and its downstream cascade [148,149,150]. In the same MCAO model, it was shown that exogenous administration of miR-1906 attenuated the inflammatory cascade, which could be a therapeutic intervention in stroke patients [148,149,150]. Moreover, in vitro studies utilizing the oxygen/glucose deprivation model revealed a therapeutic effect in downregulating the proinflammatory miR-613, which is typically upregulated after an ischemic stroke. The beneficial results were attributed to decreased reactive oxygen species, which relate to lipid peroxidation as well as DNA damage [151]. Lastly, upregulation of miR-21 decreased stroke-related cerebral inflammatory responses thereby increasing BBB integrity, neuronal cell survival, and overall better functional outcomes in a rat model of cerebral ischemia and reperfusion [152]. Preclinical studies reported the efficacy of an herbal extract of Milletia (Spatholobus suberectus [DUNN]) to ameliorate oxygen–glucose deprivation-mediated cell death in vitro as well as improve histological and biological outcomes in a mouse model of cerebral ischemia. The mechanisms were shown to be related to a decrease in miR-494 levels that led to downstream overexpression of Sox8 and activation of the mTOR and MAPK pathway [153]. Recent observations indicated clinical improvement in stroke patients after oral administration of Spatholobus suberectus [DUNN] extract [154]. This preliminary data suggests that oral administration of medications targeting miRNA may have the potential to alter gene transcription and expression leading to alteration of the inflammatory response. Randomized trials will be required to confirm these initial observations.

Cerebral Edema

A major clinical challenge is the treatment of inflammation-mediated vasogenic edema, particularly after large hemispheric strokes because of associated high mortality exceeding 70% with maximal conservative management [147]. Currently, hemicraniectomy is the only proven therapy to improve outcomes and reduce mortality; however, even with this highly invasive neurosurgical intervention that requires removal of a large aspect of the skull covering the infarcted hemisphere to relieve swelling, less than 20% of patients will be disability free 1 year after their stroke [155]. Accordingly, novel therapeutic avenues are critically needed to improve outcome in this patient population. In this respect, the water channel aquaporin-4 (AQP-4) and the sulfonylurea receptor 1 (SUR1) and transient receptor potential melastatin 4 (SUR1-TRPM4) channels have been identified as key targets that promote cerebral edema representing potential targets for early treatment in stroke. Recently, the double-blind, randomized, placebo-controlled phase 2 trial Glyburide Advantage in Malignant Edema and Stroke-Remedy Pharmaceuticals (GAMES-RP) sought to determine whether treatment with the selective SUR1 inhibitor glyburide prevents major disability and death without undergoing decompressive craniectomy in patients with large ischemic infarcts [156]. However, while this trial demonstrated feasibility and safety, there was no difference in the primary outcome between patients receiving glyburide versus placebo [156]. Therefore, further study is warranted to assess the potential clinical benefit of a reduction in swelling by targeting these water channels.

AQP-4 acts in 2 distinct patterns depending on the time and cause of edema. AQP-4 enhances early cytotoxic edema by facilitating the transport of water molecules across the cell membrane in astroglial cells. With disruption of the sodium–potassium pump, sodium accumulates in the cells and water molecules traverse the cell membrane through AQP-4 into cells following the concentration gradient. Conversely, AQP-4 has been shown to function in the reabsorption of water molecules from the parenchyma in later-developing vasogenic edema. Hence, inhibiting the AQP-4 water channel is a promising step in decreasing cytotoxic, but more importantly, vasogenic edema [157, 158]. Using an in vitro system, siRNAs have been shown to effectively halt AQP-4 translation. In this model, it was shown that water homeostasis was disrupted and downstream gene regulation altered. The silencing of the AQP-4 gene lead to upregulation of c-fos as well as nerve growth factor inducible protein-B (NGFI-B), both of which related to apoptosis [159]. Accordingly, utilizing nucleic acid-based approaches may aid our understanding of the precise mechanisms driving cerebral edema formation and thus identify novel therapeutic approaches.

Stroke Recovery

As stated initially, stroke remains a leading cause of disability worldwide [1], which relates to the fact that many patients do not reach the hospital in time to be eligible for acute reperfusion therapies as well as that even those who receive treatment often have significant residual deficits. Therefore, there is a critical need to improve rehabilitative efforts. Restorative therapies that can harness neuroplasticity are a particularly promising strategy because they would be accessible by a large proportion of affected subjects and thus benefit a substantial number of stroke survivors.

While stroke triggers the ischemic cascade leading to tissue injury and inflammation, it also triggers a number of molecular events that aid spontaneous repair via alterations in receptor expression, synaptic and dendritic growth, axonal remodeling, and angiogenesis in the perilesional as well as connected remote brain tissues [160,161,162,163,164,165,166,167]. Hence, there is hope that by modulating these endogenous pathways, such as by small molecules, functional recovery may be improved. Although pharmacological augmentation is important to help manage stroke-related complications such as spasticity, pain, depression, anxiety, and cognitive impairment, almost no strategies exist to truly enhance recovery by pharmacological means. The strongest evidence stems from the Fluoxetine for Motor Recovery After Acute Ischemic Stroke (FLAME) study, which randomized 118 patients 1:1 to receive standard rehabilitative therapies with either placebo or the serotoninergic agent fluoxetine. This trial demonstrated significant improvement in motor function in the fluoxetine group [168], and there are now several phase 3 clinical trials underway to confirm whether the routine administration of fluoxetine after an acute stroke improves patients’ functional outcome [169].

A different strategy to improve functional recovery after a stroke is based on observations that ischemic stroke induces widespread changes in gene expression within the neurovascular unit [170, 171]. Thus, there is heightened interest to determine whether it is possible to shift gene expression towards a more proregenerative state such as by modulation of miRNA, which have been shown to be highly expressed in the vasculature, subserve critical vascular cell functions, and their expression profiles are substantially altered in the wake of an ischemic stroke [172,173,174,175,176]. Indeed, several studies indicated that in vivo manipulation of cerebral miRNA activity with synthetic miRNA inhibitors and mimics can attenuate ischemic injury and has strengthened the rationale for the development of miRNA-based therapeutic drugs to treat stroke-related brain injury and promote neurological recovery [177,178,179]. For example, it has recently been shown that axonal alterations of the miR-17-92 cluster expression relate to axonal outgrowth of embryonic cortical neurons [180]. Intravenous injection of mesenchymal stromal cell exosomes containing elevated miR-17-92 cluster into rats 24 h after 2 h of middle cerebral artery occlusion stroke increased neural differentiation, plasticity, and enhanced recovery after stroke [181]. Although infarct volume was not determined in this study, the used stroke model is highly reproducible and typically causes complete infarction within 2 h [182, 183]. Accordingly, one would expect similar infarct volumes at the time of treatment initiation. Consistent with this, poststroke functional deficits prior to treatment were similar between treated and untreated animals as assessed by the modified neurological severity score and foot-fault tests [181]. A further miRNA that has been found significantly altered after brain ischemia is miR-155 [176]. It has been associated with endothelial and vascular function including a role in vascular inflammation, atherogenesis, endothelial cell morphology, and migration, as well as wound healing [179]. Hence, miR-155 may be a suitable target for both modulating postischemic inflammation and tissue regeneration [179]. Indeed, in a mouse model of focal cerebral ischemia, inhibition of miR-155 by approximately 50% by intravenous injection of an anti-miR-155 miRCURY locked nucleic acid (LNA) inhibitor 48 h after middle cerebral artery occlusion resulted in increased expression of several miR-155 target genes. This improved microvascular perfusion in the peri-infarct brain tissue and reduced vasogenic edema through increased tight junction integrity, which related to less extensive acute infarction as well as delayed neuronal damage and overall improved functional recovery and outcomes [179]. Importantly, the initial infarct volume as assessed by in vivo brain MRI showed similar infarct sizes between treated and untreated mice. This is an important observation because the infarct extent is one of the strongest predictors of functional outcome after acute ischemic stroke [184,185,186]. Together, these studies demonstrate that targeting miRNA may be a novel means to enhance neurorestorative properties of the brain.

Ischemic Stroke Biomarkers

Reliable etiologic classification is critical for effective secondary stroke prevention. However, more than 100 pathological conditions have been implicated in the pathogenesis of ischemic stroke and in a significant proportion of patients, the cause of stroke remains uncertain even after extensive diagnostic evaluation [9, 101, 187, 188]. In addition, many patients have 2 (and more) not mutually exclusive possible stroke causes [101, 187, 188]. In this respect, nucleic acids have been proposed as novel biomarkers that may aid identification of the specific ischemic stroke cause. For example, carotid plaque rupture has been linked to alterations in miRNA expression and several RNAis have been functionally associated with plaque rupture due to thinning of the fibrous cap. The thinning is induced by decreased function and/or levels of miR-221/miR-222, which function in vascular smooth muscle cell proliferation [189]. Similar to these observations, a study including a modestly sized cohort of patients with stable and unstable carotid artery plaques found increased serum miR-221 while circular RNA (circRNA)-284 was elevated in the serum of patients undergoing urgent carotid endarterectomy for symptomatic plaque rupture as compared to asymptomatic patients undergoing the same surgery for a stable plaque. If confirmed, the circRNA-284-to-miR-221 ratio could serve as a noninvasive blood marker for carotid disease [155]. Aside from potentially aiding disease monitoring including the progression of plaque pathology, the development of risk-factor-specific biomarkers could aid clinical decision-making when patients have competing potential stroke mechanisms. Presently, physicians typically resolve to taking a pragmatic approach and treat both conditions. However, this may result in avoidable adverse events such as risk for hemorrhagic complications with a combination of antithrombotic regimens or procedural risk with an intervention of an uncertain level of benefit.

In summary, this article provides an overview of the unique chances and possible advantages of nucleic acid-based therapies as promising future novel treatments across the main phases of ischemic stroke (ranging from stroke prevention to recovery after stroke), and as putative innovative noninvasive blood biomarkers in stroke. Except for antithrombotic and antiatherogenic therapies to stroke prevention, most promising compounds are still within the preclinical discovery and safety stage of development. Considering that the typical drug approval timeline takes approximately 15 years from the earliest stages to clinical approval (~ 5 years drug discovery, ~ 2 years for preclinical testing, ~ 7 years for clinical trials, and ~ 1 year for approval) [190], substantial additional work remains to be completed before most discussed nucleic acid therapies can be safely implemented in daily practice. We hope that this review will serve as an incentive to forward current scientific efforts in the field of nucleic acid therapy to attenuate the devastating consequences of ischemic stroke.

References

  1. 1.

    GBD 2016 DALYs and Hale Collaborators. Global, regional, and national disability-adjusted life-years (DALYs) for 333 diseases and injuries and healthy life expectancy (HALE) for 195 countries and territories, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet. 2017;390:1260–1344.

    Article  Google Scholar 

  2. 2.

    Feigin V, Krishnamurthi RV. Global Burden of Stroke. In: Grotta JC, Albers GW, Broderick JP, Kasner SE, Lo EH, Mendelow AD et al., editors. Stroke. Pathophysiology, Diagnosis, and Management. 6 ed.: Elsevier; 2016.

  3. 3.

    Feigin VL, Krishnamurthi RV, Parmar P et al. Update on the Global Burden of Ischemic and Hemorrhagic Stroke in 1990-2013: The GBD 2013 Study. Neuroepidemiology. 2015;45:161–176.

    Article  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Benjamin EJ, Virani SS, Callaway CW et al. Heart Disease and Stroke Statistics-2018 Update: A Report From the American Heart Association. Circulation. 2018;137:e67-e492.

    Article  PubMed  Google Scholar 

  5. 5.

    Ma VY, Chan L, Carruthers KJ. Incidence, prevalence, costs, and impact on disability of common conditions requiring rehabilitation in the United States: stroke, spinal cord injury, traumatic brain injury, multiple sclerosis, osteoarthritis, rheumatoid arthritis, limb loss, and back pain. Arch Phys Med Rehabil. 2014;95:986–995.

    Article  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Broderick JP, Palesch YY, Janis LS, National Institutes of Health StrokeNet I. The National Institutes of Health StrokeNet: A User's Guide. Stroke. 2016;47:301–303.

    Article  PubMed  Google Scholar 

  7. 7.

    Cramer SC, Wolf SL, Adams HP, Jr. et al. Stroke Recovery and Rehabilitation Research: Issues, Opportunities, and the National Institutes of Health StrokeNet. Stroke. 2017;48:813–819.

    Article  PubMed  PubMed Central  Google Scholar 

  8. 8.

    Adams HP, Jr., Bendixen BH, Kappelle LJ et al. Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment. Stroke. 1993;24:35–41.

    Article  PubMed  Google Scholar 

  9. 9.

    Hart RG, Diener HC, Coutts SB et al. Embolic strokes of undetermined source: the case for a new clinical construct. Lancet Neurol. 2014;13:429–438.

    Article  PubMed  Google Scholar 

  10. 10.

    Rothwell PM, Algra A, Chen Z, Diener HC, Norrving B, Mehta Z. Effects of aspirin on risk and severity of early recurrent stroke after transient ischaemic attack and ischaemic stroke: time-course analysis of randomised trials. Lancet. 2016;388:365–375.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Bhatt DL, Fox KA, Hacke W et al. Clopidogrel and aspirin versus aspirin alone for the prevention of atherothrombotic events. N Engl J Med. 2006;354:1706–1717.

    Article  CAS  PubMed  Google Scholar 

  12. 12.

    Lee M, Saver JL, Hong KS, Rao NM, Wu YL, Ovbiagele B. Risk-benefit profile of long-term dual- versus single-antiplatelet therapy among patients with ischemic stroke: a systematic review and meta-analysis. Ann Intern Med. 2013;159:463–470.

    Article  PubMed  Google Scholar 

  13. 13.

    Hansen ML, Sorensen R, Clausen MT et al. Risk of bleeding with single, dual, or triple therapy with warfarin, aspirin, and clopidogrel in patients with atrial fibrillation. Arch Intern Med. 2010;170:1433–1441.

    Article  CAS  PubMed  Google Scholar 

  14. 14.

    Kleinschnitz C, De Meyer SF, Schwarz T et al. Deficiency of von Willebrand factor protects mice from ischemic stroke. Blood. 2009;113:3600–3603.

    Article  CAS  PubMed  Google Scholar 

  15. 15.

    Denis C, Methia N, Frenette PS et al. A mouse model of severe von Willebrand disease: defects in hemostasis and thrombosis. Proc Natl Acad Sci U S A. 1998;95:9524–9529.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Kleinschnitz C, Pozgajova M, Pham M, Bendszus M, Nieswandt B, Stoll G. Targeting platelets in acute experimental stroke: impact of glycoprotein Ib, VI, and IIb/IIIa blockade on infarct size, functional outcome, and intracranial bleeding. Circulation. 2007;115:2323–2330.

    Article  CAS  PubMed  Google Scholar 

  17. 17.

    Diener JL, Daniel Lagasse HA, Duerschmied D et al. Inhibition of von Willebrand factor-mediated platelet activation and thrombosis by the anti-von Willebrand factor A1-domain aptamer ARC1779. J Thromb Haemost. 2009;7:1155–1162.

    Article  CAS  PubMed  Google Scholar 

  18. 18.

    Siedlecki CA, Lestini BJ, Kottke-Marchant KK, Eppell SJ, Wilson DL, Marchant RE. Shear-dependent changes in the three-dimensional structure of human von Willebrand factor. Blood. 1996;88:2939–2950.

    CAS  PubMed  Google Scholar 

  19. 19.

    Huang RH, Fremont DH, Diener JL, Schaub RG, Sadler JE. A structural explanation for the antithrombotic activity of ARC1172, a DNA aptamer that binds von Willebrand factor domain A1. Structure. 2009;17:1476–1484.

    Article  CAS  PubMed  Google Scholar 

  20. 20.

    Gilbert JC, DeFeo-Fraulini T, Hutabarat RM et al. First-in-human evaluation of anti von Willebrand factor therapeutic aptamer ARC1779 in healthy volunteers. Circulation. 2007;116:2678–2686.

    Article  CAS  PubMed  Google Scholar 

  21. 21.

    Markus HS, McCollum C, Imray C, Goulder MA, Gilbert J, King A. The von Willebrand inhibitor ARC1779 reduces cerebral embolization after carotid endarterectomy: a randomized trial. Stroke. 2011;42:2149–2153.

    Article  CAS  PubMed  Google Scholar 

  22. 22.

    Nimjee SM, Lohrmann JD, Wang H et al. Rapidly regulating platelet activity in vivo with an antidote controlled platelet inhibitor. Mol Ther. 2012;20:391–397.

    Article  CAS  PubMed  Google Scholar 

  23. 23.

    Colilla S, Crow A, Petkun W, Singer DE, Simon T, Liu X. Estimates of current and future incidence and prevalence of atrial fibrillation in the U.S. adult population. Am J Cardiol. 2013;112:1142–1147.

    Article  PubMed  Google Scholar 

  24. 24.

    Miyasaka Y, Barnes ME, Bailey KR et al. Mortality trends in patients diagnosed with first atrial fibrillation: a 21-year community-based study. J Am Coll Cardiol. 2007;49:986–992.

    Article  PubMed  Google Scholar 

  25. 25.

    Mozaffarian D, Benjamin EJ, Go AS et al. Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association. Circulation. 2016;133:e38–60.

    PubMed  Google Scholar 

  26. 26.

    European Heart Rhythm A, European Association for Cardio-Thoracic S, Camm AJ et al. Guidelines for the management of atrial fibrillation: the Task Force for the Management of Atrial Fibrillation of the European Society of Cardiology (ESC). Eur Heart J. 2010;31:2369–2429.

    Article  Google Scholar 

  27. 27.

    Patel MR, Mahaffey KW, Garg J et al. Rivaroxaban versus warfarin in nonvalvular atrial fibrillation. N Engl J Med. 2011;365:883–891.

    Article  CAS  PubMed  Google Scholar 

  28. 28.

    Connolly SJ, Ezekowitz MD, Yusuf S et al. Dabigatran versus warfarin in patients with atrial fibrillation. N Engl J Med. 2009;361:1139–1151.

    Article  CAS  PubMed  Google Scholar 

  29. 29.

    Mant J, Hobbs FD, Fletcher K et al. Warfarin versus aspirin for stroke prevention in an elderly community population with atrial fibrillation (the Birmingham Atrial Fibrillation Treatment of the Aged Study, BAFTA): a randomised controlled trial. Lancet. 2007;370:493–503.

    Article  CAS  PubMed  Google Scholar 

  30. 30.

    Barnett AS, Kim S, Fonarow GC et al. Treatment of Atrial Fibrillation and Concordance With the American Heart Association/American College of Cardiology/Heart Rhythm Society Guidelines: Findings From ORBIT-AF (Outcomes Registry for Better Informed Treatment of Atrial Fibrillation). Circ Arrhythm Electrophysiol. 2017;10.

  31. 31.

    Amroze A, McManus DD, Golden J et al. Supporting use of anticoagulation through provider profiling of oral anticoagulant therapy for atrial fibrillation (SUPPORT-AF). Heart Rhythm. 2018;15:S475.

    Article  Google Scholar 

  32. 32.

    Chan PS, Maddox TM, Tang F, Spinler S, Spertus JA. Practice-level variation in warfarin use among outpatients with atrial fibrillation (from the NCDR PINNACLE program). Am J Cardiol. 2011;108:1136–1140.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Ogilvie IM, Newton N, Welner SA, Cowell W, Lip GY. Underuse of oral anticoagulants in atrial fibrillation: a systematic review. Am J Med. 2010;123:638–645 e634.

    Article  CAS  PubMed  Google Scholar 

  34. 34.

    Seaburg L, Hess EP, Coylewright M, Ting HH, McLeod CJ, Montori VM. Shared decision making in atrial fibrillation: where we are and where we should be going. Circulation. 2014;129:704–710.

    Article  PubMed  Google Scholar 

  35. 35.

    Reynolds MR, Shah J, Essebag V et al. Patterns and predictors of warfarin use in patients with new-onset atrial fibrillation from the FRACTAL Registry. Am J Cardiol. 2006;97:538–543.

    Article  CAS  PubMed  Google Scholar 

  36. 36.

    Darrat YH, Shah J, Elayi CS et al. Regional Lack of Consistency in the Management of Atrial Fibrillation (from the RECORD-AF Trial). Am J Cardiol. 2017;119:47–51.

    Article  PubMed  Google Scholar 

  37. 37.

    Man-Son-Hing M, Nichol G, Lau A, Laupacis A. Choosing antithrombotic therapy for elderly patients with atrial fibrillation who are at risk for falls. Arch Intern Med. 1999;159:677–685.

    Article  CAS  PubMed  Google Scholar 

  38. 38.

    Steinberg BA, Kim S, Thomas L et al. Lack of concordance between empirical scores and physician assessments of stroke and bleeding risk in atrial fibrillation: results from the Outcomes Registry for Better Informed Treatment of Atrial Fibrillation (ORBIT-AF) registry. Circulation. 2014;129:2005–2012.

    Article  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Cordonnier C. Balancing risks versus benefits of anticoagulants in stroke prevention. Lancet Neurol. 2018;17:487–488.

    Article  PubMed  Google Scholar 

  40. 40.

    Deplanque D, Leys D, Parnetti L et al. Secondary prevention of stroke in patients with atrial fibrillation: factors influencing the prescription of oral anticoagulation at discharge. Cerebrovasc Dis. 2006;21:372–379.

    Article  CAS  PubMed  Google Scholar 

  41. 41.

    Piccini JP, Hammill BG, Sinner MF et al. Clinical course of atrial fibrillation in older adults: the importance of cardiovascular events beyond stroke. Eur Heart J. 2014;35:250–256.

    Article  PubMed  Google Scholar 

  42. 42.

    Poli D, Testa S, Antonucci E, Grifoni E, Paoletti O, Lip GY. Bleeding and stroke risk in a real-world prospective primary prevention cohort of patients with atrial fibrillation. Chest. 2011;140:918–924.

    Article  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Aldrugh S, Sardana M, Henninger N, Saczynski JS, McManus DD. Atrial fibrillation, cognition and dementia: A review. J Cardiovasc Electrophysiol. 2017;28:958–965.

    Article  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Panza F, Lozupone M, Solfrizzi V et al. Different Cognitive Frailty Models and Health- and Cognitive-related Outcomes in Older Age: From Epidemiology to Prevention. J Alzheimers Dis. 2018;62:993–1012.

    Article  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Patel A, Goddeau RP, Jr., Henninger N. Newer Oral Anticoagulants: Stroke Prevention and Pitfalls. Open Cardiovasc Med J. 2016;10:94–104.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Chan MY, Cohen MG, Dyke CK et al. Phase 1b randomized study of antidote-controlled modulation of factor IXa activity in patients with stable coronary artery disease. Circulation. 2008;117:2865–2874.

    Article  CAS  PubMed  Google Scholar 

  47. 47.

    Dyke CK, Steinhubl SR, Kleiman NS et al. First-in-human experience of an antidote-controlled anticoagulant using RNA aptamer technology: a phase 1a pharmacodynamic evaluation of a drug-antidote pair for the controlled regulation of factor IXa activity. Circulation. 2006;114:2490–2497.

    Article  CAS  PubMed  Google Scholar 

  48. 48.

    Rusconi CP, Scardino E, Layzer J et al. RNA aptamers as reversible antagonists of coagulation factor IXa. Nature. 2002;419:90–94.

    Article  CAS  PubMed  Google Scholar 

  49. 49.

    Rusconi CP, Roberts JD, Pitoc GA et al. Antidote-mediated control of an anticoagulant aptamer in vivo. Nat Biotechnol. 2004;22:1423–1428.

    Article  CAS  PubMed  Google Scholar 

  50. 50.

    Nimjee SM, Keys JR, Pitoc GA, Quick G, Rusconi CP, Sullenger BA. A novel antidote-controlled anticoagulant reduces thrombin generation and inflammation and improves cardiac function in cardiopulmonary bypass surgery. Mol Ther. 2006;14:408–415.

    Article  CAS  PubMed  Google Scholar 

  51. 51.

    Chan MY, Rusconi CP, Alexander JH, Tonkens RM, Harrington RA, Becker RC. A randomized, repeat-dose, pharmacodynamic and safety study of an antidote-controlled factor IXa inhibitor. J Thromb Haemost. 2008;6:789–796.

    Article  CAS  PubMed  Google Scholar 

  52. 52.

    Povsic TJ, Vavalle JP, Aberle LH et al. A Phase 2, randomized, partially blinded, active-controlled study assessing the efficacy and safety of variable anticoagulation reversal using the REG1 system in patients with acute coronary syndromes: results of the RADAR trial. Eur Heart J. 2013;34:2481–2489.

    Article  CAS  PubMed  Google Scholar 

  53. 53.

    Lincoff AM, Mehran R, Povsic TJ et al. Effect of the REG1 anticoagulation system versus bivalirudin on outcomes after percutaneous coronary intervention (REGULATE-PCI): a randomised clinical trial. Lancet. 2016;387:349–356.

    Article  CAS  PubMed  Google Scholar 

  54. 54.

    Ganson NJ, Povsic TJ, Sullenger BA et al. Pre-existing anti-polyethylene glycol antibody linked to first-exposure allergic reactions to pegnivacogin, a PEGylated RNA aptamer. J Allergy Clin Immunol. 2016;137:1610–1613 e1617.

    Article  CAS  PubMed  Google Scholar 

  55. 55.

    Povsic TJ, Lawrence MG, Lincoff AM et al. Pre-existing anti-PEG antibodies are associated with severe immediate allergic reactions to pegnivacogin, a PEGylated aptamer. J Allergy Clin Immunol. 2016;138:1712–1715.

    Article  CAS  PubMed  Google Scholar 

  56. 56.

    Chabata CV, Frederiksen JW, Sullenger BA, Gunaratne R. Emerging applications of aptamers for anticoagulation and hemostasis. Curr Opin Hematol. 2018;25:382–388.

    Article  CAS  PubMed  Google Scholar 

  57. 57.

    Kernan WN, Ovbiagele B, Black HR et al. Guidelines for the prevention of stroke in patients with stroke and transient ischemic attack: a guideline for healthcare professionals from the American Heart Association/American Stroke Association. Stroke. 2014;45:2160–2236.

    Article  PubMed  Google Scholar 

  58. 58.

    Stone NJ, Robinson JG, Lichtenstein AH et al. Treatment of blood cholesterol to reduce atherosclerotic cardiovascular disease risk in adults: synopsis of the 2013 American College of Cardiology/American Heart Association cholesterol guideline. Ann Intern Med. 2014;160:339–343.

    Article  PubMed  Google Scholar 

  59. 59.

    Amarenco P, Bogousslavsky J, Callahan A, 3rd et al. High-dose atorvastatin after stroke or transient ischemic attack. N Engl J Med. 2006;355:549–559.

    Article  CAS  PubMed  Google Scholar 

  60. 60.

    Cholesterol, diastolic blood pressure, and stroke: 13,000 strokes in 450,000 people in 45 prospective cohorts. Prospective studies collaboration. Lancet. 1995;346:1647–1653.

  61. 61.

    Pikula A, Beiser AS, Wang J et al. Lipid and lipoprotein measurements and the risk of ischemic vascular events: Framingham Study. Neurology. 2015;84:472–479.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Wilson PW, Hoeg JM, D'Agostino RB et al. Cumulative effects of high cholesterol levels, high blood pressure, and cigarette smoking on carotid stenosis. N Engl J Med. 1997;337:516–522.

    Article  CAS  PubMed  Google Scholar 

  63. 63.

    Fine-Edelstein JS, Wolf PA, O'Leary DH et al. Precursors of extracranial carotid atherosclerosis in the Framingham Study. Neurology. 1994;44:1046–1050.

    Article  CAS  PubMed  Google Scholar 

  64. 64.

    O'Leary DH, Anderson KM, Wolf PA, Evans JC, Poehlman HW. Cholesterol and carotid atherosclerosis in older persons: the Framingham Study. Ann Epidemiol. 1992;2:147–153.

    Article  CAS  PubMed  Google Scholar 

  65. 65.

    Takagi H, Umemoto T. Atorvastatin decreases lipoprotein(a): a meta-analysis of randomized trials. Int J Cardiol. 2012;154:183–186.

    Article  PubMed  Google Scholar 

  66. 66.

    Kronenberg F, Utermann G. Lipoprotein(a): resurrected by genetics. J Intern Med. 2013;273:6–30.

    Article  CAS  PubMed  Google Scholar 

  67. 67.

    Tsimikas S, Hall JL. Lipoprotein(a) as a potential causal genetic risk factor of cardiovascular disease: a rationale for increased efforts to understand its pathophysiology and develop targeted therapies. J Am Coll Cardiol. 2012;60:716–721.

    Article  CAS  PubMed  Google Scholar 

  68. 68.

    Berglund L, Ramakrishnan R. Lipoprotein(a): an elusive cardiovascular risk factor. Arterioscler Thromb Vasc Biol. 2004;24:2219–2226.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. 69.

    Utermann G. The mysteries of lipoprotein(a). Science. 1989;246:904–910.

    Article  CAS  PubMed  Google Scholar 

  70. 70.

    Tsimikas S, Viney NJ, Hughes SG et al. Antisense therapy targeting apolipoprotein(a): a randomised, double-blind, placebo-controlled phase 1 study. Lancet. 2015;386:1472–1483.

    Article  CAS  PubMed  Google Scholar 

  71. 71.

    Thanassoulis G, Campbell CY, Owens DS et al. Genetic associations with valvular calcification and aortic stenosis. N Engl J Med. 2013;368:503–512.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. 72.

    Emerging Risk Factors C, Erqou S, Kaptoge S et al. Lipoprotein(a) concentration and the risk of coronary heart disease, stroke, and nonvascular mortality. JAMA. 2009;302:412–423.

    Article  Google Scholar 

  73. 73.

    Helgadottir A, Gretarsdottir S, Thorleifsson G et al. Apolipoprotein(a) genetic sequence variants associated with systemic atherosclerosis and coronary atherosclerotic burden but not with venous thromboembolism. J Am Coll Cardiol. 2012;60:722–729.

    Article  CAS  PubMed  Google Scholar 

  74. 74.

    Kamstrup PR, Tybjaerg-Hansen A, Steffensen R, Nordestgaard BG. Genetically elevated lipoprotein(a) and increased risk of myocardial infarction. JAMA. 2009;301:2331–2339.

    Article  CAS  PubMed  Google Scholar 

  75. 75.

    Clarke R, Peden JF, Hopewell JC et al. Genetic variants associated with Lp(a) lipoprotein level and coronary disease. N Engl J Med. 2009;361:2518–2528.

    Article  CAS  PubMed  Google Scholar 

  76. 76.

    Nave AH, Lange KS, Leonards CO et al. Lipoprotein (a) as a risk factor for ischemic stroke: a meta-analysis. Atherosclerosis. 2015;242:496–503.

    Article  CAS  PubMed  Google Scholar 

  77. 77.

    Willeit P, Kiechl S, Kronenberg F et al. Discrimination and net reclassification of cardiovascular risk with lipoprotein(a): prospective 15-year outcomes in the Bruneck Study. J Am Coll Cardiol. 2014;64:851–860.

    Article  PubMed  Google Scholar 

  78. 78.

    Boerwinkle E, Leffert CC, Lin J, Lackner C, Chiesa G, Hobbs HH. Apolipoprotein(a) gene accounts for greater than 90% of the variation in plasma lipoprotein(a) concentrations. J Clin Invest. 1992;90:52–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. 79.

    Boffa MB. Emerging Therapeutic Options for Lowering of Lipoprotein(a): Implications for Prevention of Cardiovascular Disease. Curr Atheroscler Rep. 2016;18:69.

    Article  CAS  PubMed  Google Scholar 

  80. 80.

    Ranga GS, Kalra OP, Tandon H, Gambhir JK, Mehrotra G. Effect of aspirin on lipoprotein(a) in patients with ischemic stroke. J Stroke Cerebrovasc Dis. 2007;16:220–224.

    Article  PubMed  Google Scholar 

  81. 81.

    Leebmann J, Roeseler E, Julius U et al. Lipoprotein apheresis in patients with maximally tolerated lipid-lowering therapy, lipoprotein(a)-hyperlipoproteinemia, and progressive cardiovascular disease: prospective observational multicenter study. Circulation. 2013;128:2567–2576.

    Article  CAS  PubMed  Google Scholar 

  82. 82.

    Davis RA. Cell and molecular biology of the assembly and secretion of apolipoprotein B-containing lipoproteins by the liver. Biochim Biophys Acta. 1999;1440:1–31.

    Article  CAS  PubMed  Google Scholar 

  83. 83.

    Akdim F, Visser ME, Tribble DL et al. Effect of mipomersen, an apolipoprotein B synthesis inhibitor, on low-density lipoprotein cholesterol in patients with familial hypercholesterolemia. Am J Cardiol. 2010;105:1413–1419.

    Article  CAS  PubMed  Google Scholar 

  84. 84.

    Akdim F, Stroes ES, Sijbrands EJ et al. Efficacy and safety of mipomersen, an antisense inhibitor of apolipoprotein B, in hypercholesterolemic subjects receiving stable statin therapy. J Am Coll Cardiol. 2010;55:1611–1618.

    Article  CAS  PubMed  Google Scholar 

  85. 85.

    Stein EA, Dufour R, Gagne C et al. Apolipoprotein B synthesis inhibition with mipomersen in heterozygous familial hypercholesterolemia: results of a randomized, double-blind, placebo-controlled trial to assess efficacy and safety as add-on therapy in patients with coronary artery disease. Circulation. 2012;126:2283–2292.

    Article  CAS  PubMed  Google Scholar 

  86. 86.

    McGowan MP, Tardif JC, Ceska R et al. Randomized, placebo-controlled trial of mipomersen in patients with severe hypercholesterolemia receiving maximally tolerated lipid-lowering therapy. PLoS One. 2012;7:e49006.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. 87.

    Raal FJ, Santos RD, Blom DJ et al. Mipomersen, an apolipoprotein B synthesis inhibitor, for lowering of LDL cholesterol concentrations in patients with homozygous familial hypercholesterolaemia: a randomised, double-blind, placebo-controlled trial. Lancet. 2010;375:998–1006.

    Article  CAS  PubMed  Google Scholar 

  88. 88.

    Duell PB, Santos RD, Kirwan BA, Witztum JL, Tsimikas S, Kastelein JJP. Long-term mipomersen treatment is associated with a reduction in cardiovascular events in patients with familial hypercholesterolemia. J Clin Lipidol. 2016;10:1011–1021.

    Article  PubMed  Google Scholar 

  89. 89.

    Santos RD, Raal FJ, Catapano AL, Witztum JL, Steinhagen-Thiessen E, Tsimikas S. Mipomersen, an antisense oligonucleotide to apolipoprotein B-100, reduces lipoprotein(a) in various populations with hypercholesterolemia: results of 4 phase III trials. Arterioscler Thromb Vasc Biol. 2015;35:689–699.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. 90.

    Marcovina SM, Albers JJ, Wijsman E, Zhang Z, Chapman NH, Kennedy H. Differences in Lp[a] concentrations and apo[a] polymorphs between black and white Americans. J Lipid Res. 1996;37:2569–2585.

    CAS  PubMed  Google Scholar 

  91. 91.

    Refusal of the marketing authorisation for Kynamro (mipomersen). European Medicines Agency. 2012 [online]. Available at: www.ema.europa.eu/docs/en_GB/document_library/Summary_of_opinion_-_Initial_authorisation/human/002429/WC500136279.pdf. Accessed December 26, 2018.

  92. 92.

    Tan RY, Markus HS. Monogenic causes of stroke: now and the future. J Neurol. 2015;262:2601–2616.

    Article  PubMed  Google Scholar 

  93. 93.

    Tikka S, Ng YP, Di Maio G et al. CADASIL mutations and shRNA silencing of NOTCH3 affect actin organization in cultured vascular smooth muscle cells. J Cereb Blood Flow Metab. 2012;32:2171–2180.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. 94.

    Francis J, Raghunathan S, Khanna P. The role of genetics in stroke. Postgrad Med J. 2007;83:590–595.

    Article  PubMed  PubMed Central  Google Scholar 

  95. 95.

    Razvi SS, Davidson R, Bone I, Muir KW. The prevalence of cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) in the west of Scotland. J Neurol Neurosurg Psychiatry. 2005;76:739–741.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. 96.

    Chabriat H, Joutel A, Dichgans M, Tournier-Lasserve E, Bousser MG. Cadasil. Lancet Neurol. 2009;8:643–653.

    Article  PubMed  Google Scholar 

  97. 97.

    Andersson ER, Lendahl U. Therapeutic modulation of Notch signalling--are we there yet? Nat Rev Drug Discov. 2014;13:357–378.

    Article  CAS  PubMed  Google Scholar 

  98. 98.

    Bersano A, Bedini G, Oskam J et al. CADASIL: Treatment and Management Options. Curr Treat Options Neurol. 2017;19:31.

    Article  PubMed  Google Scholar 

  99. 99.

    Machuca-Parra AI, Bigger-Allen AA, Sanchez AV et al. Therapeutic antibody targeting of Notch3 signaling prevents mural cell loss in CADASIL. J Exp Med. 2017;214:2271–2282.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. 100.

    Rutten JW, Dauwerse HG, Peters DJ et al. Therapeutic NOTCH3 cysteine correction in CADASIL using exon skipping: in vitro proof of concept. Brain. 2016;139:1123–1135.

    Article  PubMed  Google Scholar 

  101. 101.

    Chung JW, Park SH, Kim N et al. Trial of ORG 10172 in Acute Stroke Treatment (TOAST) classification and vascular territory of ischemic stroke lesions diagnosed by diffusion-weighted imaging. J Am Heart Assoc. 2014;3.

  102. 102.

    Fisher M, Henninger N. Translational research in stroke: taking advances in the pathophysiology and treatment of stroke from the experimental setting to clinical trials. Curr Neurol Neurosci Rep. 2007;7:35–41.

    Article  CAS  PubMed  Google Scholar 

  103. 103.

    Saver JL. Time is brain--quantified. Stroke. 2006;37:263–266.

    Article  PubMed  Google Scholar 

  104. 104.

    Tissue plasminogen activator for acute ischemic stroke. The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group. N Engl J Med. 1995;333:1581–1587.

  105. 105.

    Hacke W, Kaste M, Fieschi C et al. Intravenous thrombolysis with recombinant tissue plasminogen activator for acute hemispheric stroke. The European Cooperative Acute Stroke Study (ECASS). JAMA. 1995;274:1017–1025.

    Article  CAS  PubMed  Google Scholar 

  106. 106.

    Hacke W, Kaste M, Fieschi C et al. Randomised double-blind placebo-controlled trial of thrombolytic therapy with intravenous alteplase in acute ischaemic stroke (ECASS II). Second European-Australasian Acute Stroke Study Investigators. Lancet. 1998;352:1245–1251.

    Article  CAS  PubMed  Google Scholar 

  107. 107.

    Hacke W, Kaste M, Bluhmki E et al. Thrombolysis with alteplase 3 to 4.5 hours after acute ischemic stroke. N Engl J Med. 2008;359:1317–1329.

    Article  CAS  PubMed  Google Scholar 

  108. 108.

    Thomalla G, Simonsen CZ, Boutitie F et al. MRI-Guided Thrombolysis for Stroke with Unknown Time of Onset. N Engl J Med. 2018;379:611–622.

    Article  PubMed  Google Scholar 

  109. 109.

    Intracerebral hemorrhage after intravenous t-PA therapy for ischemic stroke. The NINDS t-PA Stroke Study Group. Stroke. 1997;28:2109–2118.

  110. 110.

    Yaghi S, Willey JZ, Cucchiara B et al. Treatment and Outcome of Hemorrhagic Transformation After Intravenous Alteplase in Acute Ischemic Stroke: A Scientific Statement for Healthcare Professionals From the American Heart Association/American Stroke Association. Stroke. 2017;48:e343-e361.

    PubMed  Google Scholar 

  111. 111.

    Seners P, Turc G, Maier B, Mas JL, Oppenheim C, Baron JC. Incidence and Predictors of Early Recanalization After Intravenous Thrombolysis: A Systematic Review and Meta-Analysis. Stroke. 2016;47:2409–2412.

    Article  CAS  PubMed  Google Scholar 

  112. 112.

    Wang YF, Tsirka SE, Strickland S, Stieg PE, Soriano SG, Lipton SA. Tissue plasminogen activator (tPA) increases neuronal damage after focal cerebral ischemia in wild-type and tPA-deficient mice. Nat Med. 1998;4:228–231.

    Article  CAS  PubMed  Google Scholar 

  113. 113.

    Tsirka SE, Gualandris A, Amaral DG, Strickland S. Excitotoxin-induced neuronal degeneration and seizure are mediated by tissue plasminogen activator. Nature. 1995;377:340–344.

    Article  CAS  PubMed  Google Scholar 

  114. 114.

    Wang X, Lee SR, Arai K et al. Lipoprotein receptor-mediated induction of matrix metalloproteinase by tissue plasminogen activator. Nat Med. 2003;9:1313–1317.

    Article  CAS  PubMed  Google Scholar 

  115. 115.

    Bjerregaard N, Botkjaer KA, Helsen N, Andreasen PA, Dupont DM. Tissue-type plasminogen activator-binding RNA aptamers inhibiting low-density lipoprotein receptor family-mediated internalisation. Thromb Haemost. 2015;114:139–149.

    Article  PubMed  Google Scholar 

  116. 116.

    Yepes M, Sandkvist M, Moore EG, Bugge TH, Strickland DK, Lawrence DA. Tissue-type plasminogen activator induces opening of the blood-brain barrier via the LDL receptor-related protein. J Clin Invest. 2003;112:1533–1540.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  117. 117.

    Shen Q, Ren H, Cheng H, Fisher M, Duong TQ. Functional, perfusion and diffusion MRI of acute focal ischemic brain injury. J Cereb Blood Flow Metab. 2005;25:1265–1279.

    Article  PubMed  PubMed Central  Google Scholar 

  118. 118.

    Fisher M. The ischemic penumbra: a new opportunity for neuroprotection. Cerebrovasc Dis. 2006;21 Suppl 2:64–70.

    Article  PubMed  Google Scholar 

  119. 119.

    Puyal J, Ginet V, Clarke PG. Multiple interacting cell death mechanisms in the mediation of excitotoxicity and ischemic brain damage: a challenge for neuroprotection. Prog Neurobiol. 2013;105:24–48.

    Article  PubMed  Google Scholar 

  120. 120.

    Goyal M, Hill MD, Saver JL, Fisher M. Challenges and Opportunities of Endovascular Stroke Therapy. Ann Neurol. 2016;79:11–17.

    Article  PubMed  Google Scholar 

  121. 121.

    Bracard S, Ducrocq X, Mas JL et al. Mechanical thrombectomy after intravenous alteplase versus alteplase alone after stroke (THRACE): a randomised controlled trial. Lancet Neurol. 2016;15:1138–1147.

    Article  CAS  PubMed  Google Scholar 

  122. 122.

    Jovin TG, Chamorro A, Cobo E et al. Thrombectomy within 8 hours after symptom onset in ischemic stroke. N Engl J Med. 2015;372:2296–2306.

    Article  CAS  PubMed  Google Scholar 

  123. 123.

    Saver JL, Goyal M, Bonafe A et al. Stent-retriever thrombectomy after intravenous t-PA vs. t-PA alone in stroke. New England Journal of Medicine. 2015;372:2285–2295.

    Article  CAS  PubMed  Google Scholar 

  124. 124.

    Goyal M, Demchuk AM, Menon BK et al. Randomized assessment of rapid endovascular treatment of ischemic stroke. N Engl J Med. 2015;372:1019–1030.

    Article  CAS  PubMed  Google Scholar 

  125. 125.

    Berkhemer OA, Fransen PS, Beumer D et al. A randomized trial of intraarterial treatment for acute ischemic stroke. New England Journal of Medicine. 2015;372:11–20.

    Article  CAS  PubMed  Google Scholar 

  126. 126.

    Campbell BC, Mitchell PJ, Kleinig TJ et al. Endovascular therapy for ischemic stroke with perfusion-imaging selection. N Engl J Med. 2015;372:1009–1018.

    Article  CAS  PubMed  Google Scholar 

  127. 127.

    Albers GW, Marks MP, Kemp S et al. Thrombectomy for Stroke at 6 to 16 Hours with Selection by Perfusion Imaging. N Engl J Med. 2018;378:708–718.

    Article  PubMed  PubMed Central  Google Scholar 

  128. 128.

    Nogueira RG, Jadhav AP, Haussen DC et al. Thrombectomy 6 to 24 Hours after Stroke with a Mismatch between Deficit and Infarct. N Engl J Med. 2018;378:11–21.

    Article  PubMed  Google Scholar 

  129. 129.

    Henninger N, Kumar R, Fisher M. Acute ischemic stroke therapy. Expert Rev Cardiovasc Ther. 2010;8:1389–1398.

    Article  PubMed  Google Scholar 

  130. 130.

    O'Collins VE, Macleod MR, Donnan GA, Horky LL, van der Worp BH, Howells DW. 1,026 experimental treatments in acute stroke. Ann Neurol. 2006;59:467–477.

    Article  CAS  PubMed  Google Scholar 

  131. 131.

    Savitz SI, Fisher M. Future of neuroprotection for acute stroke: in the aftermath of the SAINT trials. Ann Neurol. 2007;61:396–402.

    Article  CAS  PubMed  Google Scholar 

  132. 132.

    Stroke Therapy Academic Industry R. Recommendations for standards regarding preclinical neuroprotective and restorative drug development. Stroke. 1999;30:2752–2758.

    Article  Google Scholar 

  133. 133.

    Stroke Therapy Academic Industry R, II. Recommendations for clinical trial evaluation of acute stroke therapies. Stroke. 2001;32:1598–1606.

    Article  Google Scholar 

  134. 134.

    Fisher M, Feuerstein G, Howells DW et al. Update of the stroke therapy academic industry roundtable preclinical recommendations. Stroke. 2009;40:2244–2250.

    Article  PubMed  PubMed Central  Google Scholar 

  135. 135.

    Saver JL, Jovin TG, Smith WS et al. Stroke treatment academic industry roundtable: research priorities in the assessment of neurothrombectomy devices. Stroke. 2013;44:3596–3601.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  136. 136.

    Sena E, van der Worp HB, Howells D, Macleod M. How can we improve the pre-clinical development of drugs for stroke? Trends Neurosci. 2007;30:433–439.

    Article  CAS  PubMed  Google Scholar 

  137. 137.

    Henninger N, Bratane BT, Bastan B, Bouley J, Fisher M. Normobaric hyperoxia and delayed tPA treatment in a rat embolic stroke model. J Cereb Blood Flow Metab. 2009;29:119–129.

    Article  CAS  PubMed  Google Scholar 

  138. 138.

    Bardutzky J, Meng X, Bouley J, Duong TQ, Ratan R, Fisher M. Effects of intravenous dimethyl sulfoxide on ischemia evolution in a rat permanent occlusion model. J Cereb Blood Flow Metab. 2005;25:968–977.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  139. 139.

    Terpolilli NA, Kim SW, Thal SC et al. Inhalation of nitric oxide prevents ischemic brain damage in experimental stroke by selective dilatation of collateral arterioles. Circ Res. 2012;110:727–738.

    Article  CAS  PubMed  Google Scholar 

  140. 140.

    Sorensen SS, Nygaard AB, Nielsen MY, Jensen K, Christensen T. miRNA expression profiles in cerebrospinal fluid and blood of patients with acute ischemic stroke. Transl Stroke Res. 2014;5:711–718.

    Article  CAS  PubMed  Google Scholar 

  141. 141.

    Di Y, Lei Y, Yu F, Changfeng F, Song W, Xuming M. MicroRNAs expression and function in cerebral ischemia reperfusion injury. J Mol Neurosci. 2014;53:242–250.

    Article  CAS  PubMed  Google Scholar 

  142. 142.

    Yuan Y, Kang R, Yu Y et al. Crosstalk between miRNAs and their regulated genes network in stroke. Sci Rep. 2016;6:20429.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  143. 143.

    Baczynska D, Michalowska D, Witkiewicz W. The role of microRNA in ischemic diseases--impact on the regulation of inflammatory apoptosis and angiogenesis processes, Przegl Lek. 2013;70:135–142.

    PubMed  Google Scholar 

  144. 144.

    Sun H, Zhong D, Jin J, Liu Q, Wang H, Li G. Upregulation of miR-215 exerts neuroprotection effects against ischemic injury via negative regulation of Act1/IL-17RA signaling. Neurosci Lett. 2018;662:233–241.

    Article  CAS  PubMed  Google Scholar 

  145. 145.

    Wu-Wong JR, Nakane M, Ma J. Vitamin D analogs modulate the expression of plasminogen activator inhibitor-1, thrombospondin-1 and thrombomodulin in human aortic smooth muscle cells. J Vasc Res. 2007;44:11–18.

    Article  CAS  PubMed  Google Scholar 

  146. 146.

    Hacke W, Schwab S, Horn M, Spranger M, De Georgia M, von Kummer R. 'Malignant' middle cerebral artery territory infarction: clinical course and prognostic signs. Arch Neurol. 1996;53:309–315.

    Article  CAS  PubMed  Google Scholar 

  147. 147.

    Vahedi K, Hofmeijer J, Juettler E et al. Early decompressive surgery in malignant infarction of the middle cerebral artery: a pooled analysis of three randomised controlled trials. Lancet Neurol. 2007;6:215–222.

    Article  PubMed  Google Scholar 

  148. 148.

    Xu X, Wen Z, Zhao N et al. MicroRNA-1906, a Novel Regulator of Toll-Like Receptor 4, Ameliorates Ischemic Injury after Experimental Stroke in Mice. J Neurosci. 2017;37:10498–10515.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  149. 149.

    Ouyang YB. Inflammation and stroke. Neurosci Lett. 2013;548:1–3.

    Article  CAS  PubMed  Google Scholar 

  150. 150.

    Ouyang YB, Stary CM, Yang GY, Giffard R. microRNAs: innovative targets for cerebral ischemia and stroke. Curr Drug Targets. 2013;14:90–101.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  151. 151.

    Di G, Wang Z, Wang W, Cheng F, Liu H. AntagomiR-613 protects neuronal cells from oxygen glucose deprivation/re-oxygenation via increasing SphK2 expression. Biochem Biophys Res Commun. 2017;493:188–194.

    Article  CAS  PubMed  Google Scholar 

  152. 152.

    Yao X, Wang Y, Zhang D. microRNA-21 Confers Neuroprotection Against Cerebral Ischemia-Reperfusion Injury and Alleviates Blood-Brain Barrier Disruption in Rats via the MAPK Signaling Pathway. J Mol Neurosci. 2018;65:43–53.

    Article  CAS  PubMed  Google Scholar 

  153. 153.

    Song S, Lin F, Zhu P et al. Extract of Spatholobus suberctus Dunn ameliorates ischemia-induced injury by targeting miR-494. PLoS One. 2017;12:e0184348.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  154. 154.

    Toyama T, Wada-Takahashi S, Takamichi M et al. Reactive oxygen species scavenging activity of Jixueteng evaluated by electron spin resonance (ESR) and photon emission. Nat Prod Commun. 2014;9:1755–1759.

    PubMed  Google Scholar 

  155. 155.

    Bazan HA, Hatfield SA, Brug A, Brooks AJ, Lightell DJ, Jr., Woods TC. Carotid Plaque Rupture Is Accompanied by an Increase in the Ratio of Serum circR-284 to miR-221 Levels. Circ Cardiovasc Genet. 2017;10.

  156. 156.

    Sheth KN, Elm JJ, Molyneaux BJ et al. Safety and efficacy of intravenous glyburide on brain swelling after large hemispheric infarction (GAMES-RP): a randomised, double-blind, placebo-controlled phase 2 trial. Lancet Neurol. 2016;15:1160–1169.

    Article  CAS  PubMed  Google Scholar 

  157. 157.

    Zador Z, Stiver S, Wang V, Manley GT. Role of aquaporin-4 in cerebral edema and stroke. Handb Exp Pharmacol. 2009:159–170.

  158. 158.

    Pirici I, Balsanu TA, Bogdan C et al. Inhibition of Aquaporin-4 Improves the Outcome of Ischaemic Stroke and Modulates Brain Paravascular Drainage Pathways. Int J Mol Sci. 2017;19.

  159. 159.

    Nicchia GP, Frigeri A, Liuzzi GM, Svelto M. Inhibition of aquaporin-4 expression in astrocytes by RNAi determines alteration in cell morphology, growth, and water transport and induces changes in ischemia-related genes. Faseb j. 2003;17:1508–1510.

    Article  CAS  PubMed  Google Scholar 

  160. 160.

    Cramer SC. Treatments to Promote Neural Repair after Stroke. J Stroke. 2018;20:57–70.

    Article  PubMed  PubMed Central  Google Scholar 

  161. 161.

    Zhang ZG, Chopp M. Neurorestorative therapies for stroke: underlying mechanisms and translation to the clinic. Lancet Neurol. 2009;8:491–500.

    Article  PubMed  PubMed Central  Google Scholar 

  162. 162.

    Dancause N, Barbay S, Frost SB et al. Extensive cortical rewiring after brain injury. J Neurosci. 2005;25:10167–10179.

    Article  CAS  PubMed  Google Scholar 

  163. 163.

    Chopp M, Li Y, Zhang ZG. Mechanisms underlying improved recovery of neurological function after stroke in the rodent after treatment with neurorestorative cell-based therapies. Stroke. 2009;40:S143–145.

    Article  PubMed  Google Scholar 

  164. 164.

    Jones TA, Kleim JA, Greenough WT. Synaptogenesis and dendritic growth in the cortex opposite unilateral sensorimotor cortex damage in adult rats: a quantitative electron microscopic examination. Brain Res. 1996;733:142–148.

    Article  CAS  PubMed  Google Scholar 

  165. 165.

    Ding G, Jiang Q, Li L et al. Magnetic resonance imaging investigation of axonal remodeling and angiogenesis after embolic stroke in sildenafil-treated rats. J Cereb Blood Flow Metab. 2008;28:1440–1448.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  166. 166.

    Clarkson AN, Huang BS, Macisaac SE, Mody I, Carmichael ST. Reducing excessive GABA-mediated tonic inhibition promotes functional recovery after stroke. Nature. 2010;468:305–309.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  167. 167.

    Hermann DM, Chopp M. Promoting brain remodelling and plasticity for stroke recovery: therapeutic promise and potential pitfalls of clinical translation. Lancet Neurol. 2012;11:369–380.

    Article  PubMed  PubMed Central  Google Scholar 

  168. 168.

    Chollet F, Tardy J, Albucher JF et al. Fluoxetine for motor recovery after acute ischaemic stroke (FLAME): a randomised placebo-controlled trial. Lancet Neurol. 2011;10:123–130.

    Article  CAS  PubMed  Google Scholar 

  169. 169.

    Graham C, Lewis S, Forbes J et al. The FOCUS, AFFINITY and EFFECTS trials studying the effect(s) of fluoxetine in patients with a recent stroke: statistical and health economic analysis plan for the trials and for the individual patient data meta-analysis. Trials. 2017;18:627.

    Article  PubMed  PubMed Central  Google Scholar 

  170. 170.

    Carmichael ST. Gene expression changes after focal stroke, traumatic brain and spinal cord injuries. Curr Opin Neurol. 2003;16:699–704.

    Article  PubMed  Google Scholar 

  171. 171.

    Liu XS, Zhang ZG, Zhang RL et al. Stroke induces gene profile changes associated with neurogenesis and angiogenesis in adult subventricular zone progenitor cells. J Cereb Blood Flow Metab. 2007;27:564–574.

    Article  CAS  PubMed  Google Scholar 

  172. 172.

    Costa A, Afonso J, Osorio C et al. miR-363-5p regulates endothelial cell properties and their communication with hematopoietic precursor cells. J Hematol Oncol. 2013;6:87.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  173. 173.

    Pecot CV, Rupaimoole R, Yang D et al. Tumour angiogenesis regulation by the miR-200 family. Nat Commun. 2013;4:2427.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  174. 174.

    Suarez Y, Fernandez-Hernando C, Pober JS, Sessa WC. Dicer dependent microRNAs regulate gene expression and functions in human endothelial cells. Circ Res. 2007;100:1164–1173.

    Article  CAS  PubMed  Google Scholar 

  175. 175.

    Huang X, Le QT, Giaccia AJ. MiR-210--micromanager of the hypoxia pathway. Trends Mol Med. 2010;16:230–237.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  176. 176.

    Liu DZ, Tian Y, Ander BP et al. Brain and blood microRNA expression profiling of ischemic stroke, intracerebral hemorrhage, and kainate seizures. J Cereb Blood Flow Metab. 2010;30:92–101.

    Article  CAS  PubMed  Google Scholar 

  177. 177.

    Yin KJ, Hamblin M, Chen YE. Angiogenesis-regulating microRNAs and Ischemic Stroke. Curr Vasc Pharmacol. 2015;13:352–365.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  178. 178.

    Yang X, Tang X, Sun P et al. MicroRNA-15a/16-1 Antagomir Ameliorates Ischemic Brain Injury in Experimental Stroke. Stroke. 2017;48:1941–1947.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  179. 179.

    Caballero-Garrido E, Pena-Philippides JC, Lordkipanidze T et al. In Vivo Inhibition of miR-155 Promotes Recovery after Experimental Mouse Stroke. J Neurosci. 2015;35:12446–12464.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  180. 180.

    Zhang Y, Ueno Y, Liu XS et al. The MicroRNA-17-92 cluster enhances axonal outgrowth in embryonic cortical neurons. J Neurosci. 2013;33:6885–6894.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  181. 181.

    Xin H, Katakowski M, Wang F et al. MicroRNA cluster miR-17-92 Cluster in Exosomes Enhance Neuroplasticity and Functional Recovery After Stroke in Rats. Stroke. 2017;48:747–753.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  182. 182.

    Bouley J, Fisher M, Henninger N. Comparison between coated vs. uncoated suture middle cerebral artery occlusion in the rat as assessed by perfusion/diffusion weighted imaging. Neurosci Lett. 2007;412:185–190.

    Article  CAS  PubMed  Google Scholar 

  183. 183.

    Henninger N, Sicard KM, Schmidt KF, Bardutzky J, Fisher M. Comparison of ischemic lesion evolution in embolic versus mechanical middle cerebral artery occlusion in Sprague Dawley rats using diffusion and perfusion imaging. Stroke. 2006;37:1283–1287.

    Article  PubMed  Google Scholar 

  184. 184.

    Zaidi SF, Aghaebrahim A, Urra X et al. Final infarct volume is a stronger predictor of outcome than recanalization in patients with proximal middle cerebral artery occlusion treated with endovascular therapy. Stroke. 2012;43:3238–3244.

    Article  PubMed  Google Scholar 

  185. 185.

    Yoo AJ, Chaudhry ZA, Nogueira RG et al. Infarct Volume Is a Pivotal Biomarker After Intra-Arterial Stroke Therapy. Stroke. 2012.

  186. 186.

    Schiemanck SK, Kwakkel G, Post MW, Prevo AJ. Predictive value of ischemic lesion volume assessed with magnetic resonance imaging for neurological deficits and functional outcome poststroke: A critical review of the literature. Neurorehabil Neural Repair. 2006;20:492–502.

    Article  CAS  PubMed  Google Scholar 

  187. 187.

    Ay H, Furie KL, Singhal A, Smith WS, Sorensen AG, Koroshetz WJ. An evidence-based causative classification system for acute ischemic stroke. Ann Neurol. 2005;58:688–697.

    Article  PubMed  Google Scholar 

  188. 188.

    Amarenco P, Bogousslavsky J, Caplan LR, Donnan GA, Hennerici MG. Classification of stroke subtypes. Cerebrovasc Dis. 2009;27:493–501.

    Article  CAS  PubMed  Google Scholar 

  189. 189.

    Liu X, Cheng Y, Yang J, Xu L, Zhang C. Cell-specific effects of miR-221/222 in vessels: molecular mechanism and therapeutic application. J Mol Cell Cardiol. 2012;52:245–255.

    Article  CAS  PubMed  Google Scholar 

  190. 190.

    Matthews H, Hanison J, Nirmalan N. “Omics”-Informed Drug and Biomarker Discovery: Opportunities, Challenges and Future Perspectives. Proteomes. 2016;4.

Download references

Required Author Forms

Disclosure forms provided by the authors are available with the online version of this article.

Funding

Dr. Henninger is supported by K08NS091499 from the National Institute of Neurological Disorders and Stroke of the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author information

Affiliations

Authors

Contributions

Dr. Henninger and Dr. Mayasi drafted the article.

Corresponding author

Correspondence to Nils Henninger.

Ethics declarations

Competing Interests

Dr. Henninger serves on the advisory board of Omniox, Inc. and Portola Pharmaceuticals, Inc., and as a consultant for Astrocyte Pharmaceuticals, Inc. Dr. Mayasi declares no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(PDF 433 kb)

ESM 2

(PDF 365 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Henninger, N., Mayasi, Y. Nucleic Acid Therapies for Ischemic Stroke. Neurotherapeutics 16, 299–313 (2019). https://doi.org/10.1007/s13311-019-00710-x

Download citation

Keywords

  • Stroke
  • Neuroprotection
  • Nucleic acid
  • Prevention
  • Therapy
  • Review