Optimal Treatment Parameters for Ultrasound-Stimulated Microbubbles in Upregulating Proliferation and Stemness of Bone Marrow Mesenchymal Stem Cells.

OBJECTIVES: Ultrasound-targeted microbubble disruption (UTMD) is a widely used technique to improve the differentiation and proliferation capacity of mesenchymal stem cells (MSCs), but the optimal therapeutic parameters for UTMD are unclear. In this study, we aimed to find the appropriate peak negative pressure (PNP), which is a key parameter for enhancing the stemness properties and proliferation of MSCs. METHODS: Experiments were performed in UTMD group, ultrasound (US) group under different PNP exposure conditions (0.5, 1.0, and 1.5 MPa), and control group. Apoptosis safety was analyzed by flow cytometry and MSC proliferation was measured at 12, 24, and 36 hours after irradiation by cell counting kit 8. The expression of the stemness genes NANOG, OCT-4, and SOX-2 were determined by enzyme-linked immunosorbent assay (ELISA) or reverse transcription polymerase chain reaction. RESULTS: The results showed that the 1.5 MPa UTMD-treated group had the highest proliferation capacity of MSCs at 24 hours. ELISA or quantitative reverse transcription polymerase chain reaction results showed that UTMD treatment of the 1.5 MPa group significantly upregulated the expression of the stemness genes NANOG, SOX-2, and OCT-4. CONCLUSIONS: In conclusion, the appropriate peak PNP value of UTMD was 1.5 MPa, and 1.5 MPa-mediated UTMD group obviously promoted MSCs proliferation and maintained stemness by upregulating the expression of stemness genes.

Enhancement of Tumor Perfusion and Antiangiogenic Therapy in Murine Models of Clear Cell Renal Cell Carcinoma Using Ultrasound-Stimulated Microbubbles.

OBJECTIVE: To explore the effect of ultrasound-stimulated microbubble cavitation (USMC) on enhancing antiangiogenic therapy in clear cell renal cell carcinoma. MATERIALS AND METHODS: We explored the effects of USMC with different mechanical indices (MIs) on tumor perfusion, 36 786-O tumor-bearing nude mice were randomly assigned into four groups: (i) control group, (ii) USMC0.25 group (MI = 0.25), (iii) USMC1.4 group (MI = 1.4) (iv) US1.4 group (MI = 1.4). Tumor perfusion was assessed by contrast-enhanced ultrasound (CEUS) before the USMC treatment and 30 min, 4h and 6h after the USMC treatment, respectively. Then we evaluated vascular normalization(VN) induced by low-MI (0.25) USMC treatment, 12 tumor-bearing nude mice were randomly divided into two groups: (i) control group (ii) USMC0.25 group. USMC treatment was performed, and tumor microvascular imaging and blood perfusion were analyzed by MicroFlow imaging (MFI) and CEUS 30 min after each treatment. In combination therapy, a total of 144 tumor-bearing nude mice were randomly assigned to six groups (n = 24): (i) control group, (ii) USMC1.4 group, (iii) USMC0.25 group, (iv) bevacizumab(BEV) group, (v) USMC1.4 +BEV group, (vi) USMC0.25 +BEV group. BEV was injected on the 6th, 10th, 14th, and 18th d after the tumors were inoculated, while USMC treatment was performed 24 h before and after every BEV administration. We examined the effects of the combination therapy through a series of experiments. RESULTS: Tumor blood perfusion enhanced by USMC with low MI (0.25)could last for more than 6h, inducing tumor VN and promoting drug delivery. Compared with other groups, USMC0.25+BEV combination therapy had the strongest inhibition on tumor growth, led to the longest survival time of the mice. CONCLUSION: The optimized USMC is a promising therapeutic approach that can be combined with antiangiogenic therapy to combat tumor progression.

Sononeoperfusion effect by ultrasound and microbubble promotes nitric oxide release to alleviate hypoxia in a mouse MC38 tumor model.

Tumor hypoperfusion not only impedes therapeutic drug delivery and accumulation, but also leads to a hypoxic and acidic tumor microenvironment, resulting in tumor proliferation, invasion, and therapeutic resistance. Sononeoperfusion effect refers to tumor perfusion enhancement using ultrasound and microbubbles. This study aimed to further investigate hypoxia alleviation by sononeoperfusion effect and explore the characteristics and mechanism of sononeoperfusion effect. To stimulate the sononeoperfusion effect, mice bearing MC38 colon cancers were included in this study and diagnostic ultrasound for therapy was set at a mechanical index (MI) of 0.1, 0.3, and 0.5, frequency of 3 MHz, pulse length of 5 cycles, and pulse repetition frequency of 2000 Hz. The results demonstrated that a single ultrasound and microbubble (USMB) treatment resulted in tumor perfusion enhancement at MI = 0.3, and nitric oxide (NO) concentration increased at MI = 0.3/0.5 (P < 0.05). However, there were no significant difference in the hypoxia-inducible factor-1α (HIF-1α) or D-lactate (D-LA) (P > 0.05) levels. Multiple sononeoperfusion effects were observed at MI = 0.3/0.5 (P < 0.05). For each treatment, USMB slightly but steadily improved the tumor tissue oxygen partial pressure (pO2) during and post treatment. It alleviated tumor hypoxia by decreasing HIF-1α, D-LA level and the hypoxic immunofluorescence intensity at MI = 0.3/0.5 (P < 0.05). The sononeoperfusion effect was not stimulated after eNOS inhibition. In conclusion, USMB with appropriate MI could lead to a sononeoperfusion effect via NO release, resulting in hypoxia amelioration. The tumors were not resistant to multiple sononeoperfusion effects. Repeated sononeoperfusion is a promising approach for relieving tumor hypoxia and resistance to therapy.

Development and validation of a collagen signature to predict the prognosis of patients with stage II/III colorectal cancer.

The tumor, nodes and metastasis (TNM) classification system provides useful but incomplete prognostic information and lacks the assessment of the tumor microenvironment (TME). Collagen, the main component of the TME extracellular matrix, plays a nonnegligible role in tumor invasion and metastasis. In this cohort study, we aimed to develop and validate a TME collagen signature (CSTME) for prognostic prediction of stage II/III colorectal cancer (CRC) and to compare the prognostic values of "TNM stage + CSTME" with that of TNM stage alone. Results indicated that the CSTME was an independent prognostic risk factor for stage II/III CRC (hazard ratio: 2.939, 95% CI: 2.180-3.962, p < 0.0001), and the integration of the TNM stage and CSTME had a better prognostic value than that of the TNM stage alone (AUC(TNM+CSTME) = 0.772, AUC TNM = 0.687, p < 0.0001). This study provided an application of "seed and soil" strategy for prognosis prediction and individualized therapy.

Use of anti-seizure medications in different types of autoimmune encephalitis: A narrative review.

Seizures are the main manifestation of the acute phase of autoimmune encephalitis (AE). Anti-seizure medications (ASMs) play an important role in controlling seizures in AE patients, but there is currently a lack of consensus regarding the selection, application, and discontinuation of ASMs. This narrative review focuses on the use of ASMs in patients with AE driven by different antibodies. The PubMed, Embase, and MEDLINE databases were searched up until 30 October 2022 using prespecified search terms. We identified 2,580 studies; 23 retrospective studies, 2 prospective studies and 9 case reports were evaluated based on our inclusion criteria. Anti-N-methyl-D-aspartic-acid-receptor (anti-NMDAR) encephalitis is the type of AE that responds best to ASMs, and long-term or combined use of ASMs may be not required in most patients with seizures; these results apply to both adults and children. Sodium channel blockers may be the best option for seizures in anti-leucine-rich-glioma-inactivated-1 (anti-LGI1) encephalitis, but patients with anti-LGI1 encephalitis are prone to side effects when using ASMs. Cell surface antibody-mediated AE patients are more likely to use ASMs for a long period than patients with intracellular antibody-mediated AE. Clinicians can score AE patients' clinical characteristics on a scale to identify those who may require long-or short-term use of ASMs in the early stage. This review provides some recommendations for the rational use of ASMs in encephalitis mediated by different antibodies with the aim of controlling seizures and avoiding overtreatment.

OTUB1 promotes osteoblastic bone formation through stabilizing FGFR2.

Bone homeostasis is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption. Dysregulation of this process leads to multiple diseases, including osteoporosis. However, the underlying molecular mechanisms are not fully understood. Here, we show that the global and conditional osteoblast knockout of a deubiquitinase Otub1 result in low bone mass and poor bone strength due to defects in osteogenic differentiation and mineralization. Mechanistically, the stability of FGFR2, a crucial regulator of osteogenesis, is maintained by OTUB1. OTUB1 attenuates the E3 ligase SMURF1-mediated FGFR2 ubiquitination by inhibiting SMURF1's E2 binding. In the absence of OTUB1, FGFR2 is ubiquitinated excessively by SMURF1, followed by lysosomal degradation. Consistently, adeno-associated virus serotype 9 (AAV9)-delivered FGFR2 in knee joints rescued the bone mass loss in osteoblast-specific Otub1-deleted mice. Moreover, Otub1 mRNA level was significantly downregulated in bones from osteoporotic mice, and restoring OTUB1 levels through an AAV9-delivered system in ovariectomy-induced osteoporotic mice attenuated osteopenia. Taken together, our results suggest that OTUB1 positively regulates osteogenic differentiation and mineralization in bone homeostasis by controlling FGFR2 stability, which provides an optical therapeutic strategy to alleviate osteoporosis.

Sononeoperfusion: a new therapeutic effect to enhance tumour blood perfusion using diagnostic ultrasound and microbubbles.

BACKGROUND: Hypoperfusion or resultant hypoxia in solid tumours is a main reason for therapeutic resistance. Augmenting the blood perfusion of hypovascular tumours might improve both hypoxia and drug delivery. Cavitation is known to result in microstreaming and sonoporation and to enhance drug diffusion into tumours. Here, we report the ability to enhance both tumour blood perfusion and doxorubicin (Dox) delivery using a new sononeoperfusion effect causing a cavitation effect on tumour perfusion in subcutaneous Walker-256 tumours of rats using ultrasound stimulated microbubble (USMB). METHODS: To induce the sononeoperfusion effect, USMB treatment was performed with a modified diagnostic ultrasound (DUS) system and SonoVue® microbubbles. The therapeutic pulse was operated with a peak negative pressure of 0.26 to 0.32 MPa and a pulse repetition frequency (PRF) of 50 Hz to 2 kHz. Contrast-enhanced ultrasound (CEUS) was used for tumour perfusion assessment. RESULTS: The USMB treatment of 0.26 MPa and 1 kHz could significantly enhance tumour perfusion with a 20.29% increase in the CEUS peak intensity and a 21.42% increment in the perfusion area for more than 4 hours (P < 0.05). The treatment also increased Dox delivery to tumours by approximately 3.12-fold more than that of the control (P < 0.05). Furthermore, ELISAs showed that vasodilators and inflammatory factors increased 4 hours after treatment (P < 0.05), suggesting that the inflammatory response plays an important role in the sononeoperfusion effect. CONCLUSION: The USMB-induced sononeoperfusion effect could significantly enhance the blood perfusion of Walker-256 tumours and promote drug delivery. It might be a novel physical method for overcoming the therapeutic resistance of hypoperfused or hypoxic tumours.

Mechanical destruction using a minimally invasive Ultrasound Needle induces anti-tumor immune responses and synergizes with the anti-PD-L1 blockade.

Immune checkpoint inhibitors (ICIs) have been widely used in treating various tumors; however, the objective response rate of ICIs is less than 40%. In this study, we attempted to induce anti-tumor immune responses using an improved ultrasonic horn device, Ultrasound Needle (UN). We tested its synergistic anti-tumor efficacy with an anti-PD-L1 antibody in a mouse tumor model. Under different parameters, UN treatment selectively induced mechanical destruction and thermal ablation effects on tumor tissues. The mechanical destruction effect of UN treatment increased the infiltration of CD8+ T cells in tumors and relieved the immunosuppressive tumor microenvironment. It also induced systemic anti-tumor immune responses and enhanced the therapeutic efficacy of the anti-PD-L1 antibody in both local and abscopal tumors. The mechanical destruction effect of UN treatment resulted in the release of damage-associated molecular patterns and promoted dendritic cells (DCs)-based antigen presentation. Depletion of DCs or CD8+ T cells eliminated the anti-tumor immune responses induced by UN treatment and weakened the synergistic anti-tumor efficacy with anti-PD-L1 antibody. Therefore, minimally invasive UN may provide a new therapeutic modality for ultrasound-assisted immunotherapy.

JAK/STAT3 Signaling Activation Related to Distinct Clinicopathologic Features in Systemic ALK - Anaplastic Large Cell Lymphomas : New Insights into Their Heterogeneity.

Systemic anaplastic lymphoma kinase (ALK)-negative anaplastic large cell lymphoma (ALCL) is a group of heterogenous CD30 + T-cell non-Hodgkin lymphomas. Previous studies have highlighted the importance of JAK/STAT3 signaling activation in the molecular pathogenesis of ALK - ALCLs. In the present study, we aimed to establish a potential relationship between JAK/STAT3 signaling activation and clinicopathologic features in ALK - ALCLs, and further recognize the heterogenous nature of these neoplasms. Immunohistochemistry staining of the phosphorylated-STAT3 (p-STAT3) and dual-specificity protein phosphatase 22 ( DUSP22 ) gene rearrangement analysis were performed. Forty-five cases of ALK - ALCL were divided into 3 groups, including 9 DUSP22 -rearranged ALCLs, 21 p-STAT3 + double-negative (DN) ALCLs (both ALK and DUSP22 rearrangement negative), and 15 p-STAT3 - DN-ALCLs. Morphologically, p-STAT3 + DN-ALCLs exhibited sheet-like neoplastic cells and sometimes showed large pleomorphic cells scattered in a lymphocyte-rich background more frequently than those in other ALK - ALCLs subtypes. Phenotypically, the p-STAT3 + DN-ALCLs frequently expressed cytotoxic molecules, epithelial membrane antigen, and programmed death-ligand 1, whereas CD3 and CD5 expression was not observed. Clinically, patients with p-STAT3 + DN-ALCLs had a better prognosis than those with p-STAT3 - DN-ALCLs. These observations suggest that p-STAT3 + DN-ALCLs represent a distinct subtype of ALK - ALCLs. Identifying ALK - ALCL subtypes by using p-STAT3 staining and DUSP22 rearrangement is a promising approach that may contribute to risk stratification and better treatment decisions in the future clinical practice.

OTUB2 exerts tumor-suppressive roles via STAT1-mediated CALML3 activation and increased phosphatidylserine synthesis.

Oral and esophageal squamous cell carcinomas (SCCs) are associated with high mortality, yet the molecular mechanisms underlying these malignancies are largely unclear. We show that DNA hypermethylation of otubain 2 (OTUB2), a previously recognized oncogene, drives tongue and esophageal SCC initiation and drug resistance. Mechanistically, OTUB2 promotes the deubiquitination and phosphorylation of signal transducer and activator of transcription 1 (STAT1) and subsequently regulates the transcription of calmodulin-like protein 3 (CALML3). Activation of CALML3-mediated mitochondrial calcium signaling promotes oxidative phosphorylation (OXPHOS) and the synthesis of phosphatidylserine (PS). In mouse models, orally administered soybean-derived PS inhibits SCC initiation in cells with low OTUB2 expression and increases their sensitivity to chemotherapy. Our study indicates that the OTUB2/STAT1/CALML3/PS axis plays tumor-suppressive roles and shows the potential of PS administration as a strategy for the treatment and prevention of tongue and esophageal SCCs.

Whole-Genome/Exome Sequencing Uncovers Mutations and Copy Number Variations in Primary Diffuse Large B-Cell Lymphoma of the Central Nervous System.

Background/objective: Identification of key genetic alterations is of importance in the targeted therapies of primary central nervous system lymphoma (PCNSL). However, only a small number of studies have been carried out in PCNSL. In this study, we further described the genetic mutations and copy number variations (CNVs) in PCNSL patients using whole-genome/exome sequencing (WGS/WES), as well as revealed their associations with patients' clinicopathological features and prognosis. Methods: Tumor specimens from 38 patients with primary diffuse large B-cell lymphoma of the central nervous system (CNS DLBCL) were enrolled to WGS (n = 24) or WES (n = 14). The CNVs and mutations of 24 samples (WGS) and 38 samples (WGS/WES) were characterized, respectively. The associations between CNVs and mutations with the overall survival rates of PCNSL patients were also evaluated. Results: The most common mutations were identified in IGLL5 (68%), PIM1 (63%), MYD88 (55%), CD79B (42%), BTG2 (39%), PCLO (39%), KMT2D (34%), and BTG1 (29%) genes. Among the mutated genes, EP300, ETV6, and HIST1H1E mutations were exclusively detected in the elderly, while DUSP2 mutations were associated with the immune microenvironment indicators. In addition, KMT2D mutation was associated with a poor prognosis. In addition, 488 CNVs including 91 gains and 397 deletions were observed across 24 samples from WGS results. Notably, 1q31.3 amplification was closely associated with the poor prognosis of PCNSL patients. Conclusion: This study further characterizes the genomic landscape of primary CNS DLBCL using WGS/WES, which provides insight into understanding the pathogenesis of PCNSL and fosters new ideas for the targeted treatment of PCNSL.

In Vivo and In Vitro Fibroblasts' Behavior and Capsular Formation in Correlation with Smooth and Textured Silicone Surfaces.

BACKGROUND: As the most principal complication following breast augmentation with silicone breast implants, capsular contracture is greatly influenced by surface texture. However, there have long been widespread debates on the function of smooth or textured surface implants in reducing capsular contracture. MATERIALS AND METHODS: Three commercially available silicone breast implants with smooth and textured surfaces were subjected to surface characterization, and in vitro and in vivo assessments were then implemented to investigate the effect of these different surfaces on the biological behaviors of fibroblasts and capsular formation in rat models. RESULTS: Surface characterization demonstrated that all three samples were hydrophobic with distinct roughness values. Comparing the interactions of fibroblasts or tissues with different surfaces, we observed that as surface roughness increased, the adhesion and cell spreading of fibroblasts, the level of echogenicity, the density of collagen and α-SMA-positive immunoreactivity decreased, while the proliferation of fibroblasts and capsule thickness increased. CONCLUSIONS: Our findings elucidated that the effect of silicone implant surface texture on fibroblasts' behaviors and capsular formation was associated with variations in surface roughness, and the number of myofibroblasts may have a more significant influence on the process of contracture than capsule thickness in the early stage of capsular formation. These results highlight that targeting myofibroblasts may be wielded in the prevention and treatment strategies of capsular contracture clinically. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Ultrasound-triggered microbubble destruction enhances the radiosensitivity of glioblastoma by inhibiting PGRMC1-mediated autophagy in vitro and in vivo.

BACKGROUND: Ultrasound-triggered microbubble destruction (UTMD) is a widely used noninvasive technology in both military and civilian medicine, which could enhance radiosensitivity of various tumors. However, little information is available regarding the effects of UTMD on radiotherapy for glioblastoma or the underlying mechanism. This study aimed to delineate the effect of UTMD on the radiosensitivity of glioblastoma and the potential involvement of autophagy. METHODS: GL261, U251 cells and orthotopic glioblastoma-bearing mice were treated with ionizing radiation (IR) or IR plus UTMD. Autophagy was observed by confocal microscopy and transmission electron microscopy. Western blotting and immunofluorescence analysis were used to detect progesterone receptor membrane component 1 (PGRMC1), light chain 3 beta 2 (LC3B2) and sequestosome 1 (SQSTM1/p62) levels. Lentiviral vectors or siRNAs transfection, and fluorescent probes staining were used to explore the underlying mechanism. RESULTS: UTMD enhanced the radiosensitivity of glioblastoma in vitro and in vivo (P < 0.01). UTMD inhibited autophagic flux by disrupting autophagosome-lysosome fusion without impairing lysosomal function or autophagosome synthesis in IR-treated glioblastoma cells. Suppression of autophagy by 3-methyladenine, bafilomycin A1 or ATG5 siRNA had no significant effect on UTMD-induced radiosensitization in glioblastoma cells (P < 0.05). Similar results were found when autophagy was induced by rapamycin or ATG5 overexpression (P > 0.05). Furthermore, UTMD inhibited PGRMC1 expression and binding with LC3B2 in IR-exposed glioblastoma cells (P < 0.01). PGRMC1 inhibitor AG-205 or PGRMC1 siRNA pretreatment enhanced UTMD-induced LC3B2 and p62 accumulation in IR-exposed glioblastoma cells, thereby promoting UTMD-mediated radiosensitization (P < 0.05). Moreover, PGRMC1 overexpression abolished UTMD-caused blockade of autophagic degradation, subsequently inhibiting UTMD-induced radiosensitization of glioblastoma cells. Finally, compared with IR plus UTMD group, PGRMC1 overexpression significantly increased tumor size [(3.8 ± 1.1) mm2 vs. (8.0 ± 1.9) mm2, P < 0.05] and decreased survival time [(67.2 ± 2.6) d vs. (40.0 ± 1.2) d, P = 0.0026] in glioblastoma-bearing mice. CONCLUSION: UTMD enhanced the radiosensitivity of glioblastoma partially by disrupting PGRMC1-mediated autophagy.

Neurokinin-1 receptor promotes non-small cell lung cancer progression through transactivation of EGFR.

Despite the great advances in target therapy, lung cancer remains the top cause of cancer-related death worldwide. G protein-coupled receptor neurokinin-1 (NK1R) is shown to play multiple roles in various cancers; however, the pathological roles and clinical implication in lung cancer are unclarified. Here we identified NK1R as a significantly upregulated GPCR in the transcriptome and tissue array of human lung cancer samples, associated with advanced clinical stages and poor prognosis. Notably, NK1R is co-expressed with epidermal growth factor receptor (EGFR) in NSCLC patients' tissues and co-localized in the tumor cells. NK1R can crosstalk with EGFR by interacting with EGFR, transactivating EGFR phosphorylation and regulating the intracellular signaling of ERK1/2 and Akt. Activation of NK1R promotes the proliferation, colony formation, EMT, MMP2/14 expression, and migration of lung cancer cells. The inhibition of NK1R by selective antagonist aprepitant repressed cell proliferation and migration in vitro. Knockdown of NK1R significantly slowed down the tumor growth in nude mice. The sensitivity of lung cancer cells to gefitinib/osimertinib is highly increased in the presence of the selective NK1R antagonist aprepitant. Our data suggest that NK1R plays an important role in lung cancer development through EGFR signaling and the crosstalk between NK1R and EGFR may provide a potential therapeutic target for lung cancer treatment.

The Ribosomal Protein L28 Gene Induces Sorafenib Resistance in Hepatocellular Carcinoma.

BACKGROUND: Sorafenib is the first molecular-targeted drug for the treatment of advanced hepatocellular carcinoma (HCC). However, its treatment efficiency decreases after a short period of time because of the development of drug resistance. This study investigates the role of key genes in regulating sorafenib-resistance and elucidates the mechanism of drug resistance in hepatocellular carcinoma. METHODS: The HCC HepG2 cells were used to generate a sorafenib-resistant cell model by culturing the cells in gradually increasing concentration of sorafenib. RNA microarray was applied to profile gene expression and screen key genes associated with sorafenib resistance. Specific targets were knockdown in sorafenib-resistant HepG2 cells for functional studies. The HCC model was established in ACI rats using Morris hepatoma3924A cells to validate selected genes associated with sorafenib resistance in vivo. RESULTS: The HepG2 sorafenib-resistant cell model was successfully established. The IC50 of sorafenib was 9.988µM in HepG2 sorafenib-resistant cells. A total of 35 up-regulated genes were detected by expression profile chip. High-content screening technology was used and a potential drug-resistance related gene RPL28 was filtered out. After knocking down RPL28 in HepG2 sorafenib-resistant cells, the results of cell proliferation and apoptosis illustrated that RPL28 is the key gene involving in drug resistance. Furthermore, it was found that both RNA and protein expression of RPL28 increased in HepG2 sorafenib-resistant specimens of Morris Hepatoma rats. In addition, the expression of proliferative protein Ki-67 increased in sorafenib-resistant cells. CONCLUSION: Our study suggested that RPL28 is a key gene inducing sorafenib resistance in HCC and could be a potential target for the treatment of drug-resistant HCC.

OTULIN allies with LUBAC to govern angiogenesis by editing ALK1 linear polyubiquitin.

OTULIN coordinates with LUBAC to edit linear polyubiquitin chains in embryonic development, autoimmunity, and inflammatory diseases. However, the mechanism by which angiogenesis, especially that of endothelial cells (ECs), is regulated by linear ubiquitination remains unclear. Here, we reveal that constitutive or EC-specific deletion of Otulin resulted in arteriovenous malformations and embryonic lethality. LUBAC conjugates linear ubiquitin chains onto Activin receptor-like kinase 1 (ALK1), which is responsible for angiogenesis defects, inhibiting ALK1 enzyme activity and Smad1/5 activation. Conversely, OTULIN deubiquitinates ALK1 to promote Smad1/5 activation. Consistently, embryonic survival of Otulin-deficient mice was prolonged by BMP9 pretreatment or EC-specific ALK1Q200D (constitutively active) knockin. Moreover, mutant ALK1 from type 2 hereditary hemorrhagic telangiectasia (HHT2) patients exhibited excessive linear ubiquitination and increased HOIP binding. As such, a HOIP inhibitor restricted the excessive angiogenesis of ECs derived from ALK1G309S-expressing HHT2 patients. These results show that OTULIN and LUBAC govern ALK1 activity to balance EC angiogenesis.

Ultrasound-Targeted Microbubble Destruction Accelerates Angiogenesis and Ameliorates Left Ventricular Dysfunction after Myocardial Infarction in Mice.

Failure of coronary recanalization within 12 h or no flow in the myocardium after percutaneous coronary intervention is associated with high mortality from myocardial infarction, and insufficient angiogenesis in the border zone results in the expansion of infarct area. In this study, we examined the effects of ultrasound-targeted microbubble destruction (UTMD) on angiogenesis and left ventricular dysfunction in a mouse model of myocardial infarction. Fifty-four mice with MI were treated with no UTMD, ultrasound (US) alone or UTMD four times (days 1, 3, 5 and 7), and another 18 mice underwent sham operation and therapy. Therapeutic US was generated with a linear transducer connected to a commercial diagnostic US system (VINNO70). UTMD was performed with the VINNO70 at a peak negative pressure of 0.8 MPa and lipid microbubbles. Transthoracic echocardiography was performed on the first and seventh days. The results indicated that UTMD decreased the infarct size ratio from 78.1 ± 5.3% (untreated) to 43.3 ± 6.4%, accelerated angiogenesis and ameliorated left ventricular dysfunction. The ejection fraction increased from 25.05 ± 8.52% (untreated) to 42.83 ± 9.44% (UTMD). Compared with that in other groups, expression of vascular endothelial growth factor and endothelial nitric oxide synthase and release of nitric oxide were significantly upregulated after UTMD treatment, indicating angiogenesis. Therefore, UTMD is a potential physical approach in the treatment of myocardial infarction.

Effect of diagnostic ultrasound and microbubble-enhanced chemotherapy on metastasis of rabbit VX2 tumor.

PURPOSE: Ultrasound-targeted microbubble destruction (UTMD) has been widely applied to enhance chemotherapy of tumors, yet few studies have focused on the metastatic potential induced by UTMD. This study aimed to explore the metastasis of VX2 tumors after treatment with UTMD and chemotherapy. METHODS: Forty-four New Zealand rabbits bearing subcutaneous VX2 tumors were enrolled for the treatment of UTMD with chemotherapy. For UTMD, the tumors were insonated using two pulsing protocols of diagnostic ultrasound (DUS, VINNO and ECARE) with a mechanical index (MI) of 0.29-0.33, tone burst of 8.0 cycles, and frequencies of 3-4 MHz. A total dose of 2 ml SonoVue® was injected intermittently during 10-min UTMD exposure. The combination therapy was treated using doxorubicin (DOX, 2 mg/kg) and DUS, while the tumors treated using DOX only served as the control. Tumor size was measured using the tumor volume formula. Survival time was observed until animal death or the end of the study (120 days). Specific organs (lung, liver, kidney, and brain) were removed for metastatic evaluation. RESULTS: There were no statistical differences in overall metastasis classification and individual organ metastases among all groups (P > 0.05). The tumor growth rate only showed inhibition on the 5th day (P < 0.01). The survival time did not demonstrate any significant difference between UTMD and chemotherapy only (P > 0.05). CONCLUSIONS: UTMD using long-pulse DUS with commercial microbubbles did not pose a risk of metastasis enhancement in DOX chemotherapy.

Exosomes derived from human induced pluripotent stem cell-derived neural progenitor cells protect neuronal function under ischemic conditions.

Compared with other stem cells, human induced pluripotent stem cells-derived neural progenitor cells (iPSC-NPCs) are more similar to cortical neurons in morphology and immunohistochemistry. Thus, they have greater potential for promoting the survival and growth of neurons and alleviating the proliferation of astrocytes. Transplantation of stem cell exosomes and stem cells themselves have both been shown to effectively repair nerve injury. However, there is no study on the protective effects of exosomes derived from iPSC-NPCs on oxygen and glucose deprived neurons. In this study, we established an oxygen-glucose deprivation model in embryonic cortical neurons of the rat by culturing the neurons in an atmosphere of 95% N2 and 5% CO2 for 1 hour and then treated them with iPSC-NPC-derived exosomes for 30 minutes. Our results showed that iPSC-NPC-derived exosomes increased the survival of oxygen- and glucose-deprived neurons and the level of brain-derived neurotrophic factor in the culture medium. Additionally, it attenuated oxygen and glucose deprivation-induced changes in the expression of the PTEN/AKT signaling pathway as well as synaptic plasticity-related proteins in the neurons. Further, it increased the length of the longest neurite in the oxygen- and glucose-deprived neurons. These findings validate the hypothesis that exosomes from iPSC-NPCs exhibit a neuroprotective effect on oxygen- and glucose-deprived neurons by regulating the PTEN/AKT signaling pathway and neurite outgrowth. This study was approved by the Animal Ethics Committee of Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, China (approval No. SRRSH20191010) on October 10, 2019.

The functions and regulation of Otubains in protein homeostasis and diseases.

OTU domain-containing ubiquitin aldehyde-binding proteins Otubain1 (OTUB1) and Otubain2 (OTUB2) were initially identified as OTU deubiquitinases (DUBs). Recently, Otubains have emerged as essential regulators of diverse physiological processes, such as immune signaling and DNA damage response. Dysregulation of those processes is likely to increase the risk in multiple aspects of aging-related diseases, including cancers, neurodegenerative disorders, chronic kidney diseases, bone dysplasia and pulmonary fibrosis. Consistently, Otubains are aberrantly expressed in cancers and have been identified to be both tumor suppressors and tumor promoters in different types of cancers. Therefore, the regulatory mechanism of the activity and expression of Otubains is very important for better understanding of Otubains-associated biological networks and human diseases. This review provides a comprehensive description of functions and regulatory axis of Otubains, highlighting experimental evidences indicating Otubains as potential therapeutic targets against aging-related disorders.