Control of tobacco-specific nitrosamines by the Bacillus siamensis: Strain isolation, genome sequencing, mechanism analysis and genetic engineering.

Nitrosamines are considered carcinogens that threaten human health and environment. Especially, high contents of Tobacco-specific nitrosamines (TSNAs) are generated during the fermentation process of cigar tobacco. To control the accumulation of TSNAs, one novel strain WD-32 was isolated by comprehensively evaluating the reduction characteristics of nitrate, nitrite, and TSNAs, and this strain was identified as Bacillus siamensis by 16 S rRNA gene analysis and MALDI-TOF MS evaluation. Subsequently, whole genome sequencing of B. siamensis WD-32 was carried out to excavate important genes and enzymes involved, and the possible reduction mechanism of TSNAs was explored. More importantly, the reduction of TSNAs by B. siamensis was significantly promoted by knockout of narG gene. During the practical agricultural fermentation process of the cigar tobacco leaves, the treatment by the WD-32∆narG cells resulted in a 60% reduction of the total TSNAs content compared with the control, and the concentrations of the NNN and NNK were decreased by 69% and 59%, respectively. In summary, this study offers efficient strains for reduction of the TSNAs in cigar tobacco, and provides new insights into the reduction mechanism of TSNAs, which will promote the application of microbial methods in control of TSNAs and nitrite.

Genetic identification and expression optimization of a novel protease HapR from Bacillus velezensis.

Due to the broad application and substantial market demand for proteases, it was vital to explore the novel and efficient protease resources. The aim of this study was to identify the novel protease for tobacco protein degradation and optimize the expression levels. Firstly, the tobacco protein was used as the sole nitrogen resource for isolation of protease-producing strains, and a strain with high protease production ability was obtained, identified as Bacillus velezensis WH-7. Then, the whole genome sequencing was conducted on the strain B. velezensis WH-7, and 7 proteases genes were mined by gene annotation analysis. By further heterologous expression of the 7 protease genes, the key protease HapR was identified with the highest protease activity (144.19 U/mL). Moreover, the catalysis mechanism of HapR was explained by amino acid sequence analysis. The expression levels of protease HapR were further improved through optimization of promoter, signal peptide and host strain, and the maximum protease activity reaced 384.27 U/mL in WX-02/pHY-P43-SPyfkD-hapR, increased by 167% than that of initial recombinant strain HZ/pHY-P43-SPhapR-hapR. This study identified a novel protease HapR and the expression level was significantly improved, which provided an important enzyme resource for the development of enzyme preparations in tobacco protein degradation.

Scoparone attenuates glioma progression and improves the toxicity of temozolomide by suppressing RhoA/ROCK1 signaling.

BACKGROUND: Glioma, a type of malignant brain tumor, has become a challenging health issue globally in recent years. METHODS: In this study, we investigated the potential therapeutic role of scoparone in glioma and the underlying mechanism. Initially, transcriptome sequencing was conducted to identify genes that exhibited differential expression in glioma cells treated with scoparone compared to untreated cells. Subsequently, the impact of scoparone on the proliferation, migration, and invasion of glioma cells was assessed in vitro using a range of assays including cell viability, colony formation, wound healing, and transwell assays. Moreover, the apoptotic effects of scoparone on glioma cells were evaluated through flow cytometry and western blot analysis. Furthermore, we established a glioma xenograft mouse model to assess the in vivo antitumor activity of scoparone. Lastly, by integrating transcriptome analysis, we endeavored to unravel the molecular mechanisms underlying the observed antitumor effects of scoparone by examining the expression levels of RhoA/ROCK1 signaling pathway components using western blot analysis and qRT-PCR. RESULTS: Our transcriptome sequencing results revealed that scoparone significantly downregulated RhoA/ROCK1 signaling in glioma cells. Furthermore, scoparone treatment inhibited glioma cell proliferation, migration, and invasion, and promoted cell apoptosis in vitro. Moreover, scoparone reduced tumor growth and prolonged survival in a glioma xenograft mouse model, and improved the toxicity of temozolomide. Finally, our results showed that the antitumor effects of scoparone were mediated by the suppression of RhoA/ROCK1 signaling. CONCLUSION: Scoparone could be a promising therapeutic agent for glioma by suppressing RhoA/ROCK1 signaling. These findings pave the way for future research endeavors aimed at the development and optimization of scoparone-based therapeutic strategies.

A novel rat model of chronic subdural hematoma: Induction of inflammation and angiogenesis in the subdural space mimicking human-like features of progressively expanding hematoma.

Pathophysiological mechanisms of chronic subdural hematoma (CSDH) involve localized inflammation, angiogenesis, and dysregulated coagulation and fibrinolysis. The scarcity of reproducible and clinically relevant animal models of CSDH hinders further understanding the underlying pathophysiology and improving new treatment strategies. Here, we developed a novel rat model of CSDH using extracellular matrices (Matrigel) and brain microvascular endothelial cell line (bEnd.3 cells). One hundred-microliter of Matrigel-bEnd.3 cell (106 cells per milliliter) mixtures were injected into the virtual subdural space of elderly male Sprague-Dawley rats. This approach for the first time led to a spontaneous and expanding subdural hematoma, encapsulated by internal and external neomembranes, formed as early as 3 d, reached its peak at 7 d, and lasted for more than 14 d, mimicking the progressive hemorrhage observed in patients with CSDH. The external neomembrane and hematoma fluid involved numerous inflammatory cells, fibroblasts, and highly fragile neovessels. Furthermore, a localized pathophysiological process was validated as evidenced by the increased expressions of inflammatory and angiogenic mediators in external neomembrane and hematoma fluid rather than in peripheral blood. Notably, the specific expression profiles of these mediators were closely associated with the dynamic changes in hematoma volume and neurological outcome. In summary, the CSDH model described here replicated the characteristics of human CSDH, and might serve as an ideal translational platform for preclinical studies. Meanwhile, the crucial roles of angiogenesis and inflammation in CSDH formation were reaffirmed.

Losartan Treatment Could Improve the Outcome of TBI Mice.

Traumatic brain injury frequently leads to serious mortality and physical disability, yet effective treatments remains insufficient. TBI always leads to a series of secondary brain injuries including neuronal apoptosis, continuous inflammation, endoplasmic reticulum stress, and disruption of the blood-brain barrier. Sartans that block angiotensin II type 1 receptors are strongly neuroprotective, neurorestorative and anti-inflammatory. However, whether losartan, a FDA-approved and widely used drug for regulating blood pressure, is beneficial for improving the prognosis of TBI need more evidence. Through a controlled cortical impact injury mice model, we confirmed that losartan treatment could ameliorate CCI-induced secondary brain injury. We found that losartan treatment decreased brain lesion volume, neuronal apoptosis and ER stress protein ATF4 and eIF2α. Moreover, our results showed that losartan also improved neurological and motor function. It is worth pointing out that losartan increased the expression of tight junction proteins ZO-1 and alleviated brain edema and blood brain barrier leakage. Additionally, losartan inhibited pro-inflammatory factor TNF-α and improve anti-inflammatory factor IL-10. Taken together, our data demonstrated that losartan could improve the prognosis of TBI and may be a promising therapeutic method for mitigating TBI.

Reduction of endoglin receptor impairs mononuclear cell-migration.

AIM: To test if the impairment of mononuclear cell (MNC) migration in patients with hereditary hemorrhagic telangiectasia (HHT) is due to the reduction of the endoglin (ENG) receptor on the cell surface and oxidative stress. METHODS: MNCs of HHT patients and normal controls were subjected to migration assay. Fractions of MNCs were pre-incubated with antibodies specific to HHT causative genes ENG [hereditary hemorrhagic telangiectasia type 1 (HHT1)] or activin receptor-like kinase 1 [ALK1, hereditary hemorrhagic telangiectasia type 2 (HHT2)], AMD3100 or Diprotin-A to block ENG, ALK1 C-X-C chemokine receptor 4 (CXCR4) or CD26 (increased in HHT1 MNCs) before migration assay. The MNCs were allowed to migrate toward stromal cell-derived factor-1α (SDF-1α) for 18 h. The expression of CXCR4, CD26, superoxide dismutase 1 (SOD1) and glutathione peroxidase 1 (GPX1) in MNCs and nitric oxide levels in the plasma were analyzed. RESULTS: Compared to the controls, fewer HHT1 MNCs and similar number of HHT2 MNCs migrated toward SDF-1α. Diprotin-A pre-treatment improved HHT1 MNC-migration, but had no effect on normal and HHT2 MNCs. Pre-incubation with an anti-ENG antibody reduced the migration of normal MNCs. Diprotin-A did not improve the migration of ENG antibody pre-treated MNCs. Anti-ALK1 antibody had no effect on MNC-migration. AMD3100 treatment reduced normal and HHT MNC-migration. ENG mRNA level was reduced in HHT1 and HHT2 MNCs. ALK1 mRNA was reduced in HHT2 MNCs only. CD26 expression was higher in HHT1 MNCs. Pre-treatment of MNCs with anti-ENG or anti-ALK1 antibody had no effect on CD26 and CXCR4 expression. The expression of antioxidant enzymes, SOD1, was reduced in HHT1 MNCs, which was accompanied with an increase of ROS in HHT MNCs and nitric oxide in HHT1 plasma. CONCLUSIONS: Reduction of ENG receptor on MNC surface reduced monocyte migration toward SDF-1α independent of CD26 expression. Increased oxidative stress could alter HHT MNC migration behavior.

Inhibition of intracranial hemangioma growth and hemorrhage by TNFSF15.

Hemangioblastoma (HB) is an abnormal intracranial buildup of blood vessels that exhibit a great potential for hemorrhage. Surgical options are limited, and few medications are available for treatment. We show here by immunohistochemical analysis that HB lesions display highly increased levels of VEGF expression and macrophage/microglia infiltration compared with those in normal brain tissues. In the meantime, TNF superfamily 15 (TNFSF15) (also known as vascular endothelial growth inhibitor), an antiangiogenic cytokine, is highly expressed in normal brain blood vessels but diminished in HB lesions. We set up a brain hemangioma model by using mouse bEnd.3 cells of a T antigen-transformed endothelial cell line that produce a large amount of VEGF. When implanted in mouse brains, these cells form lesions that closely resemble the pathologic characteristics of HB. Retroviral infection of bEnd.3 cells with TNFSF15 leads to inhibition of VEGF production and retardation of hemangioma formation. Similar results are obtained when wild-type bEnd.3 cells are implanted in the brains of transgenic mice overexpressing TNFSF15. Additionally, TNFSF15 treatment results in enhanced pericyte coverage of the blood vessels in the lesions together with reduced inflammatory cell infiltration and decreased hemorrhage. These findings indicate that the ability of TNFSF15 to counterbalance the abnormally highly angiogenic and inflammatory potential of the microenvironment of HB is of therapeutic value for the treatment of this disease.-Yang, G.-L., Han, Z., Xiong, J., Wang, S., Wei, H., Qin, T.-T., Xiao, H., Liu, Y., Xu, L.-X., Qi, J.-W., Zhang, Z.-S., Jiang, R., Zhang, J., Li, L.-Y. Inhibition of intracranial hemangioma growth and hemorrhage by TNFSF15.

Semaphorin 3A Contributes to Secondary Blood-Brain Barrier Damage After Traumatic Brain Injury.

Semaphorin 3A (SEMA3A) is a member of the Semaphorins family, a class of membrane-associated protein that participates in the construction of nerve networks. SEMA3A has been reported to affect vascular permeability previously, but its influence in traumatic brain injury (TBI) is still unknown. To investigate the effects of SEMA3A, we used a mouse TBI model with a controlled cortical impact (CCI) device and a blood-brain barrier (BBB) injury model in vitro with oxygen-glucose deprivation (OGD). We tested post-TBI changes in SEMA3A, and its related receptors (Nrp-1 and plexin-A1) expression and distribution through western blotting and double-immunofluorescence staining, respectively. Neurological outcomes were evaluated by modified neurological severity scores (mNSSs) and beam-walking test. We examined BBB damage through Evans Blue dye extravasation, brain water content, and western blotting for VE-cadherin and p-VE-cadherin in vivo, and we examined the endothelial cell barrier through hopping probe ion conductance microscopy (HPICM), transwell leakage, and western blotting for VE-cadherin and p-VE-cadherin in vitro. Changes in miR-30b-5p were assessed by RT-PCR. Finally, the neuroprotective function of miR-30b-5p is measured by brain water content, mNSSs and beam-walking test. SEMA3A expression varied following TBI and peaked on the third day which expressed approximate fourfold increase compared with sham group, with the protein concentrated at the lesion boundary. SEMA3A contributed to neurological function deficits and secondary BBB damage in vivo. Our results demonstrated that SEMA3A level following OGD injury almost doubled than control group, and the negative effects of OGD injury can be improved by blocking SEMA3A expression. Furthermore, the expression of miR-30b-5p decreased approximate 40% at the third day and 60% at the seventh day post-CCI. OGD injury also exhibited an effect to approximately decrease 50% of miR-30b-5p expression. Additionally, the expression of SEMA3A post-TBI is regulated by miR-30b-5p, and miR-30b-5p could improve neurological outcomes post-TBI efficiently. Our results demonstrate that SEMA3A is a significant factor in secondary BBB damage after TBI and can be abolished by miR-30b-5p, which represents a potential therapeutic target.

Increased miR-21-3p in Injured Brain Microvascular Endothelial Cells after Traumatic Brain Injury Aggravates Blood-Brain Barrier Damage by Promoting Cellular Apoptosis and Inflammation through Targeting MAT2B.

Our recent articles have reported that increased miR-21-5p in brain after traumatic brain injury (TBI) could improve the neurological outcome through alleviating blood-brain barrier (BBB) damage. miR-21-3p is another mature miRNA derived from pre-miR-21 after Dicer Procession other than miR-21-5p. Its roles in various diseases, such as tumors and myocardial disease, aroused great interest for research in recent years. To further explore the function and underlying mechanism of miR-21, especially miR-21-3p, in regulating the pathological development of BBB damage after TBI, we designed this research and focused on studying the impact of miR-21-3p on apoptosis and inflammation in brain microvascular endothelial cells (BMVECs), the major cellular component of BBB. We performed controlled cortical impact on mouse brain and employed the oxygen glucose deprivation/reoxygenation (OGD)-treated bEnd.3 cells injury model. We found that the miR-21-3p level in BMVECs from injured cerebral cortex of controlled cortical impact (CCI) mice and bEnd.3 cells with OGD treatment were both increased after injury. For in vitro experiments, downregulation on the miR-21-3p level by transfecting miR-21-3p antagomir in cultured cells alleviated OGD-induced BBB damage, characterized by decreased BBB leakage and increased expression of tight junction proteins. Besides, miR-21-3p antagomir could suppress cell death by anti-apoptosis and control inflammatory response by inhibiting the activity of NF-κB signaling. Using luciferase reporter assay and a MAT2B-silenced shRNA vector, we further proved that miR-21-3p exerted the above functions through targeting MAT2B. In addition, in vivo experiments also confirmed that intracerebroventricular infusion of miR-21-3p antagomir could alleviate BBB leakage after TBI. It reduced Evans Blue extravasation and promoted the expression of tight junction proteins, thus contributed to improve the neurological outcome of CCI mice. Taken together, increased miR-21-3p in BMVECs after TBI was bad for restoration of injured BBB. Downregulation on the miR-21-3p level in injured brain could be a promising therapeutic strategy for BBB damage after TBI.

In situ fabrication of a direct Z-scheme photocatalyst by immobilizing CdS quantum dots in the channels of graphene-hybridized and supported mesoporous titanium nanocrystals for high photocatalytic performance under visible light.

We report the considerable advantages of direct Z-scheme photocatalysts by immobilizing high-quality CdS quantum dots (QDs) in the channels of graphene-hybridized and supported mesoporous titania (GMT) nanocrystals (CdS@GMT/GR) under facile hydrothermal conditions. The photocatalysts have been characterized by XRD, PL, XPS, SEM, DRS, TEM, EIS, and N2 adsorption. CdS QDs primarily serve as photosensitizers with a unique pore-embedded structure for the effective utilization of the light source. This direct Z-scheme CdS@GMT/GR exhibits higher photocatalytic activity than CdS/GR, GMT/GR, or CdS@MT. In addition, the rate constant of CdS@GMT/GR-2 is approximately twice the sum of those of CdS@MT and GMT/GR, because GR played the role of hole-transporting and collection layer as well as the hybridization level formation in terms of hybridizing MT and serving as a support. Therefore, the GR content tunes the energy band, affects the surface area, and controls the interfacial hole transfer and collection rate of the direct Z-scheme system. Furthermore, CdS@GMT/GR retains its high performance in repeated photocatalytic processes. This can be attributed to the fact that GR prevents QDs from photocorrosion by means of the hole-transporting and collection effect. A possible reaction mechanism is proposed. This work provides a promising strategy for the construction of highly efficient visible-light-driven photocatalysts to reduce the growing menace of environmental pollution.

Activation of Alpha-7 Nicotinic Acetylcholine Receptor Reduces Brain Edema in Mice with Ischemic Stroke and Bone Fracture.

Stroke is an important risk factor for bone fracture. We showed previously that bone fracture at the acute stage of ischemic stroke worsens, and activation of α-7 nicotinic acetylcholine receptor (α-7 nAchR) improves, stroke recovery by attenuating inflammation. We hypothesized that activation of α-7 nAchR also improves the blood-brain barrier (BBB) integrity. Permanent distal middle cerebral artery occlusion (pMCAO) was performed on C57BL/6J mice followed by tibia fracture 1 day later. Mice were treated with 0.8 mg/kg PHA 568487 (PHA, α-7 nAchR-specific agonist), 6 mg/kg methyllycaconitine (MLA, α-7 nAchR antagonist), or saline 1 and 2 days after pMCAO. Brain water content, the expression of monoamine oxidase B (MAO-B), and tight junction protein (claudin-5) were assessed. We found that tibia fracture increased water content in the ischemic stroke brain (p = 0.006) and MAO-B-positive astrocytes (p < 0.001). PHA treatment reduced water content and MAO-B-positive astrocytes and increased claudin-5 expression in stroke and stroke + tibia fracture mice (p < 0.05), while MLA had the opposite effect. Our findings suggest that in addition to inhibiting inflammation, activation of α-7 nAchR also reduces brain edema, possibly through diminished astrocyte oxidative stress and improved BBB integrity. Thus, the α-7 nAchR-specific agonist could be developed into a new therapy for improving recovery of patients with stroke or stroke + bone fracture.

Persistent infiltration and pro-inflammatory differentiation of monocytes cause unresolved inflammation in brain arteriovenous malformation.

An abnormally high number of macrophages are present in human brain arteriovenous malformations (bAVM) with or without evidence of prior hemorrhage, causing unresolved inflammation that may enhance abnormal vascular remodeling and exacerbate the bAVM phenotype. The reasons for macrophage accumulation at the bAVM sites are not known. We tested the hypothesis that persistent infiltration and pro-inflammatory differentiation of monocytes in angiogenic tissues increase the macrophage burden in bAVM using two mouse models and human monocytes. Mouse bAVM was induced through deletion of AVM causative genes, Endoglin (Eng) globally or Alk1 focally, plus brain focal angiogenic stimulation. An endothelial cell and vascular smooth muscle cell co-culture system was used to analyze monocyte differentiation in the angiogenic niche. After angiogenic stimulation, the Eng-deleted mice had fewer CD68(+) cells at 2 weeks (P = 0.02), similar numbers at 4 weeks (P = 0.97), and more at 8 weeks (P = 0.01) in the brain angiogenic region compared with wild-type (WT) mice. Alk1-deficient mice also had a trend toward more macrophages/microglia 8 weeks (P = 0.064) after angiogenic stimulation and more RFP(+) bone marrow-derived macrophages than WT mice (P = 0.01). More CD34(+) cells isolated from peripheral blood of patients with ENG or ALK1 gene mutation differentiated into macrophages than those from healthy controls (P < 0.001). These data indicate that persistent infiltration and pro-inflammatory differentiation of monocytes might contribute to macrophage accumulation in bAVM. Blocking macrophage homing to bAVM lesions should be tested as a strategy to reduce the severity of bAVM.

Recombinant human erythropoietin improves the neurofunctional recovery of rats following traumatic brain injury via an increase in circulating endothelial progenitor cells.

Previous studies show that circulating endothelial progenitor cells (EPCs) promote angiogenesis, which is a process associated with improved recovery in animal models of traumatic brain injury (TBI), and that recombinant human erythropoietin (rhEPO) plays a protective role following stroke. Thus, it was hypothesized that rhEPO would enhance recovery following brain injury in a rat model of TBI via an increase in the mobilization of EPCs and, subsequently, in angiogenesis. Flow cytometry assays using CD34- and CD133-specific antibodies were utilized to identify alterations in EPC levels, CD31 and CD34 antibody-stained brain tissue sections were used to quantify angiogenesis, and the Morris water maze (MWM) test and the modified Neurological Severity Score (mNSS) test were used to evaluate behavioral recovery. Compared with saline treatment, treatment with rhEPO significantly increased the number of circulating EPCs on days 1, 4, 7, and 14 (P < 0.05), improved spatial learning ability on days 24 and 25 (P < 0.05), and enhanced memory recovery on day 26 (P < 0.05). Moreover, rhEPO treatment decreased mNSS assessment scores on days 14, 21, and 25 (P < 0.05). There was a strong correlation between levels of circulating EPCs and CD34- and CD31-positive cells within the injured boundary zone (CD34(+) r = 0.910, P < 0.01; CD31(+) r = 0.894, P < 0.01) and the ipsilateral hippocampus (CD34(+) r = 0.841, P < 0.01; CD31(+) r = 0.835, P < 0.01). The present data demonstrate that rhEPO treatment improved functional outcomes in rats following TBI via an increase in the mobilization of EPCs and in subsequent angiogenesis.

Activation of α-7 nicotinic acetylcholine receptor reduces ischemic stroke injury through reduction of pro-inflammatory macrophages and oxidative stress.

Activation of α-7 nicotinic acetylcholine receptor (α-7 nAchR) has a neuro-protective effect on ischemic and hemorrhagic stroke. However, the underlying mechanism is not completely understood. We hypothesized that α-7 nAchR agonist protects brain injury after ischemic stroke through reduction of pro-inflammatory macrophages (M1) and oxidative stress. C57BL/6 mice were treated with PHA568487 (PHA, α-7 nAchR agonist), methyllycaconitine (MLA, nAchR antagonist), or saline immediately and 24 hours after permanent occlusion of the distal middle cerebral artery (pMCAO). Behavior test, lesion volume, CD68(+), M1 (CD11b(+)/Iba1(+)) and M2 (CD206/Iba1+) microglia/macrophages, and phosphorylated p65 component of NF-kB in microglia/macrophages were quantified using histological stained sections. The expression of M1 and M2 marker genes, anti-oxidant genes and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were quantified using real-time RT-PCR. Compared to the saline-treated mice, PHA mice had fewer behavior deficits 3 and 7 days after pMCAO, and smaller lesion volume, fewer CD68(+) and M1 macrophages, and more M2 macrophages 3 and 14 days after pMCAO, whereas MLA's effects were mostly the opposite in several analyses. PHA increased anti-oxidant genes and NADPH oxidase expression associated with decreased phosphorylation of NF-kB p65 in microglia/macrophages. Thus, reduction of inflammatory response and oxidative stress play roles in α-7 nAchR neuro-protective effect.

Alpha-7 nicotinic acetylcholine receptor agonist treatment reduces neuroinflammation, oxidative stress, and brain injury in mice with ischemic stroke and bone fracture.

Bone fracture at the acute stage of stroke exacerbates stroke injury by increasing neuroinflammation. We hypothesize that activation of α-7 nicotinic acetylcholine receptor (α-7 nAchR) attenuates neuroinflammation and oxidative stress, and reduces brain injury in mice with bone fracture and stroke. Permanent middle cerebral artery occlusion (pMCAO) was performed in C57BL/6J mice followed by tibia fracture 1 day later. Mice were treated with 0.8 mg/kg PHA 568487 (PHA, α-7 nAchR-specific agonist), 6 mg/kg methyllycaconitine (α-7 nAchR antagonist), or saline 1 and 2 days after pMCAO. Behavior was tested 3 days after pMCAO. Neuronal injury, CD68(+) , M1 (pro-inflammatory) and M2 (anti-inflammatory) microglia/macrophages, phosphorylated p65 component of nuclear factor kappa b in microglia/macrophages, oxidative and anti-oxidant gene expression were quantified. Compared to saline-treated mice, PHA-treated mice performed better in behavioral tests, had fewer apoptotic neurons (NeuN(+) TUNEL(+) ), fewer CD68(+) and M1 macrophages, and more M2 macrophages. PHA increased anti-oxidant gene expression and decreased oxidative stress and phosphorylation of nuclear factor kappa b p65. Methyllycaconitine had the opposite effects. Our data indicate that α-7 nAchR agonist treatment reduces neuroinflammation and oxidative stress, which are associated with reduced brain injury in mice with ischemic stroke plus tibia fracture. Bone fracture at the acute stage of stroke exacerbates neuroinflammation, oxidative stress, and brain injury, and our study has shown that the α-7 nAchR agonist, PHA (PHA 568487), attenuates neuroinflammation, oxidative stress, and brain injury in mice with stroke and bone fracture. Hence, PHA could provide an opportunity to develop a new strategy to reduce brain injury in patients suffering from stroke and bone fracture.

Endoglin deficiency impairs stroke recovery.

BACKGROUND AND PURPOSE: Endoglin deficiency causes hereditary hemorrhagic telangiectasia-1 and impairs myocardial repair. Pulmonary arteriovenous malformations in patients with hereditary hemorrhagic telangiectasia-1 are associated with a high incidence of paradoxical embolism in the cerebral circulation and ischemic brain injury. We hypothesized that endoglin deficiency impairs stroke recovery. METHODS: Eng heterozygous (Eng+/-) and wild-type mice underwent permanent distal middle cerebral artery occlusion (pMCAO). Pial collateral vessels were quantified before pMCAO. Infarct/atrophic volume, vascular density, and macrophages were quantified in various days after pMCAO, and behavioral function was assessed using corner and adhesive removal tests on days 3, 15, 30, and 60 after pMCAO. The association between ENG 207G>A polymorphism and brain arteriovenous malformation rupture and surgery outcome was analyzed using logistic regression analysis in 256 ruptured and 157 unruptured patients. RESULTS: After pMCAO, Eng+/- mice showed larger infarct/atrophic volumes at all time points (P<0.05) and showed worse behavior performance (P<0.05) at 15, 30, and 60 days when compared with wild-type mice. Eng+/- mice had fewer macrophages on day 3 (P=0.009) and more macrophages on day 60 (P=0.02) in the peri-infarct region. Although Eng+/- and wild-type mice had similar numbers of pial collateral vessels before pMCAO, Eng+/- mice had lower vascular density in the peri-infarct region (P=0.05) on day 60 after pMCAO. In humans, ENG 207A allele has been associated with worse outcomes after arteriovenous malformation rupture or surgery of patients with unruptured arteriovenous malformation. CONCLUSIONS: Endoglin deficiency impairs brain injury recovery. Reduced angiogenesis, impaired macrophage homing, and delayed inflammation resolution could be the underlying mechanism.

De novo cerebrovascular malformation in the adult mouse after endothelial Alk1 deletion and angiogenic stimulation.

BACKGROUND AND PURPOSE: In humans, activin receptor-like kinase 1 (Alk1) deficiency causes arteriovenous malformations (AVMs) in multiple organs, including the brain. Focal Alk1 pan-cellular deletion plus vascular endothelial growth factor stimulation induces brain AVMs in the adult mouse. We hypothesized that deletion of Alk1 in endothelial cell (EC) alone plus focal vascular endothelial growth factor stimulation is sufficient to induce brain AVM in the adult mouse. METHODS: Focal angiogenesis was induced in the brain of 8-week-old Pdgfb-iCreER;Alk1(2f/2f) mice by injection of adeno-associated viral vectors expressing vascular endothelial growth factor. Two weeks later, EC-Alk1 deletion was induced by tamoxifen treatment. Vascular morphology was analyzed, and EC proliferation and dysplasia index (number of vessels with diameter>15 µm per 200 vessels) were quantified 10 days after tamoxifen administration. RESULTS: Tangles of enlarged vessels resembling AVMs were present in the brain angiogenic region of tamoxifen-treated Pdgfb-iCreER;Alk1(2f/2f) mice. Induced brain AVMs were marked by increased dysplasia index (P<0.001) and EC proliferation clustered within the dysplastic vessels. AVMs were also detected around the ear tag-wound and in other organs. CONCLUSIONS: Deletion of Alk1 in EC in adult mice leads to an increased local EC proliferation during brain angiogenesis and de novo brain AVM.

Transplantation of expanded endothelial colony-forming cells improved outcomes of traumatic brain injury in a mouse model.

BACKGROUND: Endothelial progenitor cells (EPCs) are critical for repairing injured tissue. Endothelial colony-forming cells (ECFCs) are a homogeneous subtype of EPCs. We investigated whether intravenously infused human ECFCs homed to injured brain promoted angiogenesis and ameliorate neurologic disabilities in a mouse model of traumatic brain injury. MATERIALS AND METHODS: ECFCs were generated by in vitro propagation of EPCs from human umbilical cord blood. Young female nude mice received intravenously ECFCs from human newborns (1 × 10(6)) 1 h after they were exposed to lateral fluid percussion injury. Neurologic function was evaluated by a modified neurologic severity score and Morris water maze. ECFC homing and neovascularization at the site of injury were examined by fluorescence in situ hybridization and histochemistry on days 2 and 14 after injury, respectively. RESULTS: Donor ECFCs were detected in injured brain 24 h after infusion. The modified neurologic severity score and Morris water maze tests were used to evaluate neurologic disability, and found the rate of neurologic disability was improved in mice that received ECFCs. Microvessel density and expression of the proangiogenic growth factors stromal cell-derived factor-1 and vascular endothelial growth factor were also increased in the region of injured brain from mice that received ECFCs compared with those received vehicle control. CONCLUSIONS: These data suggest that ECFCs are effective in promoting neovascularization and improving neurologic functions after traumatic brain injury.

Depletion of bone marrow-derived macrophages perturbs the innate immune response to surgery and reduces postoperative memory dysfunction.

BACKGROUND: According to rodent models of postoperative cognitive decline, activation of the innate immune response following aseptic surgical trauma results in the elaboration of hippocampal proinflammatory cytokines, which are capable of disrupting long-term potentiation, the neurobiologic correlate of memory. The authors hypothesize that hippocampal recruitment of bone marrow-derived macrophages plays a causal role in these processes, resulting in memory dysfunction. METHODS: Clodrolip injection (liposomal formulation of clodronate) before stabilized tibial fracture under general anesthesia was used to deplete bone marrow-derived macrophages. Systemic inflammation and neuroinflammation were studied on postoperative day 1, and memory in a fear-trace conditioning paradigm was assessed on postoperative day 3. CX3CR1 CCR2 mice were used to identify bone marrow-derived macrophages. RESULTS: Clodrolip effectively depleted splenic CCR2 bone marrow-derived macrophages. It also attenuated the surgery-induced increase of interleukin-6 in the serum and the hippocampus, and prevented hippocampal infiltration of CCR2 cells without affecting the number of CX3CR1 microglia. It did not alter the surgery-induced increase in hippocampal monocyte chemoattractant protein-1, the recruitment signal for CCR2 cells. Clodrolip prevented surgery-induced memory dysfunction, as evidenced by a significant increase in freezing time (29% [95% CI, 21-38%] vs. 48% [95% CI, 38-58%], n = 20, P = 0.004), but did not affect memory in nonsurgical mice. CONCLUSION: Depletion of bone marrow-derived macrophages prevents hippocampal neuroinflammation and memory dysfunction after experimental tibial fracture. These data suggest that the hippocampal recruitment of bone marrow-derived macrophages is a necessary mechanism in murine postoperative cognitive dysfunction. Interventions designed to prevent its activation and/or migration into the brain may represent a feasible preemptive strategy.