Dual-layer conduit containing VEGF-A - Transfected Schwann cells promotes peripheral nerve regeneration via angiogenesis.

Peripheral nerve injuries (PNIs) can cause neuropathies and significantly affect the patient's quality of life. Autograft transplantation is the gold standard for conventional treatment; however, its application is limited by nerve unavailability, size mismatch, and local tissue adhesion. Tissue engineering, such as nerve guidance conduits, is an alternative and promising strategy to guide nerve regeneration for peripheral nerve repair; however, only a few conduits could reach the high repair efficiency of autografts. The healing process of PNI is frequently accompanied by not only axonal and myelination regeneration but also angiogenesis, which initializes nerve regeneration through vascular endothelial growth factor A (VEGF-A). In this study, a composite nerve conduit with a poly (lactic-co-glycolic acid) (PLGA) hollow tube as the outer layer and gelatin methacryloyl (GelMA) encapsulated with VEGF-A transfected Schwann cells (SCs) as the inner layer was established to evaluate its promising ability for peripheral nerve repair. A rat model of peripheral nerve defect was used to examine the efficiency of PLGA/GelMA-SC (VA) conduits, whereas autograft, PLGA, PLGA/GelMA, and PLGA/GelMA-SC (NC) were used as controls. VEGF-A-transfected SCs can provide a stable source for VEGF-A secretion. Furthermore, encapsulation in GelMA cannot only promote proliferation and tube formation of human umbilical vein endothelial cells but also enhance dorsal root ganglia and neuronal cell extension. Previous animal studies have demonstrated that the regenerative effects of PLGA/GelMA-SC (VA) nerve conduit were similar to those of autografts and much better than those of other conduits. These findings indicate that combination of VEGF-A-overexpressing SCs and PLGA/GelMA conduit-guided peripheral nerve repair provides a promising method that enhances angiogenesis and regeneration during nerve repair. STATEMENT OF SIGNIFICANCE: Nerve guidance conduits shows promise for peripheral nerve repair, while achieving the repair efficiency of autografts remains a challenge. In this study, a composite nerve conduit with a PLGA hollow tube as the outer layer and gelatin methacryloyl (GelMA) encapsulated with vascular endothelial growth factor A (VEGF-A)-transfected Schwann cells (SCs) as the inner layer was established to evaluate its potential ability for peripheral nerve repair. This approach preserves growth factor bioactivity and enhances material properties. GelMA insertion promotes Schwann cell proliferation and morphology extension. Moreover, transfected SCs serve as a stable VEGF-A source and fostering angiogenesis. This study offers a method preserving growth factor efficacy and safeguarding SCs, providing a comprehensive solution for enhanced angiogenesis and nerve regeneration.

Angiogenesis-related immune response may be the prelude to the syndesmophyte formation in Ankylosing spondylitis.

BACKGROUND: Ankylosing spondylitis (AS) is a chronic autoimmune arthritis that mainly affects spine joints. To date, the pathogenesis of AS remains unclear, although immune cells and innate immune response cytokines have been suggested to be crucial players. METHODS: By adopting a single-cell RNA sequencing approach in the AS cynomolgus model, we profiled and characterized PBMC proportions along disease progression. RESULTS: Here, our primary focus was on the activation of an immune cascade-initiating lymphocyte subtype known as CD4+CXCR5+ T follicular helper (Tfh) cells. These Tfhs demonstrated a localized residence in AS bone lesion as an ectopic lymphoid structure. Moreover, Tfhs would serve as an upstream initiator for a pro-angiogenic cascade. Then, an expansion in CD14+ monocytes and DC cells subsets resulted in enhanced expression of angiogenesis genes in these AS cynomolgus monkeys. With a confirmed higher abundance of TNF-α accompanying H-type vascular invasion in the osteophytic region, pronounced expansion of Tfhs at such lesion site signaling for monocytes and DCs intrusion is considered as the prelude to the characteristic angiogenic bony outgrowth in AS known as syndesmophytes. CONCLUSIONS: We explored the intimate relationship between local inflammation and bone formation in AS from the perspective of nascent vascularisation. Hence, our study lays the foundation for elucidating a unified AS pathogenesis through the immune-angiogenesis-osteogenesis axis.

Follicular Helper T Cells In Peyer's Patches And Galactose-Deficient Iga1 Contribute To Iga Nephropathy.

BACKGROUND: Common primary glomerulonephritis with aberrant mucosal immunity is IgA nephropathy (IgAN). T follicular helper (TFH) cells are essential in regulating B cell differentiation. Peyer's patches (PPs) are the main site where IgA+ plasmablasts differentiate. OBJECTIVE: Our study aimed to investigate the TFH cell's potential contribution to the etiology of IgA nephropathy. MATERIALS AND METHODS: In PPs from IgAN mouse models, the ratio of the TFH cell, B220+IgA+, B220+IgM+, and B220-IgA+ lymphocytes were assessed. Then, we used Western blot to assess the expression of Bcl-6, Blimp- 1, and IL-21 proteins in PPs and used RT-PCR to assess the expression of IL-21 and TGF- 1 mRNA. TFH cells coculture with spleen cells to measure the degree of IL-21 and the ratio of activation marker CD69 on the TFH cells. Naive B cells (CD27-IgD+) from children suffering from IgAN were cultured with TFH cell-related cytokines. The supernatant was detected to assess the excretion of galactose-deficient IgA1 (Gd-IgA1). RESULTS: IgAN mice developed noticeably increased degrees of IL-21 and CD69 on TFH cells than controls did, as well as higher percentages of B220+IgA+, B220+IgM+, B220+IgA+, TGF- 1, and IL-21 mRNA and Bcl-6, IL-21 proteins in PPs. The Gd-IgA1 level in the supernatant and IgAN- positive children's serum were noticeably higher than those of the healthy controls (P < 0.05). PPs provide the microenvironment to induce the production of IgA-secreting plasmablasts. CONCLUSION: TFH cells may be a key moderator to induce B cell differentiation into IgA-secreting plasmablasts and produce Gd-IgA1, which plays a significant part in IgAN's pathogenesis. It could be a new therapeutic target in the future.

A grooved conduit combined with decellularized tissues for peripheral nerve regeneration.

Peripheral nerve injury (PNI) is a common and severe clinical disease worldwide, which leads to a poor prognosis because of the complicated treatments and high morbidity. Autologous nerve grafting as the gold standard still cannot meet the needs of clinical nerve transplantation because of its low availability and limited size. The development of artificial nerve conduits was led to a novel direction for PNI treatment, while most of the currently developed artificial nerve conduits was lack biochemical cues to promote nerve regeneration. In this study, we designed a novel composite neural conduit by inserting decellularized the rat sciatic nerve or kidney in a poly (lactic-co-glycolic acid) (PLGA) grooved conduit. The nerve regeneration effect of all samples was analyzed using rat sciatic nerve defect model, where decellularized tissues and grooved PLGA conduit alone were used as controls. The degree of nerve regeneration was evaluated using the motor function, gastrocnemius recovery, and morphological and histological assessments suggested that the combination of a grooved conduit with decellularized tissues significantly promoted nerve regeneration compared with decellularized tissues and PLGA conduit alone. It is worth to note that the grooved conduits containing decellularized nerves have a promotive effect similar to that of autologous nerve grafting, suggesting that it could be an artificial nerve conduit used for clinical practice in the future.

Bioinformatics identification of ferroptosis-related genes and therapeutic drugs in rheumatoid arthritis.

Introduction: Rheumatoid arthritis (RA) is a chronic immune disease characterized by synovial inflammation and bone destruction, with a largely unclear etiology. Evidence has indicated that ferroptosis may play an increasingly important role in the onset and development of RA. However, ferroptosis-related genes are still largely unexplored in RA. Therefore, this work focused on identifying and validating the potential ferroptosis-related genes involved in RA through bioinformatics analysis. Methods: We screened differentially expressed ferroptosis-related genes (DEFGs) between RA patients and healthy individuals based on GSE55235 dataset. Subsequently, correlation analysis, protein-protein interaction (PPI) network analysis, GO, and KEGG enrichment analyses were performed using these DEFGs. Finally, our results were validated by GSE12021 dataset. Results: We discovered 34 potential DEFGs in RA based on bioinformatics analysis. According to functional enrichment analysis, these genes were mainly enriched in HIF-1 signaling pathway, FoxO signaling pathway, and Ferroptosis pathway. Four genes (GABARPL1, DUSP1, JUN, and MAPK8) were validated to be downregulated by GSE12021 dataset and were diagnostic biomarkers and therapeutic targets for RA via the regulation of ferroptosis. Discussion: Our results help shed more light on the pathogenesis of RA. Ferroptosis-related genes in RA are valuable diagnostic biomarkers and they will be exploited clinically as therapeutic targets in the future.

Printing channels with millimeter-scale curvature and deciphering their effect on the proliferation, morphology, orientation, and migration of M-22 cells.

Complex curved structures of tissues have been recognized to influence the behavior and function of cells. Tissue curvatures sensed by cells are approximately on the millimeter scale. However, previous research mainly focused on the effect of micro- and nano-scale spatial curved structures, underestimating the significance of milli-scale curvature. Here, we employed fused deposition modeling (FDM) with two-stage temperature control, superfine cone-shaped needle, stable air pressure, and precise motion platform for the customized production of homogeneous, precise, and curved fibers; the responses of M-22 cells to FDM-printed curved channels with radii of 1.5 to 3 mm were systematically investigated. The cells aligned with these curved channels and exhibited various aspect ratios in the channels with different curvatures. Cell proliferation, migration speed of single cells, and front-end speed of collective cells were tightly regulated by these curved structures. Also, a computational model based on force equilibrium was proposed to explore the essential factors and mechanisms of curvature affecting cell behavior. Our simulation results demonstrated that the curvature and width of channels, along with the relative size of cells, can significantly impact the cell-boundary interaction force and the number of valid pseudopodia generated by cells in the process of cell migration. These results provide a comprehensive understanding of the effect of milli-scale curvature on the cells and underpin the design of scaffolds that can be produced easily with sophisticated micro- and nano-scale curved features to regulate cell behavior in tissue engineering.

3D bioprinting of multi-cellular tumor microenvironment for prostate cancer metastasis.

Prostate cancer (PCa) is one of the most lethal cancers in men worldwide. The tumor microenvironment (TME) plays an important role in PCa development, which consists of tumor cells, fibroblasts, endothelial cells, and extracellular matrix (ECM). Hyaluronic acid (HA) and cancer-associated fibroblasts (CAFs) are the major components in the TME and are correlated with PCa proliferation and metastasis, while the underlying mechanism is still not fully understood due to the lack of biomimetic ECM components and coculture models. In this study, gelatin methacryloyl/chondroitin sulfate-based hydrogels were physically crosslinked with HA to develop a novel bioink for the three-dimensional bioprinting of a coculture model that can be used to investigate the effect of HA on PCa behaviors and the mechanism underlying PCa-fibroblasts interaction. PCa cells demonstrated distinct transcriptional profiles under HA stimulation, where cytokine secretion, angiogenesis, and epithelial to mesenchymal transition were significantly upregulated. Further coculture of PCa with normal fibroblasts activated CAF transformation, which could be induced by the upregulated cytokine secretion of PCa cells. These results suggested HA could not only promote PCa metastasis individually but also induce PCa cells to activate CAF transformation and form HA-CAF coupling effects to further promote PCa drug resistance and metastasis.

CADM2 is implicated in impulsive personality and numerous other traits by genome- and phenome-wide association studies in humans and mice.

Impulsivity is a multidimensional heritable phenotype that broadly refers to the tendency to act prematurely and is associated with multiple forms of psychopathology, including substance use disorders. We performed genome-wide association studies (GWAS) of eight impulsive personality traits from the Barratt Impulsiveness Scale and the short UPPS-P Impulsive Personality Scale (N = 123,509-133,517 23andMe research participants of European ancestry), and a measure of Drug Experimentation (N = 130,684). Because these GWAS implicated the gene CADM2, we next performed single-SNP phenome-wide studies (PheWAS) of several of the implicated variants in CADM2 in a multi-ancestral 23andMe cohort (N = 3,229,317, European; N = 579,623, Latin American; N = 199,663, African American). Finally, we produced Cadm2 mutant mice and used them to perform a Mouse-PheWAS ("MouseWAS") by testing them with a battery of relevant behavioral tasks. In humans, impulsive personality traits showed modest chip-heritability (~6-11%), and moderate genetic correlations (rg = 0.20-0.50) with other personality traits, and various psychiatric and medical traits. We identified significant associations proximal to genes such as TCF4 and PTPRF, and also identified nominal associations proximal to DRD2 and CRHR1. PheWAS for CADM2 variants identified associations with 378 traits in European participants, and 47 traits in Latin American participants, replicating associations with risky behaviors, cognition and BMI, and revealing novel associations including allergies, anxiety, irritable bowel syndrome, and migraine. Our MouseWAS recapitulated some of the associations found in humans, including impulsivity, cognition, and BMI. Our results further delineate the role of CADM2 in impulsivity and numerous other psychiatric and somatic traits across ancestries and species.

Evaluation of the Effect of Fibroblasts on Melanoma Metastasis Using a Biomimetic Co-Culture Model.

Melanoma is a highly malignant tumor originating from melanocytes. The 5-year survival rate of primary melanoma is 98%, whereas the survival rate of metastatic melanoma is only 10%, which can be attributed to the insensitivity to existing treatments. Fibroblasts are the primary cells in the dermis that promote melanoma metastasis; however, the molecular mechanism underlying the fibroblast-melanoma interaction is yet to be completely understood. Herein, gelatin methacryloyl (GelMA) was used to construct a co-culture model for melanoma cells (A375) and fibroblasts. GelMA retains the good biological properties of collagen, which has been identified as the primary component of the melanoma tumor microenvironment. Fibroblasts were encapsulated in GelMA, whereas A375 cells were cultured on the GelMA surface, which realistically mimics the macrostructure of melanoma. A375 cells co-cultured with fibroblasts demonstrated a higher cellular proliferation rate, potentials of neoneurogenesis, overexpression of epithelial mesenchymal transition markers, and a faster migration rate compared with A375 cells cultured alone, which could be due to the cancer-associated fibroblast activation and the overexpression of transforming growth factor ß1 and fibroblast growth factor-2 by fibroblasts. Overall, this study revealed the possible mechanisms of fibroblast-melanoma interaction and suggested that this co-culture model could be potentially further developed as a platform for screening chemotherapies in the future.

In vitro study of decellularized rat tissues for nerve regeneration.

Peripheral nerve injuries cause an absence or destruction of nerves. Decellularized nerves, acting as a replacement for autografts, have been investigated in the promotion of nerve repair and regeneration, always being incorporated with stem cells or growth factors. However, such a strategy is limited by size availability. The potential application in heterotopic transplantation of other decellularized tissues needs to be further explored. In this study, rat decellularized kidney (dK) was selected to be compared with decellularized peripheral nerve (dN), since dK has aboundant ECM components and growth factors. The PC-12 cells were cultured on dK and dN scaffolds, as shown in the similar behaviors of cell metabolism and viability, but have a more regular arrangement on dN compared to dK, indicating that the natural structure plays an important role in guiding cell extension. However, we found significant upregulation of axon-growth-associated genes and proteins of PC-12 cells in the dK group compared to the dN group by qRT-PCR, immunofluorescence, and western blotting. Furthermore, various neurotrophic factors and growth factors of acellular kidney and nerve were evaluated by ELISA assay. The lower expression of neurotrophic factors but higher expression of growth factors such as VEGF and HGF from dK suggests that axon growth and extension for PC-12 cells may be partially mediated by VEGF and HGF expression from decellularized kidney, which further points to a potential application in nerve repair and regeneration.

The application of 3D bioprinting in urological diseases.

Urologic diseases are commonly diagnosed health problems affecting people around the world. More than 26 million people suffer from urologic diseases and the annual expenditure was more than 11 billion US dollars. The urologic cancers, like bladder cancer, prostate cancer and kidney cancer are always the leading causes of death worldwide, which account for approximately 22% and 10% of the new cancer cases and death, respectively. Organ transplantation is one of the major clinical treatments for urological diseases like end-stage renal disease and urethral stricture, albeit strongly limited by the availability of matching donor organs. Tissue engineering has been recognized as a highly promising strategy to solve the problems of organ donor shortage by the fabrication of artificial organs/tissue. This includes the prospective technology of three-dimensional (3D) bioprinting, which has been adapted to various cell types and biomaterials to replicate the heterogeneity of urological organs for the investigation of organ transplantation and disease progression. This review discusses various types of 3D bioprinting methodologies and commonly used biomaterials for urological diseases. The literature shows that advances in this field toward the development of functional urological organs or disease models have progressively increased. Although numerous challenges still need to be tackled, like the technical difficulties of replicating the heterogeneity of urologic organs and the limited biomaterial choices to recapitulate the complicated extracellular matrix components, it has been proved by numerous studies that 3D bioprinting has the potential to fabricate functional urological organs for clinical transplantation and in vitro disease models.

Promotion of Adrenal Pheochromocytoma (PC-12) Cell Proliferation and Outgrowth Using Schwann Cell-Laden Gelatin Methacrylate Substrate.

Peripheral nerve injuries cause different degrees of nerve palsy and function loss. Due to the limitations of autografts, nerve tissue engineering (TE) scaffolds incorporated with various neurotrophic factors and cells have been investigated to promote nerve regeneration. However, the molecular mechanism is still poorly understood. In this study, we co-cultured Schwann cells (SCs) and rat adrenal pheochromocytoma (PC-12) cells on 50% degrees of methacryloyl substitution gelatin methacrylate (GelMA) scaffold. The SCs were encapsulated within the GelMA, and PC-12 cells were on the surface. A 5% GelMA was used as the co-culture scaffold since it better supports SCs proliferation, viability, and myelination and promotes higher neurotrophic factors secretion than 10% GelMA. In the co-culture, PC-12 cells demonstrated a higher cell proliferation rate and axonal extension than culturing without SCs, indicating that the secretion of neurotrophic factors from SCs can stimulate PC-12 growth and axonal outgrowth. The mRNA level for neurotrophic factors of SCs in 5% GelMA was further evaluated. We found significant upregulation when compared with a 2D culture, which suggested that this co-culture system could be a potential scaffold to investigate the mechanism of how SCs affect neuronal behaviors.

Item-Level Genome-Wide Association Study of the Alcohol Use Disorders Identification Test in Three Population-Based Cohorts.

OBJECTIVE: Genome-wide association studies (GWASs) of the Alcohol Use Disorders Identification Test (AUDIT), a 10-item screen for alcohol use disorder (AUD), have elucidated novel loci for alcohol consumption and misuse. However, these studies also revealed that GWASs can be influenced by numerous biases (e.g., measurement error, selection bias), which may have led to inconsistent genetic correlations between alcohol involvement and AUD, as well as paradoxically negative genetic correlations between alcohol involvement and psychiatric disorders and/or medical conditions. The authors used genomic structural equation modeling to elucidate the genetics of alcohol consumption and problematic consequences of alcohol use as measured by AUDIT. METHODS: To explore these unexpected differences in genetic correlations, the authors conducted the first item-level and the largest GWAS of AUDIT items (N=160,824) and applied a multivariate framework to mitigate previous biases. RESULTS: The authors identified novel patterns of similarity (and dissimilarity) among the AUDIT items and found evidence of a correlated two-factor structure at the genetic level ("consumption" and "problems," rg=0.80). Moreover, by applying empirically derived weights to each of the AUDIT items, the authors constructed an aggregate measure of alcohol consumption that was strongly associated with alcohol dependence (rg=0.67), moderately associated with several other psychiatric disorders, and no longer positively associated with health and positive socioeconomic outcomes. Lastly, by conducting polygenic analyses in three independent cohorts that differed in their ascertainment and prevalence of AUD, the authors identified novel genetic associations between alcohol consumption, alcohol misuse, and health. CONCLUSIONS: This work further emphasizes the value of AUDIT for both clinical and genetic studies of AUD and the importance of using multivariate methods to study genetic associations that are more closely related to AUD.

Genome-wide association study of problematic opioid prescription use in 132,113 23andMe research participants of European ancestry.

The growing prevalence of opioid use disorder (OUD) constitutes an urgent health crisis. Ample evidence indicates that risk for OUD is heritable. As a surrogate (or proxy) for OUD, we explored the genetic basis of using prescription opioids 'not as prescribed'. We hypothesized that misuse of opiates might be a heritable risk factor for OUD. To test this hypothesis, we performed a genome-wide association study (GWAS) of problematic opioid use (POU) in 23andMe research participants of European ancestry (N = 132,113; 21% cases). We identified two genome-wide significant loci (rs3791033, an intronic variant of KDM4A; rs640561, an intergenic variant near LRRIQ3). POU showed positive genetic correlations with the two largest available GWAS of OUD and opioid dependence (rg = 0.64, 0.80, respectively). We also identified numerous additional genetic correlations with POU, including alcohol dependence (rg = 0.74), smoking initiation (rg = 0.63), pain relief medication intake (rg = 0.49), major depressive disorder (rg = 0.44), chronic pain (rg = 0.42), insomnia (rg = 0.39), and loneliness (rg = 0.28). Although POU was positively genetically correlated with risk-taking (rg = 0.38), conditioning POU on risk-taking did not substantially alter the magnitude or direction of these genetic correlations, suggesting that POU does not simply reflect a genetic tendency towards risky behavior. Lastly, we performed phenome- and lab-wide association analyses, which uncovered additional phenotypes that were associated with POU, including respiratory failure, insomnia, ischemic heart disease, and metabolic and blood-related biomarkers. We conclude that opioid misuse can be measured in population-based cohorts and provides a cost-effective complementary strategy for understanding the genetic basis of OUD.

Borneol-modified tanshinone IIA liposome improves cerebral ischemia reperfusion injury by suppressing NF-κB and ICAM-1 expression.

OBJECTIVE: To investigate the metabolism and brain tissue distribution of borneol-modified tanshinone IIA liposome (BO-TA-Lip) and its effect on NF-κB and ICAM-1 in cerebral ischemia reperfusion rats, thereby exploring the ameliorative mechanism of BO-TA-Lip on ischemic encephalopathy. METHODS: Particle size, entrapment efficiency, drug loading were measured to evaluate the preparation comprehensively. Metabolism and brain tissue distributions in vivo were measured by HPLC, and the pharmacokinetic parameters were calculated. In addition, 24 SD rats were randomly divided into sham, model, STS (sodium tanshinone IIA sulfonate, 30 mg/kg) and BO-TA-Lip groups (44 mg/kg). The middle cerebral artery occlusion (MCAO) rats were constructed with thread embolism method. Neurological deficits were scored using Zea Longa scoring standard. TTC and HE staining were used for the cerebral infarction and histopathological examination, respectively. The protein expression was examined by immunohistochemistry and Western blot. RESULTS: The average particle size, encapsulation efficiency and drug loading of BO-TA-Lip were (135.33 ± 7.25) nm, (85.95 ± 3.20)% and (4.06 ± 0.31)%, respectively. Both in the pharmacokinetic analysis of plasma and brain tissue, in BO-TA-Lip group, the peak concentration and the area under the curve increased, and the clearance rate decreased. The neurological deficit scores and infarct area of the BO-TA-Lip group were significantly lower than that of the model and STS groups. Besides, BO-TA-Lip reduced the protein expression of NF-κB, ICAM-1, IL-1ß, TNF-α and IL-6 in the brain tissue. CONCLUSION: BO-TA-Lip had higher bioavailability and better absorption in brain tissue, and could improve cerebral ischemia reperfusion injury, which might be related to the inhibitory effect of BO-TA-Lip in expression of NF-κB and ICAM-1.

Upregulation of miR-138 Increases Sensitivity to Cisplatin in Hepatocellular Carcinoma by Regulating EZH2.

Chemotherapeutic insensitivity is a major obstacle for effective treatment of hepatocellular carcinoma (HCC). Recently, new evidence showed that microRNAs (miRNAs) are closely related to drug sensitivity. This study aimed to investigate the relationship between miR-138 expression and cisplatin sensitivity of HCC cells by regulation of EZH2. CCK-8, EdU, and western blotting are determining the cell viability, proliferation, EZH2, and EMT-related protein expression. It was found that compared with normal samples, miR-138 expression was lower in cancer tissue; it was also downregulated in HCC cells. Transfected with miR-138 mimic increased sensitivity of HCC cells to cisplatin. Mechanistically, Luciferase Reporter analysis verified the interaction between miR-138 and target gene EZH2. Inhibition of EZH2 enhanced cisplatin sensitivity and transfection with EZH2 mimic mirrored the function of miR-138 in cisplatin sensitivity. Furthermore, the role of miR-138 on reversed cisplatin-induced epithelial-mesenchymal transition (EMT) was attenuated when combined with EZH2 plasmid. In conclusion, all data from this study illustrate that miR-138 may as a tumor suppressor provides a potential treatment method to treating HCC.

Mitogen-Inducible Gene 6 Inhibits Angiogenesis by Binding to SHC1 and Suppressing Its Phosphorylation.

The mitogen-inducible gene 6 (MIG6) is an adaptor protein widely expressed in vascular endothelial cells. However, it remains unknown thus far whether it plays a role in angiogenesis. Here, using comprehensive in vitro and in vivo model systems, we unveil a potent anti-angiogenic effect of MIG6 in retinal development and neovascularization and the underlying molecular and cellular mechanisms. Loss of function assays using genetic deletion of Mig6 or siRNA knockdown increased angiogenesis in vivo and in vitro, while MIG6 overexpression suppressed pathological angiogenesis. Moreover, we identified the cellular target of MIG6 by revealing its direct inhibitory effect on vascular endothelial cells (ECs). Mechanistically, we found that the anti-angiogenic effect of MIG6 is fulfilled by binding to SHC1 and inhibiting its phosphorylation. Indeed, SHC1 knockdown markedly diminished the effect of MIG6 on ECs. Thus, our findings show that MIG6 is a potent endogenous inhibitor of angiogenesis that may have therapeutic value in anti-angiogenic therapy.

A novel HPLC method for analysis of atosiban and its five related substances in atosiban acetate injection.

Consulting the national pharmacopoeia, no official quality standard was found for estimation of related substances and assay of atosiban acetate injection, of which main active component is atosiban. To solve this problem, herein, a novel high performance liquid chromatographic (HPLC) method was developed and validated in this study. A chromatographic system comprising an Inertsil ODS-2 analytical column, mobile phase-A of water (pH adjusted to 3.2 with trifluoroacetic acid)-acetonitrile-methanol (77:14:9, v/v/v), mobile phase-B of acetonitrile-methanol (65:35, v/v), a flow rate of 1.0 mL min-1 and a UV detector set at 220 nm with column temperature at 35 °C has shown simple, reproducible and specific determination for atosiban and its five related substances. Also, we combined with mass spectrometry to characterize the molecular weight and tentative structure of the impurities. Using HPLC verified methodology, results of the validation study showed that the precision, specificity and accuracy of the five impurities, good linear equation R squared was greater than 0.9993, and as such, the limit of detection and the limit of quantification have been determined. The proposed method in this study, which, to the best of our knowledge, is the most comprehensive HPLC determination applied to the routine analysis in quality control of this injection.

Dust mite-derived Der f 3 activates a pro-inflammatory program in airway epithelial cells via PAR-1 and PAR-2.

Protease activity of allergens has been suggested to be involved in the pathogenesis of allergic diseases. The major allergen Der f 3 from Dermatophagoides farinae harbors serine protease activity, but its immunopathogenesis remains unclear. This study aims to explore the effect of Der f 3 on the airway epithelial barrier and on the molecular pathways by which Der f 3 induces inflammation. RNA-seq was performed to identify differentially expressed genes in bronchial airway epithelial cells (AEC) between native Der f 3 and heat-inactivated (H) Der f 3, coupled with real-time PCR (RT-PCR) and ELISA for validation. Unlike other protease allergens such as that induce Th2-promoting alarmins (IL-25, IL-33, TSLP) in AECs, Der f 3 induced pro-inflammatory cytokines and chemokines including IL-6, IL-8 and GM-CSF, which are known to promote Th17 response. These pro-inflammatory mediators were induced by Der f 3 via the MAPK and NF-κB pathways as well as the store-operated calcium signaling. Gene silencing with small interfering RNA in A549 and BEAS-2B cells indicated that activation of AECs by Der f 3 was mainly dependent on protease-activated receptor 2 (PAR-2), while PAR-1 was also required for the full activation of AECs. Double knock-down of PAR-1 and PAR-2 largely impaired Der f 3-inducecd IL-8 production and subsequent signaling pathways. Our data suggest that Der f 3 induces pro-inflammatory mediators in human epithelial cell lines via the PARs-MAPK-NF-κB axis. Our results provide a molecular mechanism by which Der f 3 may trigger the Th17-skewed allergic response toward house dust mites.