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miR-195 Has a Potential to Treat Ischemic and Hemorrhagic Stroke through Neurovascular Protection and Neurogenesis

Tissue plasminogen activator is the only U.S. FDA-approved therapy for ischemic stroke, while there is no specific medication for hemorrhagic stroke. Therefore, the treatment of acute stroke continues to be a major unmet clinical need. We explored the effects of miR-195 on neurovascular protection and its potential in treating acute stroke. Using both cellular and animal studies, we showed that miR-195’s beneficial effects are mediated by four mechanisms: (1) anti-apoptosis for injured neural cells by directly suppressing Sema3A/Cdc42/JNK signaling, (2) neural regeneration by promoting neural stem cell proliferation and migration, (3) anti-inflammation by directly blocking the NF-kB pathway, and (4) improvement of endothelial functions. We intravenously injected miR-195 carried by nanoparticles into rats with either ischemic or hemorrhagic stroke in the acute stage. The results showed that miR-195 reduced the size of brain damage and improved functional recovery in both types of stroke rats. The reduction of injured brain volume could be up to 45% in ischemic stroke and approximately 30% in hemorrhagic stroke. The therapeutic window between stroke onset and miR-195 treatment could be up to 6 h. Our data demonstrated that miR-195 possesses the potential to become a new drug to treat acute ischemic and hemorrhagic stroke.


INTRODUCTION
Stroke is a leading cause of death and disability worldwide. Stroke can be characterized as ischemic or hemorrhagic, with ischemic stroke accounting for 85% of instances. 1 The intravenous administration of tissue plasminogen activator (tPA) within 3 h of stroke onset, and within 4.5 h of stroke onset in select patients, 2 is currently the only U.S. Food and Drug Administration (FDA)-approved therapy for ischemic stroke. Because of the time-dependent nature of tPA therapy and a fear of hemorrhagic complications, only 1%-8% of potentially eligible patients have been treated with tPA. 3 In contrast to treatment for ischemic stroke, the recommendation for managing hemorrhagic stroke is supportive treatment because no specific medication has been developed. 4,5 Therefore, treatments for acute ischemic and hemorrhagic stroke continue to be a major unmet clinical need.
We previously showed that miR-195 exerts vasculoprotective effects. 6 In addition, aberrant expression of miR-195 has been reported to play a role in neurological and cardiovascular diseases. [7][8][9][10] Stroke is a vascular disorder that leads to neural death. Because miR-195 plays roles in both the cerebrovascular and neural systems, dys-regulation of miR-195 levels may be associated with stroke event and prognosis. The primary aim of the present study is to test whether and how miR-195 is involved in stroke. To test our aim, we first investigated the mechanisms behind the of beneficial effects exhibited by miR-195 using endothelial cells, vascular smooth muscle cells, neural cells, and neural stem cells (NSCs). On the basis of the results of these cellular studies, we reasoned that miR-195 may be useful when applied to cerebrovascular events, regardless of ischemic or hemorrhagic stroke. Subsequently, we used miR-195 to treat animals with acute ischemic and hemorrhagic stroke. The results of these animal studies confirmed the potential role of miR-195 in stroke therapy.

miR-195 Exerts Neuroprotection miR-195 Increases Viability of Damaged Cells
SH-SY5Y neural cells were subjected to oxygen-glucose deprivation (OGD) for 3 or 6 h to mimic ischemic conditions in the brain.
At 24 h, and miR-195 levels were significantly reduced ( Figure 1A). In addition, OGD reduced cell viability and increased lactate dehydrogenase (LDH) release in a duration-dependent manner (Figures S1A and S1B). Transfection of miR-195 dose-dependently increased cell viability and decreased LDH release ( Figures S1C and S1D). Flow cytometry analysis revealed that OGD increased SH-SY5Y cell entry into the sub-G1 phase, while miR-195 reduced the number of cells in the sub-G1 phase and restored the normal cell distribution in the G0/G1 phase ( Figure 1B). TUNEL assay further demonstrated that miR-195 substantially decreased OGD-induced cellular apoptosis ( Figure 1C). Besides, an increase in intracellular miR-195 levels led to upregulation of anti-apoptotic factor (Bcl-2) and downregulation of two apoptotic molecules (FasL and caspase-3) ( Figure 1D). These results suggest that miR-195 possesses a neuroprotective effect by anti-apoptosis.

miR-195 Suppresses Sema3A Expression
Semaphorin 3A (Sema3A) is a potent inducer for neuronal death. 11,12 The Sema3A gene was predicted as a direct target of miR-195 by bioinformatic databases. There are two potential miR-195 binding sites at the Sema3A mRNA 3 0 UTR in human, rat, and mouse ( Figure 1E). We also experimentally confirmed that Sema3A can be knocked down by miR-195 (protein data in Figure 1F; RNA data in Figure S2). The luciferase reporter assay confirmed that miR-195 can bind to both binding sites in the Sema3A 3 0 UTR ( Figure 1G). The expression levels of Sema3A and its receptor, neuropilin (Nrp-1), were significantly increased in OGD-treated SH-SY5Y cells (protein data in Figure 1F; RNA data in Figure S2).
Knockdown of Sema3A reduced cell apoptosis ( Figure 1H), increased cell count, and reduced LDH release ( Figures S3A-S3C), confirming that miR-195's neuroprotective effect can be mediated through Sema3A repression. Cdc42 can affect cell cycle progression, regulate FasL expression, 13 activate caspase-3, and induce apoptosis. 14, 15 We previously demonstrated that Cdc42 is also a direct target of miR-195 6 ; furthermore, Cdc42 is a downstream molecule in the Sema3A signaling pathway. 16,17 Therefore, suppression of Sema3A also resulted in Cdc42 downregulation ( Figure S3D). These data suggest that miR-195 can regulate Sema3A and Cdc42 to achieve a neuroprotective effect. The miR195/Sema3A signaling pathway is schematically presented in Figure 1I.

miR-195 Improves Endothelial Functions
Given that endothelial cells (ECs) play multiple pivotal roles in stroke recovery, we tested the effects of miR-195 on endothelial functions. Transfection of miR-195 to human umbilical vein ECs (HUVECs) downregulated the expression of adhesion molecules and upregulated endothelial nitric oxide synthase (eNOS) expression (Figure 2A). To further mimic in vivo conditions, we assessed the effects of miR-195 on HUVECs in a HUVEC/human aortic smooth muscle cell (HASMC) co-culture. In the adhesion assay, oxidized low-density lipoprotein (oxLDL) treatment to HASMCs increased THP-1 adhesion to HUVECs in the HUVEC/HASMC co-culture. However, if oxLDL-treated HASMCs were also transfected with miR-195, THP-1 adhesion to HUVECs was reduced ( Figure 2B).

miR-195 Inhibits NF-kB Signaling in ECs
NF-kB signaling affects the expression of adhesion and inflammatory molecules in ECs. 18,19 We conducted a series of experiments to show that miR-195 inhibits NF-kB signaling, including IKKa and phosphorylated IkBs (p-IkBa and p-IkBb) ( Figure 2A). These inhibitory effects subsequently reduced ubiquitin-dependent IkB degradation and led to suppression of p65/p50 (p65 is also known as RelA) and RelB/p52 nuclear transposition. ( Figure S4).
CD40 is a protein expressed in the brain's neural and vascular cells that can stimulate the NF-kB pathway and was predicted to be a miR-195 direct target ( Figure S5A). 20 CD40 expression is markedly upregulated in the stroke hemisphere, and its levels are related to post-ischemia brain inflammation. 20 Our experiments confirmed that CD40 was directly suppressed by miR-195 (Figures 2A and  5B). Knockdown of CD40 in HUVECs reduced cell adhesion ( Figures  2C and 2D), decreased NF-kB signaling, and increased eNOS levels ( Figure 2D).

miR-195 Stimulates Neural Stem Cells
Proliferation and mobilization of NSCs are important factors for stroke recovery. 21 We used Nestin and SOX2 as NSC markers. Transfection of miR-195 to rat Nestin + /SOX2 + NSCs increased NSC proliferation ( Figure 2E). SDF-1 can increase stem cell proliferation and promote NSC function. SDF-1 secretion was increased by transfection of miR-195 to HASMCs ( Figure S6A), but miR-195 did not have the same effect when transfected to HUVEC, mouse brain ECs, or astrocytes ( Figures  S6A-S6C).

Exogenous miR-195 Treats Acute Ischemic Stroke
Given that miR-195 has multiple beneficial effects on the neurovascular system, we tested whether miR-195 could be used to treat acute stroke. First, we induced ischemic stroke in rats by middle cerebral artery occlusion (MCAO). 22 We used two different types of MCAO: (1) permanent MCAO, in which the artery was occluded for 24 h and the rats were sacrificed directly thereafter, and (2) transient MCAO, in which the artery was occluded for 2 h and the rats were sacrificed on day 5. miR-195 carried by the commercial PEI-based nanoparticles (called in vivo-jetPEI) was IV injected via the tail vein. Our preliminary study found that the optimal concentration was 10 nmol/kg ( Figure S7). For the rats subjected to permanent MCAO, miR-195 was injected at different time windows (0.5, 3, 4.5, and 6 h) after stroke. For the rats subjected to transient MCAO, miR-195 was given only at 6 h after stroke. NC-miR carried by in vivo-jetPEI was used as a placebo. To make the data comparable among rats subjected to transient MCAO, we calculated Garcia scores at 6 h post-stroke and selected only the rats with the scores between 6 and 8 for subsequent studies. As a result, all rats had similar levels of neurological deficiency upon enrollment for the study of transient MCAO.
The average infarct area of the treated group was significantly smaller than that of the placebo group for both permanent (Figures 3A and 3B) and transient ( Figure 3C) MCAO models. More important, miR-195 retained its therapeutic effect at the intervention time window of 6 h. Significant improvements in Garcia score were noted on days 3 and 5 in the transient MCAO rats ( Figure 3D). In the miR-195-treated rats, miR-195 levels were higher in the lesion cortex and subcortex than in the contralateral normal cortex and subcortex ( Figure 3E). Conversely, in the placebo group, miR-195 levels were significantly lower in the lesion cortex and subcortex than in their counterparts. Furthermore, there was a negative correlation between miR-195 levels in the infarct hemisphere and infarct size (Pearson correlation = À0.78, p = 0.0075; Table S1).

Pharmacokinetics and Biodistribution
Seven normal Sprague-Dawley (SD) rats were independently used to test miR-195 concentrations in the circulation (i.e., pharmacokinetic study). Five minutes after injection of nanoparticle-carried miR-195 (10 nmol/kg), circulating miR-195 levels had increased by more than 390-fold (ranging from 390-fold to 1,025-fold). The half-life (T 1/2 ) varied among animals from a minimum of 893 min to a maximum of 6,650 min (Table S2). jetPEI can interfere with miR-195 detection. The wide range of T 1/2 may be because the dissociation of miR-195 from jetPEI varied among animals. Another 18 normal SD rats received the same treatment and were used to measure the distribution of miR-195 in the rats' organs. At each of the six time points (pre-dose and 1, 6, 24, 48, and 72 h), three rats were sacrificed. As expected, the highest concentration was in the serum, followed by the heart and then the lungs ( Figure S8). Because these animals did not have stroke, the exogenous miR-195 barely affected miR-195 levels in the brain.

Confirmation of miR-195 Effects In Vivo
To replicate the effects of miR-195 demonstrated in our cellular studies, we measured anti-apoptotic markers, inflammatory substances, endothelial functions, and NSCs in the rodent brains subjected to permanent MCAO. The expression patterns of Sema3A, Nrp1, caspase-3, FasL, CdC42, and Bcl-2 in the brains were consistent with the results we obtained in the cellular studies ( Figures 3F  and 3G). Similarly, miR-195 possessed anti-inflammatory effects by decreasing TNFa, IL-1b, IL-6, and MCP-1 levels in the brains (Figure S9). Again, improvement of endothelial function by miR-195 was demonstrated through changes of the mRNA levels of inducible nitric oxide synthase (iNOS), eNOS, vascular cell adhesion molecule (VCAM), and intercellular adhesion molecule (ICAM) in the brains ( Figure S10).
To demonstrate the effect of miR-195 on NSCs in vivo, we examined the brains from rats subjected to transient MCAO. On day 5 poststroke, SDF-1 levels were higher in the miR-195-treated rats than in the NC-miR placebo rats ( Figure 4A). Most NSCs co-expressed both SOX2 and Nestin markers. Both SOX2 + /Nestin + cells in the subventricular zone (SVZ) ( Figure 4B) and peri-third ventricle (Figure 4C) were significantly increased in the miR-195-treated group compared with the placebo group. Notably, because stroke can also increases NSC proliferation, as a result there were more NSCs on the ischemic side (ipsilateral) than the uninjured contralateral side of the placebo group ( Figure 4C). GAP43 is highly expressed in neuronal growth cones during development and axonal regeneration; therefore, the GAP43 + cells indicated that miR-195 promoted neuronal regeneration in multiple areas of the infarct brain ( Figure 4D).

Exogenous miR-195 Treats Acute Hemorrhagic Stroke
We further examined the effect of miR-195 on acute hemorrhagic stroke in rats by IV injecting identically formulated miR-195 at 4 h post-stroke. The miR-195-treated group contained 31 rats, and the NC-miR placebo group contained 32 rats. All rats were evaluated for Garcia score, and the rats were sacrificed on day 3. Because the preparation of brain tissue for the measurement of edema, stroke lesion volume, and permeability differs, the rats were divided into groups of 16, 29, and 18 to be used for these three measurements, respectively. The results showed that miR-195 treatment significantly improved the functional outcome, as indicated by a higher Garcia score (all p values were 0.001 on days 1, 2, and 3; Figure 5A). miR-195 also significantly reduced brain edema (p = 0.0006; Figure 5B), lesion volume (p = 0.01; Figure 5C), and permeability (p = 0.002; Figure 5D) on day 3.

DISCUSSION
We showed that miR-195 can inhibit apoptosis in damaged neural cells, increase SDF-1 to promote NSCs for neurogenesis, block the NF-kB pathway for anti-inflammation, and improve endothelial functions. Figure 5E schematically shows the effects of miR-195 discovered in this study. These beneficial effects were also revealed by reduced brain damage and improved functional recovery in animals that had suffered from ischemic and hemorrhagic stroke. Accordingly, miR-195 possesses the potential to serve as a new drug to treat patients with acute stroke and other types of brain injury.
Current treatment and many ongoing clinical trials for ischemic stroke focus on thrombolytic therapy. However, it is important to distinguish whether the stroke is caused by ischemia or hemorrhage before using thrombolytic therapy, because this treatment may cause brain hemorrhaging that can further complicate the already injured brain. miR-195, on the other hand, has the potential to be used for both ischemic and hemorrhagic stroke; this advantage may make it more suitable for emerging treatment regardless of the stroke type. A recent breakthrough in treating acute ischemic stroke, intra-arterial clot retrieval, was validated in five clinical trials published in 2015. [23][24][25][26][27] Thus far, two strategies have been tested to treat ischemic stroke: restoring blood flow (tPA, clot retrieval) and reducing neural injury. These two strategies can have a synergistic effect. If miR-195 can be further proved effective in reducing brain injury, it may be applied to ischemic stroke patients before the procedure of clot retrieval is performed.
The change of miR-195 levels in damaged cells and tissues can be time, location, and cell type dependent. For example, the miR-195 level rapidly decreased in infarcted and border zones but dramatically increased in remote zones 1 h after myocardial ischemia. 28 On the contrary, prolonged myocardial ischemia caused the elevation of miR-195 levels in infarcted and border zones but suppression in remote zones. 28 Previous studies have reported that miR-195 can suppress antiapoptosis gene BCL2 in cancer cell lines 29 and cardiomyoctes, 30 which opposes our finding in OGD-treated SY5Y cells. We also tested for the effect of miR-195 on mouse neural cell line (N2A cells) and found that miR-195 did not suppress BCL2 expression ( Figure S11). Similar to our findings, Ai et al. 7 showed that miR-195 decreased in hypoxia-damaged neurons and reported that miR-195 prevented neuron death by suppressing the caspase pathway. 31 It should be noted that microRNA's suppressive effect is influenced by its target gene expression level, and a single microRNA can exert opposite effects under different conditions. 32,33 Therefore, the effects of miR-195 in different cells and organs may not be directly comparable.
Sema3A is upregulated when the brain is injured. 34 The increase in Sema3A signaling elevates cerebrovascular permeability, 35 and secreted Sema3A can additionally interact with its receptor Nrp-1 to inhibit neuronal regeneration and enhance neuronal death. 11,34,36 Sema3A can upregulate Cdc42 expression, and a decrease in Cdc42 expression has been reported to attenuate neuronal apoptosis in the rodent stroke model. 37 By inhibiting the cascade of Sema3A/ Nrp-1/Cdc42 and increasing Bcl-2 levels, miR-195 can achieve an anti-apoptotic effect in the injured brain and reduce neuronal death. Consistent with our results, a recent study further demonstrated a significant decrease of infarct volume in Sema3A-knockout mice subjected to ischemic stroke. 35 NSCs play a critical role in neurogenesis after stroke. NSCs need to migrate from the stem cell niches to the peri-infarct area to perform neurogenesis. We examined NSCs in the classic SVZ niche and a novel niche 38 (peri-third ventricle), and our data showed that miR-195 increased NSCs in both niches in the animals that had suffered strokes. These results were consistent with the previous report indicating that neurogenesis can be attributed to NSCs from multiple niches. 39 Numerous studies using the MCAO model have uniformly shown that proliferating NSCs are capable of migrating toward the injured sites in order to differentiate to new neurons. 38 However, NSCs can deplete over time after ischemic injury. 38 Our data revealed that miR-195 increases the number of NSCs and that these stem cells are likely to differentiate to functional neurons and glia because increased GAP43 expression was considered a marker of neuronal development and axonal regeneration. 40 Notably, GAP43 plays a role to regulate nerve sprout associated with adult plasticity. 41 Collectively, miR-195 may wake up stem cells from the niches and increase stem cell functions, leading to a better poststroke outcome.
Stroke is more prevalent in the elderly, who often have chronic disease comorbidities. In this study, we induced artificial stroke in young animals. Therefore, the beneficial effects attributed to miR-195 may be attenuated in elderly subjects who suffer stroke. In the present study we did not measure physiological parameters for the stroke rats, and also we did not conduct longer term follow-up, which must be addressed in follow-up studies. Brain stroke triggers a complex and highly interconnected cascade of cellular and molecular events. Targeting multiple components of the neurovascular unit, rather than just neurons, should be a priority in stroke research. 42 In conclusion, the present study not only demonstrated the detailed mechanisms of neurovascular protection controlled by miR-195 but also showed that miR-195 can be used to treat both acute ischemic and acute hemorrhagic stroke in experimental animals. Furthermore, the long time window for miR-195 treatment can be important for future clinical practice. Therefore, the potential of miR-195 to treat cerebral vascular accidents warrants further investigation.

MATERIALS AND METHODS
Detailed information can be found in the Supplemental Information.

Cell Studies
OGD was used to mimics ischemia in vivo. Cells were cultured in the OGD condition for 3 or 6 h, and then cells were maintained in glucosecontaining DMEM with normoxia for 21 h (if OGD duration was 3 h) or 18 h (if OGD duration was 6 h). THP-1 cells were labeled with calcein AM and were added to the culture wells containing HUVECs for the monocyte adhesion assay. The luciferase reporter assay was used to test the existence of miR-195 binding sites in the targeted genes.

Viral miR Preparation
Adenoviruses were amplified by infection into HEK293A cells. Viral MOI was estimated on the basis of in vitro HEK293A transduction efficiency: 0.5 mL of undiluted viral stocks were added to 10 6 HEK293A cells, and number of GFP-positive cells was counted 48 h after transduction.

Animal Study
The procedure for balloon injury 6 and for ischemic stroke by MCAO is described elsewhere. 22,43 In the present study's permanent MCAO model, male SD rats were subjected to occlusion for 24 h and sacrificed directly thereafter, because of the high mortality rate after 24 h MCAO. In the transient MCAO model, rats were subjected to occlusion for 2 h. In this model, rats were included for further evaluation only if their Garcia scores were between 6 and 8 at 6 h post-stroke. The eligible transient MCAO rats were then sacrificed on day 5. For hemorrhagic stroke, male SD rats were subjected to stereotaxic infusion of bacterial collagenase to induce intracranial hemorrhage (ICH). Similarly, we only included hemorrhagic stroke rats with moderate to severe neurological impairment, and these animals were sacrificed on day 3. miR-195 mimic or normal control microRNA (NC-miR) was formulated with a commercial nanoparticle called jetPEI and then intravenously (IV) administrated via the tail vein. The operators were blinded to treatment assignment of miR-195 or NC-miR. The ischemic brain slices were stained with 0.1% 2,3,5-TTC. Digital photographs of the brain slices were taken, and lesion volume was computed using ImageJ version 1.40 (NIH). The total lesion volume was calculated as the sum of lesion area in each section.
The Garcia score was used to evaluate each rat's neurological function. The researchers who conducted the Garcia assessment were not informed of whether the rats were in the treated or placebo group. The assessment was performed on days 3 and 5 for transient MCAO rats and on days 1, 2, and 3 for hemorrhagic stroke rats. Because brain edema peaked on day 3 in hemorrhagic stroke, the brain was removed www.moleculartherapy.org on day 3 and dried in an oven to obtain the dry weight. The cerebellum was used as an internal control. Water content was expressed as a percentage of wet weight: [(wet weight) À (dry weight)] (wet weight) À1 Â 100%. Blood-brain barrier permeability was assessed using a modified Evans blue extravasation method on day 3.

Immunostaining
Immunofluorescent staining was used to detect cellular expression of SDF-1, SOX2, Nestin, and GAP43. Immunohistochemistry (IHC) staining was used to stain the markers in brain slices.

Study Approval
The Animal Care and Use Committee of Kaohsiung Medical University approved the animal experimental protocols (approval number IACUC-101068), which strictly conforms to the NIH Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996).

Statistical Analysis
All values in the text and figures are expressed as mean ± SE. Statistical differences were evaluated using Student's t test or analysis of covariance (ANCOVA). A p value less than 0.05 was considered to indicate statistical significance in all experiments. Analysis of the data and plotting of the figures were performed using SigmaPlot 10 software (Systat Software, San Jose, CA, USA).