Interleukin-4 mitigates anxiety-like behavior and loss of neurons and fiber tracts in limbic structures in a microglial PPARγ-dependent manner after traumatic brain injury.

Traumatic brain injury (TBI) is commonly followed by intractable psychiatric disorders and long-term changes in affect, such as anxiety. The present study sought to investigate the effect of repetitive intranasal delivery of interleukin-4 (IL-4) nanoparticles on affective symptoms after TBI in mice. Adult male C57BL/6 J mice (10-12 weeks of age) were subjected to controlled cortical impact (CCI) and assessed by a battery of neurobehavioral tests up to 35 days after CCI. Neuron numbers were counted in multiple limbic structures, and the integrity of limbic white matter tracts was evaluated using ex vivo diffusion tensor imaging (DTI). As STAT6 is a critical mediator of IL-4-specific transcriptional activation, STAT6 knockout mice were used to explore the role of endogenous IL-4/STAT6 signaling axis in TBI-induced affective disorders. We also employed microglia/macrophage (Mi/Mϕ)-specific PPARγ conditional knockout (mKO) mice to test if Mi/Mϕ PPARγ critically contributes to IL-4-afforded beneficial effects. We observed anxiety-like behaviors up to 35 days after CCI, and these measures were exacerbated in STAT6 KO mice but mitigated by repetitive IL-4 delivery. We discovered that IL-4 protected against neuronal loss in limbic structures, such as the hippocampus and the amygdala, and improved the structural integrity of fiber tracts connecting the hippocampus and amygdala. We also observed that IL-4 boosted a beneficial Mi/Mϕ phenotype (CD206+/Arginase 1+/PPARγ+ triple-positive) in the subacute injury phase, and that the numbers of Mi/Mϕ appositions with neurons were robustly correlated with long-term behavioral performances. Remarkably, PPARγ-mKO completely abolished IL-4-afforded protection. Thus, CCI induces long-term anxiety-like behaviors in mice, but these changes in affect can be attenuated by transnasal IL-4 delivery. IL-4 prevents the long-term loss of neuronal somata and fiber tracts in key limbic structures, perhaps due to a shift in Mi/Mϕ phenotype. Exogenous IL-4 therefore holds promise for future clinical management of mood disturbances following TBI.

Genetic deletion of Krüppel-like factor 11 aggravates traumatic brain injury.

BACKGROUND: The long-term functional recovery of traumatic brain injury (TBI) is hampered by pathological events, such as parenchymal neuroinflammation, neuronal death, and white matter injury. Krüppel-like transcription factor 11 (KLF 11) belongs to the zinc finger family of transcription factors and actively participates in various pathophysiological processes in neurological disorders. Up to now, the role and molecular mechanisms of KLF11 in regulating the pathogenesis of brain trauma is poorly understood. METHODS: KLF11 knockout (KO) and wild-type (WT) mice were subjected to experimental TBI, and sensorimotor and cognitive functions were evaluated by rotarod, adhesive tape removal, foot fault, water maze, and passive avoidance tests. Brain tissue loss/neuronal death was examined by MAP2 and NeuN immunostaining, and Cresyl violet staining. White matter injury was assessed by Luxol fast blue staining, and also MBP/SMI32 and Caspr/Nav1.6 immunostaining. Activation of cerebral glial cells and infiltration of blood-borne immune cells were detected by GFAP, Iba-1/CD16/32, Iba-1/CD206, Ly-6B, and F4/80 immunostaining. Brian parenchymal inflammatory cytokines were measured with inflammatory array kits. RESULTS: Genetic deletion of KLF11 worsened brain trauma-induced sensorimotor and cognitive deficits, brain tissue loss and neuronal death, and white matter injury in mice. KLF11 genetic deficiency in mice also accelerated post-trauma astrocytic activation, promoted microglial polarization to a pro-inflammatory phenotype, and increased the infiltration of peripheral neutrophils and macrophages into the brain parenchyma. Mechanistically, loss-of-KLF11 function was found to directly increase the expression of pro-inflammatory cytokines in the brains of TBI mice. CONCLUSION: KLF11 acts as a novel protective factor in TBI. KLF11 genetic deficiency in mice aggravated the neuroinflammatory responses, grey and white matter injury, and impaired long-term sensorimotor and cognitive recovery. Elucidating the functional importance of KLF11 in TBI may lead us to discover novel pharmacological targets for the development of effective therapies against brain trauma.

Endothelium-Targeted Deletion of microRNA-15a/16-1 Promotes Poststroke Angiogenesis and Improves Long-Term Neurological Recovery.

RATIONALE: Angiogenesis promotes neurological recovery after stroke and is associated with longer survival of stroke patients. Cerebral angiogenesis is tightly controlled by certain microRNAs (miRs), such as the miR-15a/16-1 cluster, among others. However, the function of the miR-15a/16-1 cluster in endothelium on postischemic cerebral angiogenesis is not known. OBJECTIVE: To investigate the functional significance and molecular mechanism of endothelial miR-15a/16-1 cluster on angiogenesis in the ischemic brain. METHODS AND RESULTS: Endothelial cell-selective miR-15a/16-1 conditional knockout (EC-miR-15a/16-1 cKO) mice and wild-type littermate controls were subjected to 1 hour middle cerebral artery occlusion followed by 28-day reperfusion. Deletion of miR-15a/16-1 cluster in endothelium attenuates post-stroke brain infarction and atrophy and improves the long-term sensorimotor and cognitive recovery against ischemic stroke. Endothelium-targeted deletion of the miR-15a/16-1 cluster also enhances post-stroke angiogenesis by promoting vascular remodeling and stimulating the generation of newly formed functional vessels, and increases the ipsilateral cerebral blood flow. Endothelial cell-selective deletion of the miR-15a/16-1 cluster up-regulated the protein expression of pro-angiogenic factors VEGFA (vascular endothelial growth factor), FGF2 (fibroblast growth factor 2), and their receptors VEGFR2 (vascular endothelial growth factor receptor 2) and FGFR1 (fibroblast growth factor receptor 1) after ischemic stroke. Consistently, lentiviral knockdown of the miR-15a/16-1 cluster in primary mouse or human brain microvascular endothelial cell cultures enhanced in vitro angiogenesis and up-regulated pro-angiogenic proteins expression after oxygen-glucose deprivation, whereas lentiviral overexpression of the miR-15a/16-1 cluster suppressed in vitro angiogenesis and down-regulated pro-angiogenic proteins expression. Mechanistically, miR-15a/16-1 translationally represses pro-angiogenic factors VEGFA, FGF2, and their receptors VEGFR2 and FGFR1, respectively, by directly binding to the complementary sequences within 3'-untranslated regions of those messenger RNAs. CONCLUSIONS: Endothelial miR-15a/16-1 cluster is a negative regulator for postischemic cerebral angiogenesis and long-term neurological recovery. Inhibition of miR-15a/16-1 function in cerebrovascular endothelium may be a legitimate therapeutic approach for stroke recovery.

Long Noncoding RNA Malat1 Regulates Cerebrovascular Pathologies in Ischemic Stroke.

The study was designed to determine the role of long noncoding RNA (lncRNA), metastasis-associated lung adenocarcinoma transcript 1 (Malat1), in ischemic stroke outcome. Primary mouse brain microvascular endothelial cells (BMECs) were cultured and treated with Malat1 GapmeR before 16 h oxygen and glucose depravation (OGD). Cell death was assayed by LDH and MTT methods. Malat1 knock-out and wild-type mice were subjected to 1 h of middle cerebral artery occlusion (MCAO) and 24-72 h of reperfusion. To explore the underlying mechanism, apoptotic and inflammatory factors were measured by qPCR, ELISA, and Western blotting. The physical interaction between Malat1 and apoptotic or inflammatory factors was measured by RNA immunoprecipitation. Increased Malat1 levels were found in cultured mouse BMECs after OGD as well as in isolated cerebral microvessels in mice after MCAO. Silencing of Malat1 by Malat1 GapmeR significantly increased OGD-induced cell death and Caspase 3 activity in BMECs. Silencing of Malat1 also significantly aggravated OGD-induced expression of the proapoptotic factor Bim and proinflammatory cytokines MCP-1, IL-6, and E-selectin. Moreover, Malat1 KO mice presented larger brain infarct size, worsened neurological scores, and reduced sensorimotor functions. Consistent with in vitro findings, significantly increased expression of proapoptotic and proinflammatory factors was also found in the cerebral cortex of Malat1 KO mice after ischemic stroke compared with WT controls. Finally, we demonstrated that Malat1 binds to Bim and E-selectin both in vitro and in vivo Our study suggests that Malat1 plays critical protective roles in ischemic stroke.SIGNIFICANCE STATEMENT Accumulative studies have demonstrated the important regulatory roles of microRNAs in vascular and neural damage after ischemic stroke. However, the functional significance and mechanisms of other classes of noncoding RNAs in cerebrovascular pathophysiology after stroke are less studied. Here we demonstrate a novel role of Malat1, a long noncoding RNA that has been originally identified as a prognostic marker for non-small cell lung cancer, in cerebrovascular pathogenesis of ischemic stroke. Our experiments have provided the first evidence that Malat1 plays anti-apoptotic and anti-inflammatory roles in brain microvasculature to reduce ischemic cerebral vascular and parenchymal damages. Our studies also suggest that lncRNAs can be therapeutically targeted to minimize poststroke brain damage.

Sex differences in abdominal aortic aneurysms.

Abdominal aortic aneurysm (AAA) is a vascular disorder with a high case fatality rate in the instance of rupture. AAA is a multifactorial disease, and the etiology is still not fully understood. AAA is more likely to occur in men, but women have a greater risk of rupture and worse prognosis. Women are reportedly protected against AAA possibly by premenopausal levels of estrogen and are, on average, diagnosed at older ages than men. Here, we review the present body of research on AAA pathophysiology in humans, animal models, and cultured cells, with an emphasis on sex differences and sex steroid hormone signaling.

Rapid estrogen receptor-α signaling mediated by ERK activation regulates vascular tone in male and ovary-intact female mice.

Estrogen has been shown to affect vascular reactivity. Here, we assessed the estrogen receptor-α (ERα) dependency of estrogenic effects on vasorelaxation via a rapid nongenomic pathway in both male and ovary-intact female mice. We compared the effect of a primary estrogen, 17ß-estradiol (E2) or 4,4',4″-(4-propyl-[1H]pyrazole-1,3,5-triyl)tris-phenol (PPT; selective ERα agonist). We found that E2 and PPT induced greater aortic relaxation in female mice than in male mice, indicating ERα mediation, which was further validated by using ERα antagonism. Treatment with 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP dihydrochloride; ERα antagonist) attenuated PPT-mediated vessel relaxation in both sexes. ERα-mediated vessel relaxation was further validated by the absence of significant PPT-mediated relaxation in aortas isolated from ERα knockout mice. Treatment with a specific ERK inhibitor, PD-98059, reduced E2-induced vessel relaxation in both sexes but to a lesser extent in female mice. Furthermore, PD-98059 prevented PPT-induced vessel relaxation in both sexes. Both E2 and PPT treatment activated ERK as early as 5-10 min, which was attenuated by PD-98059 in aortic tissue, cultured primary vascular smooth muscle cells (VSMCs), and endothelial cells (ECs). Aortic rings denuded of endothelium showed no differences in vessel relaxation after E2 or PPT treatment, implicating a role of ECs in the observed sex differences. Here, our results are unique to show estrogen-stimulated rapid ERα signaling mediated by ERK activation in aortic tissue, as well as VSMCs and ECs in vitro, in regulating vascular function by using side-by-side comparisons in male and ovary-intact female mice in response to E2 or PPT. NEW & NOTEWORTHY Here, we assessed the estrogen receptor-α dependency of estrogenic effects in vasorelaxation of both male and ovary-intact female mice by performing side-by-side comparisons. Also, we describe the connection between estrogen-stimulated rapid estrogen receptor-α signaling and downstream ERK activation in regulating vascular function in male and ovary-intact female mice.

Long Non-Coding RNA Malat1 Regulates Angiogenesis in Hindlimb Ischemia.

Angiogenesis is a complex process that depends on the delicate regulation of gene expression. Dysregulation of transcription during angiogenesis often leads to various human diseases. Emerging evidence has recently begun to show that long non-coding RNAs (lncRNAs) may mediate angiogenesis in both physiological and pathological conditions; concurrently, underlying molecular mechanisms are largely unexplored. Previously, our lab identified metastasis associates lung adenocarcinoma transcript 1 (Malat1) as an oxygen-glucose deprivation (OGD)-responsive endothelial lncRNA. Here we reported that genetic deficiency of Malat1 leads to reduced blood vessel formation and local blood flow perfusion in mouse hind limbs at one to four weeks after hindlimb ischemia. Malat1 and vascular endothelial growth factor receptor 2 (VEGFR2) levels were found to be increased in both cultured mouse primary skeletal muscle microvascular endothelial cells (SMMECs) after 16 h OGD followed by 24 h reperfusion and in mouse gastrocnemius muscle that underwent hindlimb ischemia followed by 28 days of reperfusion. Moreover, Malat1 silencing by locked nucleic acid (LNA)-GapmeRs significantly reduced tube formation, cell migration, and cell proliferation in SMMEC cultures. Mechanistically, RNA subcellular isolation and RNA-immunoprecipitation experiments demonstrate that Malat1 directly targets VEGFR2 to facilitate angiogenesis. The results suggest that Malat1 regulates cell-autonomous angiogenesis through direct regulation of VEGFR2.

MicroRNA-15a/16-1 Antagomir Ameliorates Ischemic Brain Injury in Experimental Stroke.

BACKGROUND AND PURPOSE: Dysregulation of the miR-15a/16-1 cluster in plasma has been reported in patients with stroke as a potential biomarker for diagnostic and prognostic use. However, the essential role and therapeutic potential of the miR-15a/16-1 cluster in ischemic stroke are poorly understood. This study is aimed at investigating the regulatory role of the miR-15a/16-1 cluster in ischemic brain injury and insight mechanisms. METHODS: Adult male miR-15a/16-1 knockout and wild-type mice, or adult male C57 BL/6J mice injected via tail vein with the miR-15a/16-1-specific inhibitor (antagomir, 30 pmol/g), were subjected to 1 hour of middle cerebral artery occlusion and 72 hours of reperfusion. The neurological scores, brain infarct volume, brain water content, and neurobehavioral tests were then evaluated and analyzed. To explore underlying signaling pathways associated with alteration of miR-15a/16-1 activity, major proinflammatory cytokines were measured by quantitative polymerase chain reaction or ELISA and antiapoptotic proteins were examined by Western blotting. RESULTS: Genetic deletion of the miR-15a/16-1 cluster or intravenous delivery of miR-15a/16-1 antagomir significantly reduced cerebral infarct size, decreased brain water content, and improved neurological outcomes in stroke mice. Inhibition of miR-15a/16-1 significantly decreased the expression of the proinflammatory cytokines interleukin-6, monocyte chemoattractant protein-1, vascular cell adhesion molecule 1, tumor necrosis factor alpha, and increased Bcl-2 and Bcl-w levels in the ischemic brain regions. CONCLUSIONS: Our data indicate that pharmacological inhibition of the miR-15a/16-1 cluster reduces ischemic brain injury via both upregulation of antiapoptotic proteins and suppression of proinflammatory molecules. These results suggest that the miR-15a/16-1 cluster is a novel therapeutic target for ischemic stroke.

Sex differences in vascular physiology and pathophysiology: estrogen and androgen signaling in health and disease.

Sex differences between women and men are often overlooked and underappreciated when studying the cardiovascular system. It has been long assumed that men and women are physiologically similar, and this notion has resulted in women being clinically evaluated and treated for cardiovascular pathophysiological complications as men. Currently, there is increased recognition of fundamental sex differences in cardiovascular function, anatomy, cell signaling, and pathophysiology. The National Institutes of Health have enacted guidelines expressly to gain knowledge about ways the sexes differ in both normal function and diseases at the various research levels (molecular, cellular, tissue, and organ system). Greater understanding of these sex differences will be used to steer future directions in the biomedical sciences and translational and clinical research. This review describes sex-based differences in the physiology and pathophysiology of the vasculature, with a special emphasis on sex steroid receptor (estrogen and androgen receptor) signaling and their potential impact on vascular function in health and diseases (e.g., atherosclerosis, hypertension, peripheral artery disease, abdominal aortic aneurysms, cerebral aneurysms, and stroke).

The long noncoding RNA Malat1: Its physiological and pathophysiological functions.

Recent studies suggest that in humans, DNA sequences responsible for protein coding regions comprise only 2% of the total genome. The rest of the transcripts result in RNA transcripts without protein-coding ability, including long noncoding RNAs (lncRNAs). Different from most members in the lncRNA family, the metastasis-associated lung adenocarcinoma transcript 1 (Malat1) is abundantly expressed and evolutionarily conserved throughout various mammalian species. Malat1 is one of the first identified lncRNAs associated with human disease, and cumulative studies have indicated that Malat1 plays critical roles in the development and progression of various cancers. Malat1 is also actively involved in various physiologic processes, including alternative splicing, epigenetic modification of gene expression, synapse formation, and myogenesis. Furthermore, extensive evidences show that Malat1 plays pivotal roles in multiple pathological conditions as well. In this review, we will summarize latest findings related to the physiologic and pathophysiological processes of Malat1 and discuss its therapeutic potentials.

Krüpple-like factors in the central nervous system: novel mediators in stroke.

Transcription factors play an important role in the pathophysiology of many neurological disorders, including stroke. In the past three decades, an increasing number of transcription factors and their related gene signaling networks have been identified, and have become a research focus in the stroke field. Krüppel-like factors (KLFs) are members of the zinc finger family of transcription factors with diverse regulatory functions in cell growth, differentiation, proliferation, migration, apoptosis, metabolism, and inflammation. KLFs are also abundantly expressed in the brain where they serve as critical regulators of neuronal development and regeneration to maintain normal brain function. Dysregulation of KLFs has been linked to various neurological disorders. Recently, there is emerging evidence that suggests KLFs have an important role in the pathogenesis of stroke and provide endogenous vaso-or neuro-protection in the brain's response to ischemic stimuli. In this review, we summarize the basic knowledge and advancement of these transcriptional mediators in the central nervous system, highlighting the novel roles of KLFs in stroke.

KLF11 mediates PPARγ cerebrovascular protection in ischaemic stroke.

Peroxisome proliferator-activated receptor gamma (PPARγ) is emerging as a major regulator in neurological diseases. However, the role of (PPARγ) and its co-regulators in cerebrovascular endothelial dysfunction after stroke is unclear. Here, we have demonstrated that (PPARγ) activation by pioglitazone significantly inhibited both oxygen-glucose deprivation-induced cerebral vascular endothelial cell death and middle cerebral artery occlusion-triggered cerebrovascular damage. Consistent with this finding, selective (PPARγ) genetic deletion in vascular endothelial cells resulted in increased cerebrovascular permeability and brain infarction in mice after focal ischaemia. Moreover, we screened for (PPARγ) co-regulators using a genome-wide and high-throughput co-activation system and revealed KLF11 as a novel (PPARγ) co-regulator, which interacted with (PPARγ) and regulated its function in mouse cerebral vascular endothelial cell cultures. Interestingly, KLF11 was also found as a direct transcriptional target of (PPARγ). Furthermore, KLF11 genetic deficiency effectively abolished pioglitazone cytoprotection in mouse cerebral vascular endothelial cell cultures after oxygen-glucose deprivation, as well as pioglitazone-mediated cerebrovascular protection in a mouse middle cerebral artery occlusion model. Mechanistically, we demonstrated that KLF11 enhanced (PPARγ) transcriptional suppression of the pro-apoptotic microRNA-15a (miR-15a) gene, resulting in endothelial protection in cerebral vascular endothelial cell cultures and cerebral microvasculature after ischaemic stimuli. Taken together, our data demonstrate that recruitment of KLF11 as a novel (PPARγ) co-regulator plays a critical role in the cerebrovascular protection after ischaemic insults. It is anticipated that elucidating the coordinated actions of KLF11 and (PPARγ) will provide new insights into understanding the molecular mechanisms underlying (PPARγ) function in the cerebral vasculature and help to develop a novel therapeutic strategy for the treatment of stroke.

Role of regulatory non-coding RNAs in traumatic brain injury.

Traumatic brain injury (TBI) is a potentially fatal health event that cannot be predicted in advance. After TBI occurs, it can have enduring consequences within both familial and social spheres. Yet, despite extensive efforts to improve medical interventions and tailor healthcare services, TBI still remains a major contributor to global disability and mortality rates. The prompt and accurate diagnosis of TBI in clinical contexts, coupled with the implementation of effective therapeutic strategies, remains an arduous challenge. However, a deeper understanding of changes in gene expression and the underlying molecular regulatory processes may alleviate this pressing issue. In recent years, the study of regulatory non-coding RNAs (ncRNAs), a diverse class of RNA molecules with regulatory functions, has been a potential game changer in TBI research. Notably, the identification of microRNAs (miRNAs), long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and other ncRNAs has revealed their potential as novel diagnostic biomarkers and therapeutic targets for TBI, owing to their ability to regulate the expression of numerous genes. In this review, we seek to provide a comprehensive overview of the functions of regulatory ncRNAs in TBI. We also summarize regulatory ncRNAs used for treatment in animal models, as well as miRNAs, lncRNAs, and circRNAs that served as biomarkers for TBI diagnosis and prognosis. Finally, we discuss future challenges and prospects in diagnosing and treating TBI patients in the clinical settings.

Loss of microRNA-15a/16-1 function promotes neuropathological and functional recovery in experimental traumatic brain injury.

The diffuse axonal damage in white matter and neuronal loss, along with excessive neuroinflammation, hinder long-term functional recovery after traumatic brain injury (TBI). MicroRNAs (miRs) are small noncoding RNAs that negatively regulate protein-coding target genes in a posttranscriptional manner. Recent studies have shown that loss of function of the miR-15a/16-1 cluster reduced neurovascular damage and improved functional recovery in ischemic stroke and vascular dementia. However, the role of the miR-15a/16-1 cluster in neurotrauma is poorly explored. Here, we report that genetic deletion of the miR-15a/16-1 cluster facilitated the recovery of sensorimotor and cognitive functions, alleviated white matter/gray matter lesions, reduced cerebral glial cell activation, and inhibited infiltration of peripheral blood immune cells to brain parenchyma in a murine model of TBI when compared with WT controls. Moreover, intranasal delivery of the miR-15a/16-1 antagomir provided similar brain-protective effects conferred by genetic deletion of the miR-15a/16-1 cluster after experimental TBI, as evidenced by showing improved sensorimotor and cognitive outcomes, better white/gray matter integrity, and less inflammatory responses than the control antagomir-treated mice after brain trauma. miR-15a/16-1 genetic deficiency and miR-15a/16-1 antagomir also significantly suppressed inflammatory mediators in posttrauma brains. These results suggest miR-15a/16-1 as a potential therapeutic target for TBI.

Vascular endothelial cell-specific microRNA-15a inhibits angiogenesis in hindlimb ischemia.

The effects and potential mechanisms of the vascular endothelial cell (EC)-enriched microRNA-15a (miR-15a) on angiogenesis remain unclear. Here, we show a novel finding that EC-selective miR-15a transgenic overexpression leads to reduced blood vessel formation and local blood flow perfusion in mouse hindlimbs at 1-3 weeks after hindlimb ischemia. Mechanistically, gain- or loss-of-miR-15a function by lentiviral infection in ECs significantly reduces or increases tube formation, cell migration, and cell differentiation, respectively. By FGF2 and VEGF 3'-UTR luciferase reporter assays, Real-time PCR, and immunoassays, we further identified that the miR-15a directly targets FGF2 and VEGF to facilitate its anti-angiogenic effects. Our data suggest that the miR-15a in ECs can significantly suppress cell-autonomous angiogenesis through direct inhibition of endogenous endothelial FGF2 and VEGF activities. Pharmacological modulation of miR-15a function may provide a new therapeutic strategy to intervene against angiogenesis in a variety of pathological conditions.

[Effects of Different Carbon Inputs on Soil Stoichiometry in Tianshan Mountains].

Soil C, N, and P elements are important components of the forest ecosystem. Studying the influence of exogenous carbon input change on the stoichiometry of the forest soil can reveal the element recycling process and the balanced feedback mechanism of the forest ecosystem. In this study, using the research object of a spruce forest in Tianshan Mountain, the short-term effect of exogenous carbon input on soil C, N, and P in the soil was analyzed through Detritus Input and Removal Treatment (DIRT), and then the interrelationship between soil stoichiometry and other soil physicochemical factors under different treatments was discussed. The results showed that:① the soil C, N, and P contents in most soil layers were the highest double litter (DL) treatment, soil ω(C) by soil depth from shallow to deep was 168.92, 119.88, 103.33, and 64.23 g·kg-1; soil ω(N) was 10.60, 9.32, 8.78, and 8.07 g·kg-1; soil ω(P) was 0.50, 0.45, 0.37, and 0.36 g·kg-1; in the no input (NI) treatment, soil ω(C) by soil depth from shallow to deep was 104.56, 89.24, 48.08, and 43.96 g·kg-1; soil ω(N) was 6.83, 2.60, 2.63, and 2.22 g·kg-1; soil ω(P) was 0.40, 0.34, 0.32, and 0.22 g·kg-1; and a decreased trend was shown with the deepening of the soil layer. Except in the NI treatment, C:N was 0-10 cm and significantly higher than that in other soils (P<0.05), NL soil C:P at 30-50 cm was significantly higher than that in other soils, and NI soil N:P was 0-10 cm and significantly higher than that in other soils (P<0.05). ② Microbial carbon, nitrogen, and phosphorus were significantly higher from 0-10 cm than that in other soil layers (P<0.05). ③ Redundancy analysis results showed that soluble organic carbon and microbial nitrogen at different carbon input levels were important factors affecting the stoichiometric characteristics of soil C, N, and P.

Vascular PPARδ protects against stroke-induced brain injury.

OBJECTIVE: To investigate the effects of peroxisome proliferator-activated receptor (PPAR)δ in the cerebral vasculature following stroke-induced brain injury. METHODS AND RESULTS: Here, we report a novel finding that selective PPARδ genetic deletion in vascular smooth muscle cells (VSMCs) resulted in increased cerebrovascular permeability and brain infarction in mice after middle cerebral artery occlusion (MCAO). Mechanistically, we revealed for the first time that PPARδ expression is reduced, but matrix metalloproteinase (MMP)-9 activity is increased in cultured VSMCs after oxygen-glucose deprivation and also in the cerebral cortex of mice following MCAO. Moreover, gain- and loss of PPARδ function in VSMCs significantly reduces and increases oxygen-glucose deprivation-induced MMP-9 activity, respectively. We have further identified that MMP-9 is a direct target of PPARδ-mediated transrepression by chromatin immunoprecipitation and PPARδ transcriptional activity assays. Furthermore, inhibition of MMP-9 activity by lentiviral MMP-9 short hairpin RNA effectively improves cerebrovascular permeability and reduces brain infarction in VSMC-selective PPARδ conditional knockout mice after MCAO. CONCLUSIONS: Our data demonstrate that PPARδ in VSMCs can prevent ischemic brain injury by inhibition of MMP-9 activation and attenuation of postischemic inflammation. The pharmacological activation of PPARδ may provide a new therapeutic strategy to treat stroke-induced vascular and neuronal damage.

Non-coding RNAs in the regulation of blood-brain barrier functions in central nervous system disorders.

The blood-brain barrier (BBB) is an essential component of the neurovascular unit that controls the exchanges of various biological substances between the blood and the brain. BBB damage is a common feature of different central nervous systems (CNS) disorders and plays a vital role in the pathogenesis of the diseases. Non-coding RNAs (ncRNAs), such as microRNAs (miRNAs), long non-coding RNA (lncRNAs), and circular RNAs (circRNAs), are important regulatory RNA molecules that are involved in almost all cellular processes in normal development and various diseases, including CNS diseases. Cumulative evidences have demonstrated ncRNA regulation of BBB functions in different CNS diseases. In this review, we have summarized the miRNAs, lncRNAs, and circRNAs that can be served as diagnostic and prognostic biomarkers for BBB injuries, and demonstrated the involvement and underlying mechanisms of ncRNAs in modulating BBB structure and function in various CNS diseases, including ischemic stroke, hemorrhagic stroke, traumatic brain injury (TBI), spinal cord injury (SCI), multiple sclerosis (MS), Alzheimer's disease (AD), vascular cognitive impairment and dementia (VCID), brain tumors, brain infections, diabetes, sepsis-associated encephalopathy (SAE), and others. We have also discussed the pharmaceutical drugs that can regulate BBB functions via ncRNAs-related signaling cascades in CNS disorders, along with the challenges, perspective, and therapeutic potential of ncRNA regulation of BBB functions in CNS diseases.

Genetic Deficiency of MicroRNA-15a/16-1 Confers Resistance to Neuropathological Damage and Cognitive Dysfunction in Experimental Vascular Cognitive Impairment and Dementia.

Chronic cerebral hypoperfusion-derived brain damage contributes to the progression of vascular cognitive impairment and dementia (VCID). Cumulative evidence has shown that microRNAs (miRs) are emerging as novel therapeutic targets for CNS disorders. In this study, it is sought to determine the regulatory role of miR-15a/16-1 in VCID. It is found that miR-15a/16-1 knockout (KO) mice exhibit less cognitive and sensorimotor deficits following VCID. Genetic deficiency of miR-15a/16-1 in VCID mice also mitigate myelin degeneration, axonal injury, and neuronal loss. Mechanistically, miR-15a/16-1 binds to the 3'-UTR of AKT3 and IL-10RA. Genetic deletion of miR-15a/16-1 increases AKT3 and IL-10RA expression in VCID brains, and intranasal delivery of AKT3 and IL-10RA siRNA-loaded nanoparticles partially reduce brain protection and cognitive recovery in miR-15a/16-1 KO mice after VCID. In conclusion, the miR-15a/16-1-IL/10RA/AKT3 axis plays a critical role in regulating vascular brain damage and cognitive decline after VCID. Targeting miR-15a/16-1 is a novel therapeutic approach for the treatment of VCID.

Peroxisome proliferator-activated receptor delta regulation of miR-15a in ischemia-induced cerebral vascular endothelial injury.

Cerebral vascular endothelial cell (CEC) degeneration significantly contributes to blood-brain barrier (BBB) breakdown and neuronal loss after cerebral ischemia. Recently, emerging data suggest that peroxisome proliferator-activated receptor delta (PPARdelta) activation has a potential neuroprotective role in ischemic stroke. Here we report for the first time that PPARdelta is significantly reduced in oxygen-glucose deprivation (OGD)-induced mouse CEC death. Interestingly, PPARdelta overexpression can suppress OGD-induced caspase-3 activity, Golgi fragmentation, and CEC death through an increase of bcl-2 protein levels without change of bcl-2 mRNA levels. To explore the molecular mechanisms, we have identified that upregulation of PPARdelta can alleviate ODG-activated microRNA-15a (miR-15a) expression in CECs. Moreover, we have demonstrated that bcl-2 is a translationally repressed target of miR-15a. Intriguingly, gain- or loss-of-miR-15a function can significantly reduce or increase OGD-induced CEC death, respectively. Furthermore, we have identified that miR-15a is a transcriptional target of PPARdelta. Consistent with the in vitro findings, we found that intracerebroventricular infusion of a specific PPARdelta agonist, GW 501516 (2-[2-methyl-4-[[4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl]methylsulfanyl]phenoxy]acetic acid), significantly reduced ischemia-induced miR-15a expression, increased bcl-2 protein levels, and attenuated caspase-3 activity and subsequent DNA fragmentation in isolated cerebral microvessels, leading to decreased BBB disruption and reduced cerebral infarction in mice after transient focal cerebral ischemia. Together, these results suggest that PPARdelta plays a vascular-protective role in ischemia-like insults via transcriptional repression of miR-15a, resulting in subsequent release of its posttranscriptional inhibition of bcl-2. Thus, regulation of PPARdelta-mediated miR-15a inhibition of bcl-2 could provide a novel therapeutic strategy for the treatment of stroke-related vascular dysfunction.