An MMP-9 exclusive neutralizing antibody attenuates blood-brain barrier breakdown in mice with stroke and reduces stroke patient-derived MMP-9 activity.

Rapid upregulation of matrix metalloproteinase 9 (MMP-9) leads to blood-brain barrier (BBB) breakdown following stroke, but no MMP-9 inhibitors have been approved in clinic largely due to their low specificities and side effects. Here, we explored the therapeutic potential of a human IgG monoclonal antibody (mAb), L13, which was recently developed with exclusive neutralizing specificity to MMP-9, nanomolar potency, and biological function, using mouse stroke models and stroke patient samples. We found that L13 treatment at the onset of reperfusion following cerebral ischemia or after intracranial hemorrhage (ICH) significantly reduced brain tissue injury and improved the neurological outcomes of mice. Compared to control IgG, L13 substantially attenuated BBB breakdown in both types of stroke model by inhibiting MMP-9 activity-mediated degradations of basement membrane and endothelial tight junction proteins. Importantly, these BBB-protective and neuroprotective effects of L13 in wild-type mice were comparable to Mmp9 genetic deletion and fully abolished in Mmp9 knockout mice, highlighting the in vivo target specificity of L13. Meanwhile, ex vivo co-incubation with L13 significantly neutralized the enzymatic activities of human MMP-9 in the sera of ischemic and hemorrhagic stroke patients, or in the peri-hematoma brain tissues from hemorrhagic stroke patients. Overall, we demonstrated that MMP-9 exclusive neutralizing mAbs constitute a potential feasible therapeutic approach for both ischemic and hemorrhagic stroke.

Generation of Vgll4-DreER transgenic mouse for visualizing and manipulating VGLL4-expressing cells in vivo.

Vestigial like family member 4 (VGLL4), a member of the Hippo pathway, is a transcriptional cofactor involved in many biological processes, such as tumor progression, postnatal heart growth, and muscle regeneration. However, the VGLL4 expression pattern in vivo remains unclear. To detect and trace Vgll4-expressing cells and their progeny, we generated and characterized a new tamoxifen-inducible Dre knock-in mouse line, Vgll4-DreER. This mouse line expressed DreER (Dre recombinase fused to the estrogen receptor) under the control of the endogenous Vgll4 promoter. After crossing the Vgll4-DreER mouse line with the Dre-responsive reporter H11-rRFP, Dre-mediated recombination in the tissue was monitored on the basis of red fluorescent protein (RFP) signals, which indicated the distribution of VGLL4-positive cells in vivo. Our data revealed that VGLL4 is widely expressed in various cell types at embryonic and neonatal stages. After comparison with our previously reported Vgll4-GFP mouse, we found that the RFP signal profile was wider than the green fluorescent protein (GFP) pattern, indicating that Vgll4-DreER is more sensitive for labeling VGLL4-expressing cells. We next used a dual-recombination system to simultaneously label VGLL4- and keratin 5 (KRT5)-positive cell populations, and no crosstalk was observed in the Krt5-CreER;Vgll4-DreER;R26-rGlR mice. Taken together, the Vgll4-DreER mouse line is a valuable new tool for examining the precise VGLL4 expression profile and conditional manipulating of VGLL4-expressing cells and their progeny.

Integrated transcriptomic and proteomic profiling reveals the key molecular signatures of brain endothelial reperfusion injury.

BACKGROUND: Reperfusion therapy after ischemic stroke often causes brain microvascular injury. However, the underlying mechanisms are unclear. METHODS: Transcriptomic and proteomic analyses were performed on human cerebral microvascular endothelial cells following oxygen-glucose deprivation (OGD) or OGD plus recovery (OGD/R) to identify molecules and signaling pathways dysregulated by reperfusion. Major findings were further validated in a mouse model of cerebral ischemia and reperfusion. RESULTS: Transcriptomic analysis identified 390 differentially expressed genes (DEGs) between the OGD/R and OGD group. Pathway analysis indicated that these genes were mostly associated with inflammation, including the TNF signaling pathway, TGF-ß signaling pathway, cytokine-cytokine receptor interaction, NOD-like receptor signaling pathway, and NF-κB signaling pathway. Proteomic analysis identified 201 differentially expressed proteins (DEPs), which were primarily associated with extracellular matrix destruction and remodeling, impairment of endothelial transport function, and inflammatory responses. Six genes (DUSP1, JUNB, NFKBIA, NR4A1, SERPINE1, and THBS1) were upregulated by OGD/R at both the mRNA and protein levels. In mice with cerebral ischemia and reperfusion, brain TNF signaling pathway was activated by reperfusion, and inhibiting TNF-α with adalimumab significantly attenuated reperfusion-induced brain endothelial inflammation. In addition, the protein level of THBS1 was substantially upregulated upon reperfusion in brain endothelial cells and the peri-endothelial area in mice receiving cerebral ischemia. CONCLUSION: Our study reveals the key molecular signatures of brain endothelial reperfusion injury and provides potential therapeutic targets for the treatment of brain microvascular injury after reperfusion therapy in ischemic stroke.

Emergence of eravacycline heteroresistance in carbapenem-resistant Acinetobacter baumannii isolates in China.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is resistant to almost all antibiotics. Eravacycline, a newer treatment option, has the potential to treat CRAB infections, however, the mechanism by which CRAB isolates develop resistance to eravacycline has yet to be clarified. This study sought to investigate the features and mechanisms of eravacycline heteroresistance among CRAB clinical isolates. A total of 287 isolates were collected in China from 2020 to 2022. The minimum inhibitory concentration (MIC) of eravacycline and other clinically available agents against A. baumannii were determined using broth microdilution. The frequency of eravacycline heteroresistance was determined by population analysis profiling (PAP). Mutations and expression levels of resistance genes in heteroresistant isolates were determined by polymerase chain reaction (PCR) and quantitative real-time PCR (qRT-PCR), respectively. Antisense RNA silencing was used to validate the function of eravacycline heteroresistant candidate genes. Twenty-five eravacycline heteroresistant isolates (17.36%) were detected among 144 CRAB isolates with eravacycline MIC values ≤4 mg/L while no eravacycline heteroresistant strains were detected in carbapenem-susceptible A. baumannii (CSAB) isolates. All eravacycline heteroresistant strains contained OXA-23 carbapenemase and the predominant multilocus sequence typing (MLST) was ST208 (72%). Cross-resistance was observed between eravacycline, tigecycline, and levofloxacin in the resistant subpopulations. The addition of efflux pump inhibitors significantly reduced the eravacycline MIC in resistant subpopulations and weakened the formation of eravacycline heteroresistance in CRAB isolates. The expression levels of adeABC and adeRS were significantly higher in resistant subpopulations than in eravacycline heteroresistant parental strains (P < 0.05). An ISAba1 insertion in the adeS gene was identified in 40% (10/25) of the resistant subpopulations. Decreasing the expression of adeABC or adeRS by antisense RNA silencing significantly inhibited eravacycline heteroresistance. In conclusion, this study identified the emergence of eravacycline heteroresistance in CRAB isolates in China, which is associated with high expression of AdeABC and AdeRS.

Lithium attenuates blood-brain barrier damage and brain edema following intracerebral hemorrhage via an endothelial Wnt/ß-catenin signaling-dependent mechanism in mice.

BACKGROUND: Vasogenic cerebral edema resulting from blood-brain barrier (BBB) damage aggravates the devastating consequences of intracerebral hemorrhage (ICH). Although augmentation of endothelial Wnt/ß-catenin signaling substantially alleviates BBB breakdown in animals, no agents based on this mechanism are clinically available. Lithium is a medication used to treat bipolar mood disorders and can upregulate Wnt/ß-catenin signaling. METHODS: We evaluated the protective effect of lithium on the BBB in a mouse model of collagenase IV-induced ICH. Furthermore, we assessed the effect and dependency of lithium on Wnt/ß-catenin signaling in mice with endothelial deletion of the Wnt7 coactivator Gpr124. RESULTS: Lithium treatment (3 mmol/kg) significantly decreased the hematoma volume (11.15 ± 3.89 mm3 vs. 19.97 ± 3.20 mm3 in vehicle controls, p = 0.0016) and improved the neurological outcomes of mice following ICH. Importantly, lithium significantly increased the BBB integrity, as evidenced by reductions in the levels of brain edema (p = 0.0312), Evans blue leakage (p = 0.0261), and blood IgG extravasation (p = 0.0009) into brain tissue around the hematoma. Mechanistically, lithium upregulated the activity of endothelial Wnt/ß-catenin signaling in mice and increased the levels of tight junction proteins (occludin, claudin-5 and ZO-1). Furthermore, the protective effect of lithium on cerebral damage and BBB integrity was abolished in endothelial Gpr124 knockout mice, suggesting that its protective effect on BBB function was mainly dependent on Gpr124-mediated endothelial Wnt/ß-catenin signaling. CONCLUSION: Our findings indicate that lithium may serve as a therapeutic candidate for treating BBB breakdown and brain edema following ICH.

Lithium alleviates blood-brain barrier breakdown after cerebral ischemia and reperfusion by upregulating endothelial Wnt/ß-catenin signaling in mice.

Although upregulation of endothelial Wnt/ß-catenin signaling may be used to treat blood-brain barrier (BBB) breakdown caused by cerebral ischemia/reperfusion injury, no agents based on this mechanism are available clinically. Lithium, a medication used for treating bipolar mood disorders, upregulates Wnt/ß-catenin signaling, but whether lithium alleviates BBB breakdown after ischemic stroke by upregulating endothelial Wnt/ß-catenin signaling is unclear. Here, we evaluated the BBB-protective effect of lithium in adult mice with 1-h middle cerebral artery occlusion and 48-h reperfusion (MCAO/R) by determining neurological outcomes, BBB function and related molecular components. Furthermore, we assessed the effect and dependence of lithium on Wnt/ß-catenin signaling in brain microvascular endothelial cells in cell culture and in mice with conditional endothelial knockout of Wnt7 co-receptor Gpr124. Our data show that lithium treatment (3 mmol/kg) significantly decreased infarct volume (34.1 ± 1.8% versus 58.3 ± 2.8% in vehicle controls, P < 0.0001) and improved neurological outcomes of mice following MCAO/R. Importantly, lithium significantly increased BBB integrity shown by reduction of Evans blue leakage (by 45.7%, P = 0.0064) and blood IgG extravasation (by 65.8%, P < 0.0001) into infarcted brain tissue. Mechanistically, lithium upregulated the activity of endothelial Wnt/ß-catenin signaling in vivo and in vitro, increased the protein levels of tight junctions (Claudin-5 and ZO-1), and reduced MMP-9 expression. Furthermore, the protective effect of lithium on cerebral damage and BBB integrity was abolished in endothelial Gpr124 knockout mice, indicating the protection of lithium on BBB was mainly dependent on the Gpr124-mediated endothelial Wnt/ß-catenin signaling. Taken together, our findings indicate that lithium may serve as a therapeutic candidate for treating the BBB breakdown in the early stage of ischemic stroke following reperfusion therapy.

LINC00052 regulates the expression of NTRK3 by miR-128 and miR-485-3p to strengthen HCC cells invasion and migration.

Long non-coding RNAs (LncRNAs) are a group of RNAs that are more than 200 nt in length but cannot encode proteins. Accumulating evidences showed that abnormal LncRNA expressions are highly involved in many kinds of tumor. By using gene trap methods which could knockdown gene expression to find important genes, we found one LncRNA which called intergenic non-protein coding RNA 52 (LINC00052) has the ability to inhibit invasion and migration of hepatocarcinoma cells. We found that invasion, migration and proliferation abilities in SMMC7721 cell were inhibited after up-expressing LINC00052. We identified that NTRK3 was the target gene of LINC00052. Down-expression of NTRK3 could increase SMMC7721 cell invasion, migration and proliferation. Meanwhile, we discovered that LINC00052 could regulate NTRK3 expression by forming complementary base pairing with miR-128 and miR-485-3p to reduce the luciferase activity of NTRK3 3'UTR. These results reveal a new mechanism for understanding hepatocarcinoma cells invasion and migration.

Upregulated in Hepatitis B virus-associated hepatocellular carcinoma cells, miR-331-3p promotes proliferation of hepatocellular carcinoma cells by targeting ING5.

UNLABELLED: Hepatitis B virus (HBV) is a major risk factor for development and progression of hepatocellular carcinoma (HCC). It has been reported that viral infection can interfere with cellular microRNA (miRNA) expression and participate in the pathogenesis of oncogenicity. Our miRNAs array data indicated that miR-331-3p expression in HCC cell lines increased, but the relationship between miR-331-3p expression and HBV activity is unclear. Here, we observed elevated expression of miR-331-3p in different HCC cell lines expressing HBV. HBV, especially HBx, promotes miR-331-3p expression by enhancing its promoter activity. Using a miRNA target prediction database miRBase, we identified ING5 to be a novel target gene of miR-331-3p. miR-331-3p could inhibit ING5 expression by directly targeting its 3'-untranslated region (3'-UTR). As predicted, HBV was confirmed to repress ING5 at both mRNA and protein levels by promoting miR-331-3p expression. Our result indicated that miR-331-3p expression promotes proliferation of SMMC7721 cells by inhibiting ING5. ING5 overexpression promoted cell apoptosis in HCC cell lines. We also found ING5 expression was decreased in tumor tissue of HCC patient with HBV infection compared to its expression in para-carcinoma tissues. CONCLUSION: These results showed that miR-331-3p is upregulated by HBV and promotes proliferation of HCC cells though repression of ING5 expression. These data provide new insights for understanding the mechanisms of HBV-related HCC pathogenesis.